The Interactive Fly

Genes involved in tissue and organ development

Mesoderm and Muscle

Mesoderm Development

Motoneurons regulate myoblast proliferation and patterning in Drosophila
A nutrient sensor mechanism controls Drosophila growth
Mononuclear muscle cells in Drosophila ovaries revealed by GFP protein traps
Variation in mesoderm specification across drosophilids is compensated by different rates of myoblast fusion during body wall musculature development
Founder cells regulate fiber number but not fiber formation during adult myogenesis in Drosophila
Surface apposition and multiple cell contacts promote myoblast fusion in Drosophila flight muscles
Identification and functional characterization of muscle satellite cells in Drosophila
Nanoscopy reveals the layered organization of the sarcomeric H-zone and I-band complexes
Flying high-muscle-specific underreplication in Drosophila

Gene Expression during Mesoderm Development
Mesoderm migration in Drosophila is a multi-step process requiring FGF signaling and integrin activity
Gene expression during mesoderm development
A core transcriptional network for early mesoderm development
A machine learning approach for identifying novel cell type-specific transcriptional regulators of myogenesis
Restricted gene expression patterns in somatic muscles
Notch and Ras signaling pathway effector genes expressed in fusion competent and founder cells during Drosophila myogenesis
Shadow enhancers are pervasive features of developmental regulatory networks
The formin Diaphanous regulates myoblast fusion through actin polymerization and Arp2/3 regulation
A non-signaling role of Robo2 in tendons is essential for Slit processing and muscle patterning
The bHLH transcription factor Hand is required for proper wing heart formation in Drosophila
Noncanonical roles for Tropomyosin during myogenesis
Tet protein function during Drosophila development
The scaffolding protein Cnk binds to the receptor tyrosine kinase Alk to promote visceral founder cell specification in Drosophila
Drosophila Hsp67Bc hot-spot variants alter muscle structure and function
The glutamic acid-rich long C-terminal extension of troponin T has a critical role in insect muscle functions
The cis-regulatory dynamics of embryonic development at single-cell resolution
Transcriptional silencers in Drosophila serve a dual role as transcriptional enhancers in alternate cellular contexts
Binding partners of the kinase domains in Drosophila obscurin and their effect on the structure of the flight muscle
Characterizing the actin-binding ability of Zasp52 and its contribution to myofibril assembly
A Novel Mechanism for Activation of Myosin Regulatory Light Chain by Protein Kinase C-Delta in Drosophila
Drosophila NUAK functions with Starvin/BAG3 in autophagic protein turnover

Muscle derived Myoglianin regulates wing growth
Muscle-derived Myoglianin regulates Drosophila imaginal disc growth

The neuromuscular system
Deconstruction of the beaten Path-Sidestep interaction network provides insights into neuromuscular system development
Drosophila adult muscle precursor cells contribute to motor axon pathfinding and proper innervation of embryonic muscles

Muscle growth, physiology, movement and cell death
A model of muscle atrophy based on live microscopy of muscle remodelling in Drosophila metamorphosis
Genetic screen in Drosophila muscle identifies autophagy-mediated T-tubule remodeling and a Rab2 role in autophagy
Glycolysis supports embryonic muscle growth by promoting myoblast fusion
The Dmca1D channel mediates Ca inward currents in Drosophila embryonic muscles
The Drosophila formin Fhos is a primary mediator of sarcomeric thin-filament array assembly
Spatial pattern analysis of nuclear migration in remodelled muscles during Drosophila metamorphosis
Distortion of the Actin A-triad results in contractile disinhibition and cardiomyopathy
Live imaging and analysis of muscle contractions in Drosophila embryo
Drosophila Nedd4-long reduces Amphiphysin levels in muscles and leads to impaired T-tubule formation
Transcriptome analysis of IFM-specific actin and myosin nulls in Drosophila melanogaster unravels lesion-specific expression blueprints across muscle mutations
Five Alternative Myosin converter domains influence muscle power, stretch activation, and kinetics
Cell death regulates muscle fiber number
A comprehensive anatomical map of the peripheral octopaminergic/tyraminergic system of Drosophila melanogaster
Myofibril diameter is set by a finely tuned mechanism of protein oligomerization in Drosophila
Reverse engineering forces responsible for dynamic clustering and spreading of multiple nuclei in developing muscle cells
Microtubules provide guidance cues for myofibril and sarcomere assembly and growth
Diet-MEF2 interactions shape lipid droplet diversification in muscle to influence Drosophila lifespan
Systemic muscle wasting and coordinated tumour response drive tumourigenesis

Moss-Taylor, L., Upadhyay, A., Pan, X., Kim, M. J. and O'Connor, M. B. (2019). Body size and tissue-scaling is regulated by motoneuron-derived activinbeta in Drosophila melanogaster. Genetics. PubMed ID: 31585954

Upadhyay, A., Peterson, A. J., Kim, M. J. and O'Connor, M. B. (2020). Muscle-derived Myoglianin regulates Drosophila imaginal disc growth. Elife 9:e51710. PubMed ID: 32633716

Muscle-derived Myoglianin regulates Drosophila imaginal disc growth

Organ growth and size are finely tuned by intrinsic and extrinsic signaling molecules. In Drosophila, the BMP family member Dpp is produced in a limited set of imaginal disc cells and functions as a classic morphogen to regulate pattern and growth by diffusing throughout imaginal discs. However, the role of TGFβ/Activin-like ligands in disc growth control remains ill-defined. This study demonstrates that Myoglianin (Myo), an Activin family member, and a close homolog of mammalian Myostatin (Mstn), is a muscle-derived extrinsic factor that uses canonical dSmad2-mediated signaling to regulate wing size. It is proposed that Myo is a myokine that helps mediate an allometric relationship between muscles and their associated appendages. Although Babo/dSmad2 signaling has been previously implicated in imaginal disc growth control, the ligand(s) responsible and their production sites(s) have not been identified. Previous in situ hybridization and RNAi knockdown experiments suggested that all three Activin-like ligands contribute to control of wing size. However, no expression of these Activin-like ligands was found in imaginal discs, with the exception of Actβ which is expressed in differentiating photoreceptors of the eye imaginal disc. It is concluded that the small wing phenotypes caused by RNAi knockdown of Actβ or daw are likely the result of off-target effects and that Myo is the only Activin-type ligand that regulates imaginal disc growth (Upadhyay, 2020).

Although Babo/dSmad2 signaling has been previously implicated in imaginal disc growth control, the ligand(s) responsible and their production sites(s) have not been identified. Previous in situ hybridization and RNAi knockdown experiments suggested that all three Activin-like ligands (Myoglianin, Activinβ, and Dawdle) contribute to control of wing size. However, this study found no expression of these Activin-like ligands in imaginal discs, with the exception of Actβ which is expressed in differentiating photoreceptors of the eye imaginal disc. More importantly, using genetic null mutants, this study showed that only loss of myo affects imaginal disc size. The discrepancy in phenotypes between tissue-specific knockdown results and the genetic nulls is often noted and not fully understood. In addition to simple off-target effects within the wing disc itself, one possible explanation is that many GAL4 drivers are expressed in tissues other than those reported, potentially resulting in deleterious effects for the animal that indirectly affect imaginal disc size. Another possibility is that in Actβ and daw genetic null backgrounds a non-autonomous compensatory signal is generated by another tissue and this signal is not activated in the case of tissue-specific knockdown. Both of these explanations are thought unlikely in this instance since it was demonstrated that only the Babo-A receptor isoform is expressed and required in discs. Since it was previously shown that Daw only signals through isoform Babo-C, it is unclear why knockdown of daw in the wing disc would result in a small wing phenotype as previously reported. It is concluded that the small wing phenotypes caused by RNAi knockdown of Actβ or daw are likely the result of off-target effects and that Myo is the only Activin-type ligand that regulates imaginal disc growth (Upadhyay, 2020).

The signaling ability of TGFβ ligands is modulated by the specific combinations of receptors and co-receptors to which they bind. In Drosophila, the receptor requirements for effective signaling through dSmad2 likely vary for each ligand and tissue. This study found that Myo signaling in the wing disc requires Punt as the type II receptor and Babo-A as the type I receptor. Furthermore, it was establish that Myo is the exclusive Activin-like ligand signaling to the discs since loss of Myo eliminated detectable phosphorylation of dSmad2 in the wing imaginal disc. Since Babo-A is the only isoform expressed in wing discs, it is also concluded that Myo is able to signal through this isoform in the absence of other isoforms. Whether Myo can also signal through Babo-B or C is not yet clear, but in the context of mushroom body remodeling Babo-A also appears to be the major receptor isoform utilized. The co-receptor Plum (see Plum, an Immunoglobulin Superfamily Protein, Regulates Axon Pruning by Facilitating TGF-β Signaling) is also required for mushroom body remodeling, suggesting that Plum and Babo-A are both necessary for efficient Myo signaling. However, it is noteworthy that Plum null mutants are viable while Myo null mutants are not. This observation suggests that Plum is not required for all Myo signaling during development. Further studies will be required to evaluate whether Plum is essential to mediate Myo signaling in imaginal discs (Upadhyay, 2020).

The requirement of Punt as a type II receptor for production of an efficient signaling complex with Myo may be context dependent. In the mushroom body, indirect genetic evidence suggests that the two Type II receptors function redundantly. Although both punt and wit are expressed in imaginal discs, only loss of punt produces a phenotype in the brk reporter assay. To date, clear signaling has not been seen in S2 cells expressing Punt and Babo-A when Myo is added. It is also notable that a previous attempt to study Myo signaling in a heterotypic cell culture model also failed. In that study, Myo was found to form a complex with Wit and Babo-A in COS-1 cells but no phosphorylation of dSmad2 was reported. One explanation is that effective signaling by Myo requires Punt, and Babo-A, and perhaps another unknown co-receptor that substitutes for Plum. Despite this caveat, the current results provide in vivo functional evidence for a Myo signaling complex that requires Babo-A and Punt to phosphorylate dSmad2 for regulation of imaginal disc growth (Upadhyay, 2020).

Final tissue size is determined by several factors including cell size, proliferation, death rates, and duration of the growth period. While cell size changes were observed upon manipulation of Myo signaling, the direction of change depended on the genotype. In myo mutants, estimation of cell size via apical surface area indicates that the cells are ~20% smaller than wild-type. Although this measurement does not indicate the actual volume of the cells, it gives an indication of cell density in the epithelial sheet of the wing pouch, which is analogous to counting cells in the adult wing. RNAi knock down of babo-a in the entire disc produced smaller adult wings with larger (less dense) cells. This result differs from the myo mutant, but is similar to the reported adult wing phenotypes of babo mutants and larval disc phenotypes of dSmad2 mutants. When babo-a is knocked down in one compartment, that compartment is reduced in size with smaller cells. It is concluded that tissue size reduction is the consistent phenotype upon loss of Myo signaling, but cell size changes depend on the specific type of manipulation (Upadhyay, 2020).

While cell size effects may be context dependent, it is notable that neither reduction in size of imaginal discs nor adult wing surface area can be explained solely by a cell size defect. Since no apoptotic increase was seen in myo mutant discs, and because dSmad2 knockdown also fails to alter apoptotic rate, the mostly likely cause is an altered proliferation rate. Consistent with this view is that the large disc phenotype exhibited by dSmad2 protein null mutants is clearly dependent on Myo and it has been previously shown that this is the result of enhanced proliferation. Similarly earlier studies also showed that expression of activated Babo or activated dSmad2 in wing discs also leads to larger wings with slightly smaller cells which is most easily explained by an enhanced proliferation rate. It is worth noting that this proposed enhanced proliferation rate is difficult to detect since cell division is random with regard to space and time during development. Thus a ~ 20% reduction in adult wing size caused by a proliferation defect translates into about 1/5th of disc cells dividing on average one less time throughout the entire time course of larval development. Therefore, without prolonged live imaging, this small reduction in proliferation rate will not be detectable using assays that provide only static snapshots of cell division. It is worth noting, however, that previous clonal studies also concluded that dSmad2 or Babo loss in wing disc clones resulted in a reduced proliferation rate (Upadhyay, 2020).

One attempt to shed light on the transcriptional output of TGFβ signaling responsible for wing disc size employed microarray mRNA profiling of wild-type versus dSmad2 gain- and loss-of-function wing discs. However, this study did not reveal a clear effect on any class of genes including cell cycle components, and it was concluded that the size defect is the result of small expression changes of many genes. Consistent with this view are dSmad2 Chromatin Immunoprecipitation experiments in Kc cells which revealed that dSmad2 is associated with many genomic sites and thus may regulate a myriad of genes (Upadhyay, 2020).

Insect myoglianin is a clear homolog of vertebrate Myostatin (Mstn/GDF8), a TGFβ family member notable for its role in regulating skeletal muscle mass. Mstn loss-of-function mutants lead to enlarged skeletal muscles. Mstn is thought to affect muscle size through autocrine signaling that limits muscle stem cell proliferation, as well as perturbing protein homeostasis via the Insulin/mTOR signaling pathways. Similarly, Gdf11, a Mstn paralog, also regulates size and proliferation of muscles and adipocytes, and may promote healthy aging. Mstn and Gdf11 differ in where they are expressed and function. Mstn is highly expressed in muscles during development while Gdf11 is weakly expressed in many tissues. Both molecules are found to circulate in the blood as latent complexes in which their N-terminal prodomains remain associated with the ligand domain. Activation requires additional proteolysis of the N-terminal fragment by Tolloid-like metalloproteases to release the mature ligand for binding to its receptors. Interestingly in Drosophila, the Myoglianin N-terminal domain was also found to be processed by Tolloid-like factors, but whether this is a prerequisite for signaling has not yet been established. In terms of functional conservation in muscle size control, the results of both null mutants and RNAi depletion indicates that it has little effect on muscle size. This contradicts a previous study in which muscle-specific RNAi knockdown of myo was reported to produce larger muscles similar to the vertebrate observation. The discrepancy between the tissue-specific RNAi knockdown and previous studies is not clear, but the current null mutant analysis strongly argues that Drosophila Myo does not play a role in muscle size control. Intriguingly however, this study found that loss of Actβ, another ligand that signals through Babo and dSmad2, results in a smaller muscles) contrary to that produced by loss of vertebrate Mstn and various other vertebrate Activin family members. Recent data has shown that Drosophila Actβ is the only Activin-like ligand that affects muscle growth, and it does so, in part, by regulating Insulin/Tor signaling in the opposite direction compared to vertebrates. Thus, in Drosophila the Myo/Activin pathway promotes muscle growth while in vertebrates it inhibits muscle growth (Upadhyay, 2020).

The most intriguing finding of this study is that muscle-derived Myo acts non-autonomously to regulate imaginal disc growth. This is in stark contrast to the two BMP ligands, Dpp and Gbb, which are produced by disc cells and act autonomously within the disc itself to regulate both growth and pattern. The fact that a TGFβ ligand can act in an endocrine-like manner is not particularly novel since many vertebrate members of the TGFβ family, including Myostatin, the closest homolog to Drosophila Myoglianin, are found in the blood. Even the disc intrinsic molecule Dpp has been recently shown to be secreted into the hemolymph where it circulates and signals to the prothoracic gland to regulate a larval nutritional checkpoint. Several additional reports indicate that ligands from the Drosophila Activin-like subfamily also circulate in the hemolymph and function as inter-organ signals. For example, muscle-derived Actβ and Myo signal to the fat body to regulate mitochondrial function and ribosomal biogenesis, respectively. In addition, Daw produced from many tissue sources can signal to the Insulin producing cells and the midgut to stimulate Insulin secretion and repress expression of sugar metabolizing genes, respectively. Thus, many TGFβ type factors act as both paracrine and endocrine signals depending on the tissue and process involved (Upadhyay, 2020).

The phenotype of the myo mutant animal supports the claim that endogenous Myo contributes to imaginal disc growth. The ectopic expression assay produced various wing disc sizes when Myo was expressed in different tissues, indicating that the growth response likely depends on the level of Myo being produced in the distal tissue. Loss of glial derived Myo is not sufficient to suppress overgrowth of dSmad2 mutant discs, but overexpression of Myo in glia did partially rescue size of myo null wing discs, likely because the repo-Gal4 driven overexpression produces more ligand than endogenous glia. Likewise, expression from a large tissue such as muscle or fat body likely produces more Myo than glia leading to normal disc growth or even overgrowth. It is also possible that Myo signaling activity is modified depending on the tissue source. Like other TGFβ family members, Myo requires cleavage by a furin protease at its maturation site to separate the C-terminal ligand from the prodomain. Myo may also require a second cleavage by a Tolloid protease family member to achieve full dissociation of the prodomain from the ligand to ensure complete activation. Either of these cleavage reactions, or any other step impacting the bioavailability of active Myo ligand, may vary with tissue or may be modulated by environmental conditions (Upadhyay, 2020).

What is the rationale for larval muscle regulating imaginal discs size? A possible reason is that for proper appendage function, the muscle and the structure (leg, wing, and haltere) that it controls should be appropriately matched to ensure optimal organismal fitness for the environmental niche the adult occupies. For example, a large muscle powering a small wing might result in diminished fine motor control. Conversely, a small muscle may not be able to power a large wing to support flight. However, the multi-staged nature of muscle and appendage development complicates this picture. Larval muscles are histolysed during metamorphosis and do not contribute to the adult muscle. However, remnants of larval muscles in the thoracic segment are preserved as fibers that act as scaffolds upon which the larval myoblasts infiltrate and fuse to become the adult indirect flight muscles. Thus, at least for the indirect flight muscles, the size of the larval muscle scaffold might contribute to the building of a bigger adult muscle. Another possibility invokes a signal relay system. Wg and Serrate/Notch signaling from the wing disc epithelial cells control myoblast proliferation during larval development. Thus it may be that Myo signaling from the larval muscles stimulates proliferation of the disc epithelial layer which in turn enhances Wnt and Serrate/Notch signaling to myoblasts to increase their number thereby coordinating the adult appendage size with muscle size. A final scenario is that, since muscle is a major metabolic and endocrine organ, Myo production may be regulated by the general metabolic state of the larva. If healthy, high levels of Myo, in concert with other growth signals such as insulin, leads to a bigger fly with large wings, and if the metabolic state is poor then lower Myo levels leads to diminished proliferation and a smaller cell size resulting in a smaller fly with small wings (Upadhyay, 2020).

Regardless of the precise mechanism, the ability of the muscle to control appendage size has interesting implications in terms of evolutionary plasticity. The proportionality of insect wing size to body size can vary over a large range, but the mechanism responsible for determining this particular allometric relationship for a given species is not understood. It was recently demonstrated that in Drosophila, motor neuron derived Actβ, another TGFβ superfamily member, can dramatically affect muscle/body size (Moss-Taylor, 2019). Therefore, it is tempting to speculate that evolutionary forces might modulate the activity of these two genes to produce an appropriate body-wing allometry that is optimal for that species' ecological niche (Upadhyay, 2020).

Fat Body
Fat Body
Genetic control of the distinction between fat body and gonadal mesoderm
Role and regulation of starvation-induced autophagy in the Drosophila fat body

Development of Haemocytes and the Lymph Gland
The two origins of hemocytes in Drosophila
The Drosophila lymph gland as a developmental model of hematopoiesis
Subdivision and developmental fate of the head mesoderm in Drosophila
Mechanical tension and spontaneous muscle twitching precede the formation of cross-striated muscle in vivo
Dynamics of transcriptional (re)-programming of syncytial nuclei in developing muscles

Genes expressed in mesoderm and mesodermal derivatives including muscle

Please note: c, s, or v = expression in cardiac, somatic or visceral musculature; h = expression in head mesoderm, d = expression in dorsal medial cells, f = expression in fat body, h = expression in hemocytes

Mesoderm migration in Drosophila is a multi-step process requiring FGF signaling and integrin activity

Migration is a complex, dynamic process that has largely been studied using qualitative or static approaches. As technology has improved, it is now possible to take quantitative approaches towards understanding cell migration using in vivo imaging and tracking analyses. In this manner, a four-step model of mesoderm migration during Drosophila gastrulation was establised: (I) mesodermal tube formation, (II) collapse of the mesoderm, (III) dorsal migration and spreading and (IV) monolayer formation. The data provide evidence that these steps are temporally distinct and that each might require different chemical inputs. To support this, the role was analyzed of fibroblast growth factor (FGF) signaling, in particular the function of two Drosophila FGF ligands, Pyramus and Thisbe, during mesoderm migration. It was determined that FGF signaling through both ligands controls movements in the radial direction. Thisbe is required for the initial collapse of the mesoderm onto the ectoderm, whereas both Pyramus and Thisbe are required for monolayer formation. In addition, it was uncovered that the GTPase Rap1 regulates radial movement of cells and localization of the beta-integrin subunit, Myospheroid, which is also required for monolayer formation. These analyses suggest that distinct signals influence particular movements, since it was found that FGF signaling is involved in controlling collapse and monolayer formation but not dorsal movement, whereas integrins are required to support monolayer formation only and not earlier movements. This work demonstrates that complex cell migration is not necessarily a fluid process, but suggests instead that different types of movements are directed by distinct inputs in a stepwise manner (McMahon, 2010).

Mesoderm migration was found to be a combination of complex three-dimensional movements involving many molecular components. live imaging, coupled with quantitative analyses, is important for studying complex cell movements, as it allowed migration to be decomposed into different movement types and thus has allowed description of subtle phenotypes. First, analysis of the directional movements of mesoderm cells within wild-type embryos was extended, focusing on the temporal sequences of events. Cells were found follow a sequential and distinct set of trajectories: movement in the radial direction (tube collapse: -5 to 15 minutes, 0=onset of germband elongation), followed by movement in the angular direction (dorsal migration: 15 to 75 minutes) and ending with small intercalation movements in the radial direction (monolayer formation: 75 to 110 minutes). These movements appear temporally distinct (i.e. stepwise), and thus molecular signals controlling each process were sought (McMahon, 2010).

Which mesoderm movements were FGF-dependent were investigated and, in particular, either Ths- or Pyr-dependent. The interaction between Htl and its two ligands provides a simpler system relative to vertebrates (which exhibit over 120 receptor-ligand interactions) in which to study how and why multiple FGF ligands interact with the same receptor. Previously, it was found that FGF signaling via the Htl FGFR controls collapse of the mesodermal tube but not dorsal-directed spreading (McMahon, 2008). This study demonstrated that FGF signaling is also required for monolayer formation. In addition, distinct, non-redundant roles were defined for the FGF ligands: Ths (but not Pyr) is required for collapse of the mesodermal tube, whereas both Pyr and Ths are required for proper intercalation of mesoderm cells after dorsal spreading (McMahon, 2010).

This analysis raises questions about ligand choice during collapse and monolayer formation. Within the mesodermal tube, cells at the top require a long-range signal in order to orient towards the ectoderm during tube collapse, whereas the signals controlling intercalation during monolayer formation can be of shorter range. It is suggested that the ligands have different activities that are appropriately tuned for these processes. In fact, recent studies of the functional domains of these proteins suggest that Ths has a longer range of action than Pyr, in agreement with the analysis that Pyr does not support tube collapse, but does have a hand in monolayer formation (McMahon, 2010).

This study has demonstrated that Rap1 mutants have a similar mesoderm phenotype to the FGFR htl mutant, with defects in collapse and monolayer formation. It was not possible to establish whether Rap1 acts downstream of FGF signaling, as the complete loss of Mys in Rap1 mutants is more severe than the patchy expression of Mys seen in htl mutants. Therefore, Rap1 could be working in parallel to or downstream of FGF signaling during mesoderm migration. Rap1 has been implicated in several morphogenetic events during Drosophila gastrulation and probably interacts with many different signaling pathways. Further study of Rap1, along with other GTPases, will shed light onto their role during mesoderm migration, how they interact with one another and what signaling pathways control them (McMahon, 2010).

Focus was placed on the more specific phenotype of mys mutants, as its localization is affected in htl mutants and it exhibits a monolayer defect that is similar to pyr and ths mutants. Integrins are important for cell adhesion, so it is not surprising that cells fail to make stable contact with the ectoderm through intercalation in mys mutants. However, some cells do contribute to monolayer formation in the absence of Mys, implying that other adhesion molecules are involved in maintaining contact between the mesoderm and ectoderm. These other adhesion molecules might be activated downstream of FGF signaling as the htl mutant monolayer phenotype is more severe than the mys mutant. Discovering the downstream targets of Htl, which might regulate cell adhesion properties, will help to shed light on the mechanisms supporting collapse of the mesodermal tube (which is not dependent on Mys) and monolayer formation (which is Mys-dependent) (McMahon, 2010).

Cell protrusions, such as filopodia, are important for sensing chemoattractants and polarizing movement during migration. Previous studies have focused on protrusive activity at the leading edge during mesoderm migration in Drosophila and shown that these protrusions are FGF-dependent. In this study, it was found that protrusions exist in all mesoderm cells, not just the leading edge, and that these protrusions also extend into the ectoderm (McMahon, 2010).

The study demonstrates that FGF signaling, as well as integrin activity, is required to support protrusive activity into the ectoderm; this is a potential mechanism by which FGF signaling and Mys could control movement toward the ectoderm during monolayer formation. The function of protrusions at the leading edge remains unclear, as they appear to be reduced in pyr and mys mutants, but migration in the dorsal direction still occurs in both mutant backgrounds. One interpretation is that FGF and Mys are important for generalized protrusive activity and that extensive protrusions are required for intercalation but not dorsal migration (McMahon, 2010).

Based on this study, it is proposed that mesoderm migration is a stepwise process, with each event requiring different molecular cues to achieve collective migration. Invagination of the mesoderm is the first step in this process and is dependent on Snail, Twist, Concertina, Fog and several other genes. Next, collapse of the mesoderm tube onto the ectoderm requires Htl activation via Ths. Rap1 might be involved in this process as well but the phenotype of Rap1 mutants is complex and it is unclear which phenotypes are primary defects (McMahon, 2010).

Following collapse, mesoderm cells spread dorsally by an unknown mechanism. Dorsal migration is unaffected in pyr and ths mutants and occurs in all cells that contact the ectoderm in htl mutants, implying that FGF signaling is, at most, indirectly involved in this step owing to the earlier tube collapse defect (McMahon, 2008). Whether dorsal migration requires chemoattractive signals or whether the cells simply move in this direction because it is the area of least resistance remains unclear (McMahon, 2010).

Finally, after dorsal spreading is complete, any remaining cells not contacting the ectoderm intercalate to form a monolayer. This process is controlled by a combination of both Pyr and Ths interacting through Htl and also by Rap1 and Mys. In other systems, intercalation can lead to changes in the properties of the cell collective, for instance, lengthening of a body plan. However, this study has shown that dorsal migration and spreading are not a result of intercalation, as intercalation occurs after spreading has finished (McMahon, 2010).

Coordination of these signals to control collective migration enables the mesoderm to form a symmetrical structure, which is essential for embryo survival. This model begins to address the question of how hundreds of cells move in concerted fashion and is relevant for a generalized understanding of embryogenesis and organogenesis. It was found that mesoderm migration is accomplished through sequential movements in different directions, implying that collective migration might be best achieved by distinct phases of movement (McMahon, 2010).

Gene expression during mesoderm development

The transcription factor Twist initiates Drosophila mesoderm development, resulting in the formation of heart, somatic muscle, and other cell types. Using a Drosophila embryo sorter, enough homozygous twist mutant embryos were isolated to perform DNA microarray experiments. Transcription profiles of twist loss-of-function embryos, embryos with ubiquitous twist expression, and wild-type embryos were compared at different developmental stages. The results implicate hundreds of genes, many with vertebrate homologs, in stage-specific processes in mesoderm development. One such gene, gleeful/lame duck, related to the vertebrate Gli genes, is essential for somatic muscle development and is sufficient to cause neural cells to express a muscle marker (Furlong, 2001).

Formation of muscles during embryonic development is a complex process that requires coordinate actions of many genes. Somatic, visceral, and heart muscle are all derived from mesoderm progenitor cells. The Drosophila twist gene, which encodes a bHLH transcription factor, is essential for multiple steps of mesoderm development: invagination of mesoderm precursors during gastrulation, segmentation, and specification of muscle types. The role of twist in mesoderm development has been conserved during evolution, perhaps because it controls conserved regulatory mesoderm genes. For example, tinman and dMef2 are regulated by Twist in flies and are highly conserved in sequence and function in vertebrates (Furlong, 2001).

In Drosophila, somatic muscle forms from progenitor cells that divide to become muscle founder cells. Founder cells acquire unique identities controlled by transcription factors including Krüppel, S59, vestigial, and apterous. Each of the 30 body wall muscles in an abdominal hemisegment is initiated by a single founder cell and has unique attachments and innervations. To further clarify mechanisms underlying founder cell specification, myoblast fusion, and muscle patterning, Drosophila mutants together with microarrays of cDNA clones were used (Furlong, 2001).

twist mutant embryos develop no mesoderm. A population of mRNA species isolated from twist homozygous embryos was compared with that of stage-matched wild-type embryos. Drosophila lethal mutations are maintained as heterozygotes, in trans to balancer chromosomes. A twist mutation was established in trans to a balancer chromosome carrying a transgene encoding green fluorescent protein (GFP). Embryos were collected from wild-type and twist/GFP-balancer fly stocks at specific developmental stages. The twist/GFP-balancer collections contain a mixed population of embryos: one-quarter twist homozygotes lacking GFP, half heterozygotes with one copy of GFP, and one-quarter homozygous for the balancer chromosome with two copies of GFP. Homozygous twist mutant embryos were separated from their siblings using an embryo sorter. Putative homozygous twist embryos were assessed by immunostaining with an antibody to dMef2. More than 99% of the selected embryos had the twist phenotype (Furlong, 2001).

Three different periods of mesoderm development were analyzed: stages 9-10, 11, and 11-12 . During stage 9-10, the earliest time GFP is detectable in the balancer embryos, mesoderm cells migrate dorsally and become specified as somatic, visceral, cardiac, and fat body mesoderm. twist and its direct targets tinman and dMef2 are expressed throughout stage 9-10 mesoderm. The middle period contains stage 11 embryos and is a transition between the first period (stage 9-10) and the third period (late stage 11-12). During late stage 11 and stage 12, myoblast fusion begins and twist expression remains prominent in only a subset of the somatic muscle cells (Furlong, 2001).

For each developmental period, three independent embryo collections, embryo sortings, and microarray hybridizations were conducted. The microarrays used for the analysis contained over 8500 cDNAs corresponding to 5081 unique genes plus a variety of controls. Each embryonic RNA sample was compared with a reference sample, containing RNA made from all stages of the Drosophila life cycle to allow direct comparisons among all the experiments (Furlong, 2001).

To determine how transcription was affected by the twist mutation, SAM (significance analysis of microarrays) analysis was used. Genes that are normally highly expressed in mesoderm should have lower transcript levels in twist homozygotes. Genes in other tissues whose expression depends on signals from the mesoderm might also have reduced expression. Transcripts of 130 genes, the 'Twist-low' group, were significantly lower in twist mutants than in wild type. Conversely, cells that would have formed mesoderm may take on other fates in the absence of twist, such as neuroectoderm; therefore, many transcript levels could increase in twist mutants. Genes whose transcription is repressed by signals from the mesoderm would also be enriched in twist mutants. One hundred fifty genes, called the 'Twist-high' group, have increased levels of RNA in twist mutant embryos (Furlong, 2001).

In total, 280 of ~5000 genes had significant changes in transcript levels, with 10 false positives. The genes on the array include 15 previously characterized mesoderm-specific genes, all of which are significantly reduced in twist mutant embryos. The arrays also contain genes known to be transcribed in both mesoderm and other cell types. Significant changes in expression were detected for many of these genes (Furlong, 2001).

The 130 Twist-low genes were divided into three groups with similar trends of expression by a self-organizing map (SOM) clustering program. The 24 group A genes, which include tinman, dMef2, and bagpipe, have reduced transcript levels in twist mutants at all developmental stages assayed. Most of the Twist-low genes fall into the B and C groups. The 62 group B 'early genes' encode transcripts with reduced levels of expression in twist mutants only during stages 9-10, not later. One member of group B, stumps (dof/hbr) is essential for mesoderm cell migration. stumps RNA is abundant in the mesoderm at stages 9-10 and is strongly reduced by stage 11. At stage 11, stumps RNA accumulates in trachea, which is largely unaffected in twist mutants (Furlong, 2001).

The 44 group C genes have reduced transcript levels in twist mutant embryos only during late stage 11 and stage 12. These 'lategenes' include blown fuse, a gene essential for myoblast fusion; delilah, a gene required for somatic muscle attachment, and genes such as kettin, which is required to form contractile muscle. Given the predominantly early expression of twist, the early genes in groups A and B are the best candidates for direct transcription targets of Twist, though some indirectly activated genes may be present within these groups. Group C late genes are likely to be regulated by products of genes that are activated by Twist (Furlong, 2001).

In situ hybridizations were done using a previously uncharacterized representative of each Twist-low group. In each case, the hybridization pattern was consistent with the predicted time of transcription. A group A gene, CG15015 (GH16741), is transcribed in somatic muscle throughout stages 9-12. A group B gene, CG12177 (GH22706), is transcribed during early mesoderm development, but not later. CG14848 (GH21860), a group C gene, is expressed in the stomodeum but not the mesoderm during stages 9-10. Its mesoderm expression initiates during stage 11, the latest period of the twist experiment. Thus, combining loss-of-function mutant embryo analysis with staged embryo collections provides gene expression information for both tissue specificity and temporal expression (Furlong, 2001).

The mis-expression of twist in the ectoderm is sufficient to convert both neuronal and epidermal tissues to a myogenic cell fate. RNA from embryos with ubiquitous twist expression was used to evaluate the ability of Twist to initiate mesoderm-like gene expression in cells that would normally form other tissue types. Genes whose transcript levels decrease in twist loss-of-function embryos and increase when twist is ubiquitous are excellent candidates for regulators of mesoderm development or differentiation (Furlong, 2001).

To ectopically express twist, a dominant gain-of-function mutation of the maternal gene Toll (Toll10B) was used. Activated Toll induces the expression of twist and snail in early embryos and of immune response genes in older embryos. Thus, the effects of Toll10B on gene expression reflect the activities of Twist as well as Snail and Dorsal, or their combined actions. Toll10B embryos are essentially bags of mesoderm; epidermal structures are absent or greatly reduced, and they have been used successfully in subtractive hybridization screens to identify mesoderm genes. The gene transcription profile of Toll10B embryos was compared with that of wild-type embryos during four periods of development, using the reference sample to normalize experiments. The earliest period, stage 5, is when twist is initially expressed in presumptive mesoderm. The other three periods analyzed were those used in the twist mutant analysis: stages 9-10, 11, and 11-12 (Furlong, 2001).

In Toll10B embryos, 447 genes had significant changes in RNA levels compared with stage-matched wild-type embryos, 16 of which are predicted to be false positives. Transcripts from 166 genes were reduced in Toll10B embryos compared with wild type. These genes may be involved in neuroectoderm events that are blocked when cells are turned into mesoderm. Transcripts of 281 'Toll-high' genes were increased in Toll10B embryos. Of the 21 previously characterized mesoderm-specific genes on the arrays, 18 have significantly increased transcript levels in Toll10B embryos. The remainder may require activators other than Toll, such as signals from the severely altered ectoderm (Furlong, 2001).

Genes with altered transcription in twist and Toll10B mutants were analyzed with a hierarchical clustering program to identify similar transcription profiles. The genes were divided into putative 'mesoderm' and 'non-mesoderm' groups. Non-mesoderm genes were defined as having increased transcript levels in mesoderm-deficient (twist) embryos and/or decreased expression in mesoderm-enriched (Toll10B) embryos. Mesoderm genes were defined as having decreased transcript levels in twist mutants and/or increased transcripts in Toll10B mutants. The mesoderm genes group would also contain genes expressed in other tissues in a mesoderm-dependent manner (Furlong, 2001).

Non-mesoderm genes in clusters B and C are repressed in Toll10B mutants. Cluster B genes have increased RNA levels in twist mutant embryos, whereas cluster C genes do not change significantly. The overexpression of twist in the presumptive ectoderm in Toll10B embryos results in a conversion of ectodermal cell fate into mesoderm. snail and dorsal are ectopically expressed in Toll10B embryos and transcriptionally repress the expression of neuroectoderm and ectoderm genes. The conversion of ectoderm to mesoderm due to twist misexpression, and the ability of Snail and Dorsal to repress ectoderm genes, suggests that the B and C clusters should contain primarily neuroectodermal genes. Indeed, the non-mesoderm genes include 31 previously characterized neuroectodermal genes. One previously unknown cluster B gene that encodes a putative cell adhesion protein is transcribed in the ventral nerve cord. Another previously unknown gene within cluster C is transcribed within the developing brain (Furlong, 2001).

The Twist-low and Toll-high genes have in common 51 genes that are highly likely to be involved mesoderm development. For example, transcription of the genes in cluster D is reduced in twist mutants and increased in Toll10B mutants during most or all time periods (Furlong, 2001).

A complete overlap between Twist-low and Toll-high gene sets is not expected for three reasons: (1) development of dorsal mesoderm, and of muscle founder cells marked by apterous and connectin, requires the Decapentaplegic signal and perhaps others. Changed characteristics of cells that form ectoderm in Toll10B embryos interfere with these signaling events. A significant reduction is observed in dpp RNA levels in Toll10B embryos. (2) During midgut development, endoderm cells migrate along the mesoderm. Midgut endoderm development is affected in twist mutants. Some Twist-low genes with unchanged expression in Toll10B embryos are transcribed in the midgut. (3) Ectopic Twist inhibits visceral mesoderm and heart development and promotes excess somatic muscle development. Toll10B embryos produce high levels of Twist throughout the embryo, so genes that have reduced RNA levels in both twist and Toll10B mutant embryos are likely to be visceral muscle and heart genes. Indeed, bagpipe and connectin, genes expressed in visceral mesoderm, are among the 79 Twist-low genes not induced by ectopic Twist (Furlong, 2001).

Of the 281 Toll-high genes, 230 are unaffected in twist mutants. Some of the 230 are normally expressed late in embryogenesis in wild-type embryos but are expressed prematurely in Toll10B embryos due to ectopic Twist. These include Myo61F, MSP-300, and Paramyosin, genes normally active in terminally differentiated muscle (stage 16). Ectopic Snail and Dorsal in Toll10B embryos may activate genes that are unaffected in twist mutants. Snail can repress neuroectodermal genes and may also activate mesoderm genes. Dorsal activates immune response genes later in development. relish, drosomycin, and metchnikowin genes -- all immuneresponse genes -- have higher transcript levels in Toll10B embryos (Furlong, 2001).

Data from loss- and gain-of-function experiments, combined with careful staging, yield a useful picture of genes that are likely to be required for mesoderm specification and muscle differentiation. Of 360 identified mesoderm genes, 273 have not been the focus of developmental studies. The predicted proteins encode transcription factors, signal transduction molecules, kinases, and pioneer proteins. The stage at which each gene is active is one criterion for assigning possible functions. Another key criterion will be finding a mutant phenotype. As a pilot, this additional step was undertaken for the gene CG4677 (LD47926). Changes in CG4677 transcript levels were also observed in a Toll10B subtractive hybridization screen (Furlong, 2001).

CG4677 is transcribed in the visceral mesoderm at stages 10-13 and the somatic mesoderm during stages 11-13. This gene encodes a C2H2 zinc finger transcription factor with high sequence similarity to vertebrate Gli proteins: the gene has been named gleeful (gfl). Mammalian Gli proteins act downstream of Hedgehog signaling proteins to control target gene transcription (Furlong, 2001).

The role of gfl in mesoderm development was assessed by disrupting its function using RNA interference. Injection of a double-stranded RNA (dsRNA) control sequence had no effect on mesoderm development. In contrast, gfl dsRNA injection causes severe loss and disorganization of somatic muscle cells, whereas heart and visceral muscle are unaffected. A similar phenotype is seen in Df(3R)hh homozygous embryos: the deficiency removes gfl but not the nearby hedgehog gene (Furlong, 2001).

To determine whether gfl can induce muscle cell development, a UAS-gfl transgenic fly strain was generated. Ectopic expression of gfl using an en-GAL4 driver results in lethality and induction of ectopic dMEF2 expression in the ventral nerve cord. Remarkably, Gfl is sufficient to induce expression of a muscle gene in neuronal cells. Previous studies have shown an essential role for Sonic hedgehog signaling in the formation of slow muscle in avian and zebrafish embryos. gfl may be performing a similar role in Drosophila somatic muscle development (Furlong, 2001).

A core transcriptional network for early mesoderm development

To delineate the combinatorial relationships between Twist and other TFs, an initial transcriptional network was generated for early mesoderm development. The temporal binding map for Twist was integrated with in vivo binding data for Mef2, Dorsal, and Tinman. A previous study of Mef2-bound enhancers offers the largest collection of regulatory regions bound at this stage of development to date. As it is difficult to visualize all 494 Twist target genes, focus was placed on TFs whose CRMs are cobound by two or more regulators during these stages of development. Therefore, all links in this network represent direct connections to the same CRM at the same stages of development (Sandmann, 2007).

The resulting core network of 51 TFs is already relatively complex, with nine genes [nau, E(spl), eve, bap, Ubx, lbe, odd, hth, and Ptx1] being targeted by three out of the four examined regulators. The topology of the network provides several insights into how Twist functions to regulate multiple aspects of early mesoderm development. Extensive combinatorial binding and feed-forward regulation are abundant features. Dorsal activates twist, which in turn coregulates the majority of known direct Dorsal targets. This network motif is even more prominent within the mesoderm: Twist regulates the expression of Mef2 and tinman, and cobinds with these TFs to many of their targets' enhancers. In fact, Twist co-occupies 42% of all Mef2-bound enhancers during early mesoderm development. Depending on the logical inputs from the two upstream regulators (transcriptional repression or activation), feed-forward loops can aid in cellular decision making by filtering out noisy regulatory inputs or control the timing of a transcriptional response. For example, early gene expression in the mesoderm (e.g., activation of tin) depends on Twist alone, while transcription of other genes initiated at a later stage may require the input from both Twist and Tinman proteins (Sandmann, 2007).

A machine learning approach for identifying novel cell type-specific transcriptional regulators of myogenesis

Transcriptional enhancers integrate the contributions of multiple classes of transcription factors (TFs) to orchestrate the myriad spatio-temporal gene expression programs that occur during development. A molecular understanding of enhancers with similar activities requires the identification of both their unique and their shared sequence features. To address this problem, phylogenetic profiling was combined with a DNA-based enhancer sequence classifier that analyzes the TF binding sites (TFBSs) governing the transcription of a co-expressed gene set. A small number of enhancers were assembled that are active in Drosophila melanogaster muscle founder cells (FCs) and other mesodermal cell types. Using phylogenetic profiling, the number of enhancers was increased by incorporating orthologous but divergent sequences from other Drosophila species. Functional assays revealed that the diverged enhancer orthologs were active in largely similar patterns as their D. melanogaster counterparts, although there was extensive evolutionary shuffling of known TFBSs. A classifier using this enhancer set was then built and trained, and additional related enhancers were identified based on the presence or absence of known and putative TFBSs. Predicted FC enhancers were over-represented in proximity to known FC genes; and many of the TFBSs learned by the classifier were found to be critical for enhancer activity, including POU homeodomain, Myb, Ets, Forkhead, and T-box motifs. Empirical testing also revealed that the T-box TF encoded by org-1 is a previously uncharacterized regulator of muscle cell identity. Finally, extensive diversity was found in the composition of TFBSs within known FC enhancers, suggesting that motif combinatorics plays an essential role in the cellular specificity exhibited by such enhancers. In summary, machine learning combined with evolutionary sequence analysis is useful for recognizing novel TFBSs and for facilitating the identification of cognate TFs that coordinate cell type-specific developmental gene expression patterns (Busser, 2012).

There are three main approaches for the prediction of tissue-specific regulatory elements that are based on high-throughput sequencing coupled with chromatin immunoprecipitation (ChIP-Seq), DNA sequence pattern analysis, or hybrid methods that combine both of these strategies. ChIP-Seq for p300 using mouse embryonic tissue has proven to be an accurate means for identifying enhancers and their associated activities, with in vivo validation rates varying from 62% to 88%. Computational analysis of whole-genome histone modification profiles using hidden Markov models and machine learning techniques has also been highly successful at linking chromatin signatures with regulatory elements. Finally, computational models that identify tissue-specific enhancers relying on sequence motifs and linear regression and support vector machines have been similarly effective, with in vivo validation rates of de novo predictions ranging from 62% for heart enhancers to 91% for brain enhancers. Although experimental techniques are often preferred for identifying enhancers on a genome-wide scale, ChIP-Seq has several limitations. For example, ChIP-Seq experiments are typically carried out in only one species and for individual cell types, and are currently not sufficiently precise for low-quality genome sequences. Thus, de novo prediction of regulatory elements based on ChIP-Seq data critically depends on the availability of relevant data for the species, cell type and genomic regions of interest. Currently, computational analysis of DNA sequence patterns shared by a set of regulatory elements with the same or similar biological activity remains a highly effective method for the de novo discovery of tissue-specific enhancers, and the simultaneous elucidation of cell type-specific regulatory codes. The method presented in this study further extends the usefulness of computational sequence analysis by exploring phylogenetic information that can be used to improve the classification accuracy, a strategy that promises to be advantageous in the large number of cases where comparative genomics data are available (Busser, 2012).

Computational approaches for predicting cis-regulatory modules are commonly based on machine learning of arrangements of TFBSs in enhancers that have common functions. These methods rely heavily on a training set of related enhancers to detect over-represented TFBS combinations. Unfortunately, in the vast majority of cases -- including the present study of Drosophila muscle FC enhancers -- the size of the training set is limited by the lack of experimentally validated tissue- and cell type-specific enhancers, which results in overfitting of computational models and poor accuracy of predictions. To overcome this problem, and to provide a generalizable approach for increasing the size of the training set, a phylogenetic profiling strategy was developed based on a search for diverged orthologous counterparts of available enhancers from distantly related species. Twenty-four Drosophila orthologs were identified using this approach, which more than doubled the size of the training set. The ability to accurately distinguish FC enhancers was assessed in a cross-validation framework using the extended training set, and it was determined that the classifier accuracy is 89% as assessed by the AUC approach. This classifier was then developed to scan the entire genome of D. melanogaster for novel FC enhancers, retrieving 5,500 high-scoring predictions at a FPR of 5%. These predictions were significantly associated with genes expressed in FCs, demonstrating that the model was able to capture essential features of FC gene co-regulation. A similar machine learning approach could be applied to a diverse array of datasets, including experimentally-verified regulatory elements from co-expressed targets at either a germ layer, organ, tissue or cellular level from invertebrate and vertebrate databases. Alternatively, a similar approach could be coupled to a training set of predicted regulatory elements derived from genome-wide analyses of chromatin marks or DNAse hypersensitive sites in active enhancers associated with a co-expressed gene set (Busser, 2012).

Evolutionary constraint of functional sequences is routinely employed as an effective filter to improve the prediction of regulatory elements. Furthermore, cross-species comparisons have been successfully exploited to obtain evidence for functional TFBSs. For example, Rouault (2010) used twelve Drosophila species to identify over-represented motifs in the regulatory elements of genes expressed in neural progenitor cells, with sequence orthologs used to enrich the training set and to give prominence to conserved motifs. However, this method extends this approach by including suitably diverged orthologous enhancers from other Drosophila species in the dataset used to train the classifier. The purpose in designing this strategy was two-fold. First, it was desired to enrich for relevant sequence motifs in the training data, allowing for a level of variation that would improve the generalization of the model. Second, a potentially wider variety of TFBS arrangements would be provided that characterize the architecture of authentic FC enhancers. In essence, the addition of orthologous sequences boosts the statistical power of the significance tests, revealing patterns of TFBSs that otherwise could have been neglected (Busser, 2012).

Of note, when 5 of these orthologous sequences were tested in transgenic reporter assays in D. melanogaster, the overall expression pattern generated was similar to the D. melanogaster counterpart despite extensive evolutionary shuffling of known TFBSs. Similar binding site reorganization has been documented for the enhancers that regulate both the segmentation and mesodermal patterns of eve expression. Numerous other studies have shown that the order and spacing of TFBSs is critical for enhancer function. These results suggest that regulatory elements can direct similar expression patterns provided that the overall composition and order of collaborating TFs is maintained. The finding that enhancer function is preserved in the orthologous sequences examined in this study establishes the validity of the sequence conservation thresholds chosen for the present studies, and suggests that the incorporation of orthologous sequences to increase a training set without over-fitting the data will be a generally applicable approach (Busser, 2012).

To assess the accuracy of this method, 12 predicted FC enhancers were selected and their in vivo functions were tested. Seventy-five percent of the putative enhancers were experimentally validated as having transcriptional activity, demonstrating the effectiveness of this approach to identify regulatory sequences. However, of the sequences showing regulatory functions, only 4 of 9 were active in the mesoderm—including 2 in FCs—and 3 of 9 had nervous system activity. These data suggest that the model has been able to reliably recognize general properties of tissue-specific enhancers without specifically distinguishing an overall muscle FC code, even though numerous individual FC-specific motifs were identified. The former finding is similar to the results of Sinha and colleagues (Kantorovitz, 2009) who found that the majority of their classifier predictions were active enhancers, but only a minority were expressed in the predicted pattern. A number of confounding factors can explain this outcome (Busser, 2012).

First, most members of the enhancer training set are active in both FCs and other cell types, including additional mesodermal cells such as the cardiac and visceral mesoderm, as well as some cells of the nervous system. For example, the enhancer responsible for the FC activity of the hunchback gene is also active in the longitudinal visceral mesoderm, and enhancers directing the FC expression of the vestigial, big brain and king-tubby genes are also active in the peripheral nervous system. These results suggest that the regulatory networks specifying the somatic and visceral mesoderm share common features, which is consistent with both the available genetic and genomic evidence for the diverse developmental functions of key mesodermal transcription factors. Second, different members of a given TF family bind to similar motifs but have distinct tissue-specific expression patterns and developmental activities. Thus, combinations of motifs involved in the specification of muscle FCs and the nervous system may overlap. For example, this situation occurs with E-box and NK-homeodomain motifs. Third, some TFs are expressed and functional in the derivatives of more than one germ layer. Fourth, the sequence features characteristic of cell type-specific enhancers, such as those active in muscle FCs, are expected to be under-represented in available training sets owing to the diversity of combinatorial TF models required to specify such a heterogeneous cell type. Identification of many examples of a particular cell-specific signature is a major challenge since each of the approximately 30 FCs in each Drosophila hemisegement expresses a unique combination of cell-specific muscle identity TFs and downstream target genes. Thus, 30 distinct cell states exist, each governed by a different but partially overlapping set of regulatory TFs. In contrast to the difficulties involved in dissecting regulatory codes at single cell resolution, shared features that direct activity to the general level of tissues and organs have been more readily identified using a machine learning approach, as was found in this study for enhancers having mesodermal, although not necessarily FC, activity. This likely reflects the dominant role that some TFs play in the regulatory network specifying the identities of numerous tissues. Fifth, since there appears to be a regulatory signature for enhancers, it is likely that these aspects of enhancer structure will be more significantly over-represented than those features that specify individual FC activity patterns. Sixth, the use of phylogenetic profiling might have expanded the biological function of the training dataset by introducing additional enhancer functions acquired by the orthologs of the original D. melanogaster sequences during their evolution. While this study has been able to show that the phylogenetic profiling approach improves the accuracy of the classifier, one drawback of its use might be that the final classifier recognizes a broader biological domain than the function of the original training set of sequences derived from the reference species. Finally, classifier predictions may represent cis-regulatory elements other than enhancers, for example, silencers and insulators, which would not be detected by the transgenic reporter assays (Busser, 2012).

In summary, a number of confounding factors influenced the ability to identify an enhancer signature that is specific for individual muscle FCs. However, despite these challenges, the successful identification of novel TF binding motifs responsible for the cell type-specific activity of FC enhancers is encouraging the idea that this is a tractable problem that can be solved by an iterative approach to the computational analysis of this and other complex developmental systems. Thus, future studies must focus on obtaining a larger training set of sequences in which enhancers are categorized based on their activities at single cell resolution, combined with the appropriate weighting of newly validated motifs that contribute to the expression pattern of interest. In this manner, each experimental round would improve the accuracy of the classifier (Busser, 2012).

The motifs ranked by the classifier used in this study as having the highest discriminatory power are part of a large regulatory network that is known to be critical for mesoderm specification and myogenesis. These motifs include binding sites for JAK/STAT, Ets, bHLH, Wingless/Tcf, homeodomain and forkhead proteins. Furthermore, it has previously been suggested that Ets is part of a transcriptional code regulating the C1 subset of FC genes, which was validated in this study using site-directed mutational analysis of the Ndg enhancer, a previously characterized regulatory element associated with a C1 FC gene (Busser, 2012).

To extend the components of the myogenic regulatory network beyond these known TFs and motifs, the function was examined of the classifier-defined sequence motifs recognized by POU homeodomain and Myb proteins, transcription factors having no previously known role in Drosophila myogenesis. Mutagenesis of POUHD motifs attenuated the activity of the Ndg enhancer in many mesodermal cells. However, a zygotic loss-of-function mutation in acj6, the only POUHD that was found to be expressed in the mesoderm, had no effect on Ndg gene expression . Given the strong maternal contribution to this gene, RNAi was used to knock down both maternal and zygotic acj6 transcripts, but this manipulation had no effect on Ndg-GFP reporter activity. These findings leave unresolved the identity of the TF that binds to the motif in question. The future characterization of this TF, including exploring the possibility that it is not a POUHD protein, will require searching functional motifs against larger TF databases, combined with analysis of the embryonic expression and function of any new candidates that emerge (Busser, 2012).

Inactivating mutations of the Myb binding sites in the Ndg enhancer led to extensive de-repression of the reporter in other mesodermal cells. Myb is a ubiquitously-expressed DNA binding protein which plays a critical role in controlling regulatory decisions during proliferation and differentiation of progenitor cells. Identifying a putative role for Myb in myogenesis documents the power of this approach, since functional studies tend to focus on genes with restricted expression patterns. However, a definitive assessment requires examining the effect of loss-of-function mutations in Myb. In any event, as myogenesis in Drosophila occurs through a series of asymmetric and symmetric cell divisions, a role for Myb in regulating FC gene expression is entirely consistent with a transcriptional regulator acting at the interface between replication and transcription. Alternatively, Myb may cooperate with other TFs to activate cell or tissue-specific gene expression (Busser, 2012).

Interestingly, T-box motifs scored well in the classification, yet no role for T-box TFs has previously been described in Drosophila somatic muscle development, despite widespread functions of this TF class in mesoderm specification and myogenesis in vertebrates, as well as cardiogenesis in Drosophila and vertebrates. This study shows using both cis and trans tests of TF function, along with gene co-expression, that Optomotor-blind-related-gene-1 (Org-1) is a muscle identity TF. In particular, the cis effects of Org-1 were documented in the FC enhancers associated with two known muscle identity TFs, Slou and Lbl, and org-1 expression localizes to the SBM and VT1, muscles in which the lb genes and slou, respectively, are the only previously described determinants of muscle identity. Slou function is critical for the proper development of muscles LO1 and VT1 and is further required to repress the lb genes in these cells, suggesting a co-regulatory relationship between slou and lb. It is likely that org-1 acts upstream of slou and lb in this regulatory hierarchy since org-1 expression precedes slou and lb, and the ectopic expression of org-1 causes increased expression of slou and lb. In addition, the essential role of org-1 in this regulatory network is revealed by the effects of org-1 overexpression and RNAi knockdown on development of lb- and slou-expressing muscles. Interestingly, the mouse orthologs of org-1 and lb genes, Tbx1 and Lbx1, respectively, have been suggested to regulate myogenic differentiation in the limb. Given the high degree of sequence similarity, and the close correspondence of expression patterns and functions in Drosophila and mouse, the collaborative roles of these two TFs in myogenesis appear to have been conserved through evolution (Busser, 2012).

Computational prediction of regulatory elements requires a thorough understanding of the TFs and motifs that orchestrate gene co-expression patterns. In prior studies, it was established that 5-way and 3-way 'AND' combinations of 3 signal-activated (Tcf, Mad and Pnt) plus 2 tissue-restricted (Twi and Tin) TFs constitute distinct regulatory models for different FC enhancers. The present study significantly extend these prior combinatorial codes for FC gene regulation by identifying four additional classes of TFBSs that are critical for accurate FC enhancer activity, namely POUHD, Myb, Fkh and T-box motifs. Moreover, these findings provided an opportunity to examine the complete spectrum of regulatory motif usage across a collection of regulatory elements that are active in different muscle FCs, which led to the identification of 18 unique combinations of 11 TFBSs for the entire set of 18 known FC enhancers. Thus, unlike other cases that have been studied, a single enhancer archetype does not appear to exist for this subpopulation of myoblasts. This finding likely reflects the fact that although these elements all display FC activity, with some overlap at the level of individual cells, no two FC gene expression patterns directed by this enhancer set are identical (Busser, 2012).

The marked heterogeneity of FC enhancer architecture uncovered in this study reflects not only distinct combinations of various TF classes (including signal-activated, ubiquitous and both tissue- and cell type-specific TFs), but also diversity at other biological levels, including the unique identities of the thirty muscle FCs and their differentiated derivatives in each abdominal hemisegment, and the different gene expression patterns exhibited by those particular cells. Thus, TFBS combinatorics provide a plausible molecular explanation for the functional complexity of enhancers having related but non-identical activites at the resolution of individual cells in the context of the developing embryo (Busser, 2012).

This study has investigated the transcriptional regulatory network specifying individual muscle FCs using an integrated genomics approach that includes identification of orthologous enhancers, de novo motif discovery, classification of enhancer sequence features, empirical testing of candidate enhancers, and cis-trans tests of target gene regulation. It was also established that a small set of training sequences can be expanded with orthologous sequences. Moreover, motifs learned by the classifier were empirically found to be critical for the appropriate spatio-temporal activities of FC enhancers, and suggested new candidate TFs in the myogenic regulatory network. Using this approach, one such candidate TF, Org-1, was identified as a novel muscle identity TF, and further found that no two enhancers with related activities contain the same combination of TFBSs. The tools and strategy used in this study can be readily applied to other cell types to identify the motifs and trans-acting factors regulating a set of co-expressed genes. Finally, it is anticipated that an iterative application of this approach, which could include training on datasets of different epigenetic marks associated with active enhancers or previous ChIP studies of known mesodermally-relevant TFs , will lead to further refinements in the determination of cell type-specific transcriptional codes (Busser, 2012).

Notch and Ras signaling pathway effector genes expressed in fusion competent and founder cells during Drosophila myogenesis

Drosophila muscles originate from the fusion of two types of myoblasts -- founder cells (FCs) and fusion-competent myoblasts (FCMs). To better understand muscle diversity and morphogenesis, a large-scale gene expression analysis was performed to identify genes differentially expressed in FCs and FCMs. Embryos derived from Toll10b mutants were employed to obtain primarily muscle-forming mesoderm, and activated forms of Ras or Notch were expressed to induce FC or FCM fate, respectively. The transcripts present in embryos of each genotype were compared by hybridization to cDNA microarrays. Among the 83 genes differentially expressed, genes known to be enriched in FCs or FCMs, such as heartless or hibris, previously characterized genes with unknown roles in muscle development, and predicted genes of unknown function, were found. These studies of newly identified genes revealed new patterns of gene expression restricted to one of the two types of myoblasts, and also striking muscle phenotypes. Whereas genes such as phyllopod play a crucial role during specification of particular muscles, others such as tartan are necessary for normal muscle morphogenesis (Artero, 2003).

The Toll10b mutation gives rise to embryos composed primarily of somatic mesoderm. In these embryos FCs and FCMs are readily detected, and they respond to the Ras and Notch signaling pathways in the same way as their wild-type counterparts. Advantage was taken of this fact to enrich Toll10b mutant embryos for FCs or FCMs, which allowed a concentration on the transcription in these two specific cell types within the context of the entire embryo. Genes known to be expressed and regulated in FCs or FCMs emerged from the screen in the proper categories. Not all known FC/FCM genes were detected in the screen for several reasons: the high stringency set for interpretation of the array data; the presence of only about one-third of the genome on the arrays; the loss of Dpp in the Toll10b background, and the specific window of myogenesis (5- to 9-hours) that was the focus of this investigation. However, a plethora of potential new muscle regulators were uncovered, including known genes with no previously recognized function in the mesoderm (such as phyl and asteroid), and genes predicted from the Drosophila genome sequence but not previously analyzed (Artero, 2003).

Various tests were applied to ascertain the validity of the results. Available databases were analyzed to find evidence that the known and predicted genes were expressed at the correct time and place. In addition, Northern analysis with eleven genes tested the reliability of the microarray detection and selection criteria; the results from all genes tested agreed with the array data (Artero, 2003).

A Toll10b sample on the Northern blots allowed ascertainment of why a gene is enriched in a particular condition. For example, in the case of FC enriched genes, the signal in the Ras and Notch lanes can be compared with Toll10b alone to determine whether the Ras/Notch ratio for a gene is due to activation by Ras or repression by Notch. Those genes that are 'enriched under Notch conditions', for example, could reflect a variety of transcription mechanisms that would result in a ratio of less than 0.6. By Northern analysis, many of the 'Notch-regulated' genes, and hence the predicted FCM genes, were found to be repressed by Ras signaling and slightly activated by Notch. As a case in point, hibris is induced by Notch (2-fold) and repressed by Ras (10-fold), both by Northern analysis and by in situ hybridization in embryos (Artero, 2003).

A combination of in situ hybridization, immunostaining and confocal microscopy was used to verify that the differential expression changes that were observed in these overexpression embryos reflected true differential expression in the wild-type situation. The expression of nine genes from different functional categories was analyzed. For seven of these, expression was detected in the predicted type of myoblast. For two, asteroid (ast) and cadmus, no specific staining in embryos was detected by in situ hybridization. For those genes that fell into the category of 'specific role in muscle development uncertain', in situ hybridization of several (28%) showed expression in tissues other than somatic mesoderm that are present in the Toll10b background. These genes changed their expression levels in response to Ras or Notch, and may be Ras and Notch targets in non-mesodermal tissues (Artero, 2003).

The most stringent test, mutational analysis, was applied to a set of genes for which mutations are available. Preliminary analyses of another four FCM-enriched genes was carried out: Elongation factor Tu mitochondrial (EfTuM), Glutamine synthetase 1, cadmus and parcas. All four mutants have muscle defects, including muscle losses and aberrant muscle morphologies. Thus all the genes tested show some muscle defect, supporting the usefulness of the genetic and genomic approach (Artero, 2003).

Taken together, these data suggest that the majority of genes detected play important roles in FCs or FCMs during muscle development. Some of these genes might not have been found in traditional forward genetic screens because of partial or complete genetic redundancy. The data complement traditional forward genetic approaches for finding genes crucial for muscle morphogenesis (Artero, 2003).

Each of the thirty FCs per abdominal hemisegment is hypothesized to produce its own unique combination of transcriptional regulators, though the evidence for this is limited. In turn the combination of regulators would control the morphology of the final muscle. Although several transcriptional regulators have been linked to FC identity, the molecular description is far from complete. This screen contributed two more FC-specific genes. Previously known markers, such as slouch or eve, once induced in the muscle FC, are maintained throughout the remainder of development. Ubx, which emerged from this screen, is a similarly simple case, as its transcripts are steadily present in most FCs. By contrast, more complex patterns of gene expression have been identified in FCs, such as the transient transcription of asense in a subset of FCs. The subsequent transcriptional inactivation of asense may underlie temporal changes in cell properties (Artero, 2003).

Even less is known about transcriptional regulators controlling FCM differentiation. Only one gene, lame duck, has been shown to have a role in FCMs. This screen has uncovered three more potential players: delilah, E(spl)mß and CG4136, confirming that FCMs follow their own, distinct, myogenic program. Discovering what aspects of FCM biology are controlled by these transcriptional regulators awaits analysis of the loss-of-function phenotypes (Artero, 2003).

Notch and Ras signaling pathways interact during muscle progenitor segregation. The results suggest that phyl and polychaetoid (pyd) may be additional links between the two signaling pathways in FCs. phyl and pyd both interact genetically with Notch and Delta. The transcription of phyl, which promotes neural differentiation, is negatively regulated by Notch signaling during specification of SOPs and their progeny. This study shows a similar regulation in muscle cells, where Notch signaling represses phyl expression and Ras signaling increases phyl expression. Likewise, in the nervous system, the segregation of SOPs requires pyd, a Ras target gene, to negatively regulate ac-sc complex expression. Similarly, Pyd may restrict the muscle progenitor fate to a single cell, perhaps by regulating lethal of scute transcription. Thus, Pyd would collaborate with Notch signaling to restrict muscle progenitor fate to one cell (Artero, 2003).

FCMs appear to integrate Ras and Notch signaling differently. Two genes whose transcripts were enriched under activated Notch conditions, parcas and asteroid (ast), have been implicated in Ras signaling in other tissues, directly (ast) or indirectly (parcas). These data are suggestive of a role for Ras signaling in the FCMs, in addition to its role in FC specification. In addition, Notch signaling to FCMs may prime cells for subsequent Ras signaling during muscle morphogenesis, much as occurs in FCs where Ras signaling primes the cell for subsequent Notch signaling during asymmetric division of the muscle progenitor (Artero, 2003).

Embryos that lack or ectopically express phyl have morphological defects in specific muscles, for example, in LL1 and DO4 in response to diminished phyl function, and in DT1 and LT4 in response to increased phyl function. The morphological defects in the loss-of-function embryos appear to be due to a failure to specify particular FCs, a conclusion that is based upon missing or abnormal production of the FC marker Kr. In eye development and SOP specification, Phyl directs degradation of the transcriptional repressor Tramtrack. In a subset of the primordial muscle cells, Phyl may work similarly, targeting Tramtrack for degradation. The presence of Tramtrack would contribute to the specific identity program of the muscle. Since Tramtrack is expressed in the mesoderm, this possibility is likely. Alternatively, Phyl may be required for targeted degradation of some other protein in a subset of FCs. The molecular partner for Phyl during muscle differentiation is unknown, although preliminary data suggest that sina is also expressed in somatic mesoderm and thus may be its partner. These studies have identified a new role for Phyl in muscle progenitor specification and suggest the importance of targeted ubiquitination for proper muscle patterning (Artero, 2003).

A role for ubiquitination in muscle differentiation is further reinforced by the identification of the RING finger-containing protein Goliath (Gol), induced by activated Notch conditions, and CG17492, induced by activated Ras conditions. Several RING-containing proteins function as E3 ubiquitin ligases, with the ligase activity mapping to the RING motif itself. Ligase function has been experimentally confirmed for the Gol ortholog GREUL1 in Xenopus. Thus, targeted protein degradation during muscle morphogenesis could serve a host of crucial functions, such as protein turnover, vesicle sorting, transcription factor activation and signal degradation (Artero, 2003).

The simplest view of the 'founder cell' hypothesis is that each FC contains all the information for the development of a particular muscle. By contrast, FCMs have been seen as a naïve group of myoblasts, entrained to a particular muscle program upon fusion to the FC. This work indicates that these two groups of myoblasts have distinct transcriptional profiles. These data raise the possibility of a greater role for FCMs in determining the final morphology of the muscle and emphasize a need to characterize fully those FCM genes. For example, this screen identified a protein kinase of the SR splice site selector factors (SRPK) whose transcripts are enriched in FCMs, suggesting that regulation of the splicing machinery is important for muscle morphogenesis. The Mhc gene undergoes spatially and temporally regulated alternative splicing in body wall muscles conferring different physiological properties on these muscles. This FCM-specific expression of SRPK may indicate that the production of a particular Mhc isoform is regulated by the FCMs that contribute to that muscle, rather than by the particular FC that seeds the muscle. In addition, a number of observations suggest that FCMs may be a diverse population of myoblasts, with different subsets having different potential to contribute to the final muscle pattern. For example, hbs expression suggests that only a subset of FCMs express the gene, and twist expression in lame duck mutant embryos persists in a subset of FCMs. This study provides additional genes for exploring whether FCMs are a heterogeneous population of myoblasts as well as determining the nature of FCM contribution to the final muscle (Artero, 2003).

The molecular events underlying complex morphological changes, such as migration, cell fusion, cell shape changes or changes in the physiology of a cell, require a rich and dynamic program of transcription changes. This study has described approximately one-third of this transcriptional profile. The FC- or FCM-specific transcription of seven genes, and the mutant phenotype of four selected genes, allowed the definition of new muscle mutations that specifically affect the morphological traits of a subset of muscles (Artero, 2003).

Shadow enhancers are pervasive features of developmental regulatory networks

Embryogenesis is remarkably robust to segregating mutations and environmental variation; under a range of conditions, embryos of a given species develop into stereotypically patterned organisms. Such robustness is thought to be conferred, in part, through elements within regulatory networks that perform similar, redundant tasks. Redundant enhancers (or "shadow" enhancers), for example, can confer precision and robustness to gene expression, at least at individual, well-studied loci. However, the extent to which enhancer redundancy exists and can thereby have a major impact on developmental robustness remains unknown. This study systematically assessed and identifies over 1,000 predicted shadow enhancers during Drosophila mesoderm development. The activity of 23 elements, associated with five genes, was examined in transgenic embryos, while natural structural variation among individuals was used to assess their ability to buffer against genetic variation. Data reveal three clear properties of enhancer redundancy within developmental systems. First, it is much more pervasive than previously anticipated, with 64% of loci examined having shadow enhancers. Their spatial redundancy is often partial in nature, while the non-overlapping function may explain why these enhancers are maintained within a population. Second, over 70% of loci do not follow the simple situation of having only two shadow enhancers-often there are three (rols), four (CadN and ade5), or five (Traf1), at least one of which can be deleted with no obvious phenotypic effects. Third, although shadow enhancers can buffer variation, patterns of segregating variation suggest that they play a more complex role in development than generally considered (Cannavò, 2015).

The formin Diaphanous regulates myoblast fusion through actin polymerization and Arp2/3 regulation

The formation of multinucleated muscle cells through cell-cell fusion is a conserved process from fruit flies to humans. Numerous studies have shown the importance of Arp2/3, its regulators, and branched actin for the formation of an actin structure, the F-actin focus, at the fusion site. This F-actin focus forms the core of an invasive podosome-like structure that is required for myoblast fusion. The formin Diaphanous (Dia), which nucleates and facilitates the elongation of actin filaments, was found to be essential for Drosophila myoblast fusion. Following cell recognition and adhesion, Dia is enriched at the myoblast fusion site, concomitant with, and having the same dynamics as, the F-actin focus. Through analysis of Dia loss-of-function conditions using mutant alleles but particularly a dominant negative Dia transgene, it was demonstrated that reduction in Dia activity in myoblasts leads to a fusion block. Significantly, no actin focus is detected, and neither branched actin regulators, SCAR or WASp, accumulate at the fusion site when Dia levels are reduced. Expression of constitutively active Dia also causes a fusion block that is associated with an increase in highly dynamic filopodia, altered actin turnover rates and F-actin distribution, and mislocalization of SCAR and WASp at the fusion site. Together these data indicate that Dia plays two roles during invasive podosome formation at the fusion site: it dictates the level of linear F-actin polymerization, and it is required for appropriate branched actin polymerization via localization of SCAR and WASp. These studies provide new insight to the mechanisms of cell-cell fusion, the relationship between different regulators of actin polymerization, and invasive podosome formation that occurs in normal development and in disease (Deng, 2015).

This study provides the first evidence that Dia is essential for Drosophila myoblast fusion. Dia is expressed in all myoblasts and is recruited to the myoblast fusion site. The spatial and temporal distribution of Dia at the fusion site parallels that of the F-actin focus, which forms the core of an invasive podosome. This actin rich podosome is critical for FCM invasion of the FC/Myotube during fusion. In keeping with its expression pattern, Dia is essential for myoblast fusion progression: both loss and gain of Dia function lead to a fusion block. Under both conditions, the integrity of the F-actin focus and hence the invasive podosome is compromised; myoblasts expressing DiaDN fail to form the focus, whereas myoblasts expressing DiaCA have many filopodia and have a diffuse organization of F-actin, both of which contribute to a failure in invasive podosome formation and fusion. Dia activity is required after FC/Myotube and FCM recognition and adhesion but upstream of Arp2/3 activity. It is required, in parallel with PI(4,5)P2 signaling, to build a functional F-actin focus at the fusion site. These experiments further indicate that Dia activity is critical for actin dynamics at the fusion site, which, in turn, regulate fusion progression. Moreover, the aberrant F-actin organization at the fusion site in both loss and gain of function is also due to altered localization of the Arp2/3 regulators, SCAR and WASp. Taken together, these data support a role for the formin Dia in a critical first step of actin polymerization at the fusion site, downstream of cell-cell recognition and adhesion, and link its activity to the formation of F-actin foci, required for myoblast fusion (Deng, 2015).

Actin remodeling is critical for myoblast fusion, but Arp2/3 was the only known actin polymerization factor that was shown to be necessary for myoblast fusion. This study now shows that the formin Dia is also required during myoblast fusion. Whereas Arp2/3 preferably binds to pre-existing actin filaments and generates uncapped F-actin, formins nucleate F-actin both de novo and from the barbed ends of pre-existing actin filaments. Thus, Dia can generate actin filaments de novo, which Arp2/3 can bind or elongate (Deng, 2015).

This study also shows that the level of Dia activity is critical for myoblast fusion. Too much actin polymerization leads to too many filopodia and absence of an invasive podosome with its characteristic F-actin core. Too little polymerization leads no actin focus and no podosome formation. FRAP data with DiaCA also hint at whether a limited pool of actin is available for the actin polymerization factors during myoblast fusion. Despite the high rates of actin turnover with expression of DiaCA, the final fluorescence levels of actin returns to the same value as in controls. Additional actin monomers are not recruited to the site, even with high levels of polymerization activity. Interestingly, the rate of actin turnover has also been measured in mutants that affect Arp2/3 activity: specifically, mutations in blow, which regulates the Arp2/3 NPF WASp, show lower rates of actin exchange than in controls, due to a reduced exchange rate for WASp on the barbed ends of actin at the fusion site. Together these data suggest future experiments aimed at examination of whether rates of actin polymerization regulated by both Dia and Arp2/3 are optimized for the available actin pool and tightly controlled for myoblast fusion to properly occur (Deng, 2015).

Both cooperative and antagonistic functions between Dia and Arp2/3 have been reported. This study demonstrates that the coordinated and cooperative activities of these two actin polymerization factors leads to the formation of the F-actin focus. With the exception of sltr/Dwip/vrp mutants that form a focus of wild-type size, single mutants in the Arp2/3 NPF pathways, WASp and SCAR, lead to enlarged foci; however, double mutants in WASp and SCAR pathways do not form foci. This is the same phenotype that is seen in myoblasts expressing the DiaDN. The data support Dia activity being upstream of WASp and SCAR activation of Arp2/3 at the fusion site. This suggests that, at the fusion site, Dia initially provides the necessary context upon which Arp2/3 can act and not vice versa, as has been suggested in other contexts in which linear actin filaments emerge from Arp2/3 based structures. Nevertheless, both sets of actin regulators are necessary for F-actin focus formation that provides the core of the invasive podosome. Neither Dia nor Arp2/3 alone are sufficient (Deng, 2015).

The interplay between Dia and Arp2/3 at the fusion site is also reflected by the localization studies. Too little or too much Dia activity resulted in improper localization and, by extension, improper activity of Arp2/3 NPFs. How could Dia regulate this localization? One possibility is that Dia indirectly regulates Arp2/3 localization. Dia could nucleate linear actin filaments, which then would provide the necessary substrate for recruitment, maintenance and /or activation of Arp2/3 and its regulators, such as the WASp-WIP complex. Another possibility is that Dia, through its interactions with members of the SCAR/WAVE complex such as Abi, may directly localize and/or maintain the localization of Arp2/3 regulators, which are then activated at the fusion site. Abi has been reported to bind directly with Dia in vitro, and this interaction is required for the formation and stabilization of cell-cell junctions. Dia likely changes the localization and integrity of the SCAR/WAVE complex by competitively binding to the N-terminal part of Abi, dissociating Kette/Nap1 from the complex, and thus changing the stability and localization of SCAR/WAVE. It has also been established that the recognition and adhesion receptor, Sns, is capable of recruiting the Arp2/3 NPFs, such as WASp, to the fusion site. While Sns is still clustered at the fusion site in DiaDN and DiaCA, its recruitment activity appears not sufficient for focus formation capable of supporting an invasive podosome (Deng, 2015).

This study has shown that localization of Arp2/3 NPFs is affected in Dia loss and gain of function. In addition to this spatial control, another important way of controlling Arp2/3 activity is through activation of the NPFs via small GTPases. SCAR is activated through Rac-dependent dissociation from SCAR inhibitory complex. WASp is activated by binding to Cdc42, which releases it from auto-inhibited state. This study did not examine the localization of these activated GTPases. However, previous work has shown that PI(4,5)P2 signaling is required for proper localization of activated Rac at the fusion site. How the localization and activity of small GTPases at the fusion site contribute to the spatial and temporal interplay between Dia and Arp2/3 regulation of actin polymerization requires further investigation (Deng, 2015).

It remains unresolved how Dia itself is recruited to the fusion site. The data suggest that the recognition and adhesion receptors Duf and Sns would be involved either directly or indirectly in recruiting Dia to the fusion site, as embryos that fail to express either of these adhesion receptors fail to recruit Dia to the fusion site. In addition, recent data from Drosophila epithelial tubes indicate that PI(4,5)P2 serves as a localization cue for Dia. Previous work has shown that PI(4,5)P2 accumulates at the fusion site after FC-FCM recognition and adhesion; sequestering of PI(4,5)P2 results in a significant fusion block. Therefore, whether PI(4,5)P2 regulates Dia localization at the fusion site was tested. Dia was found to be recruited to the fusion site in the PI(4,5)P2 sequestered myoblasts, suggesting that, in this context, PI(4,5)P2 signaling is not required for Dia localization. These data provide possible explanations for why in PI(4,5)P2 sequestering embryos, smaller actin foci are detected: the localized Dia may be sufficient to recruit low levels of Arp2/3 and its NPFs, which, upon activation, lead to the formation of small F-actin foci. Nevertheless, in the absence of PI(4,5)P2 signaling, Dia that is recruited to the fusion site is not sufficient to produce functional actin focus, capable of directing a fusion event. Recent work also indicates that charged residues in the N- and C-termini of mDia1 are sufficient both for mDia's clustering of PI(4,5)P2 and its own membrane anchorage. This interaction between mDia1 and PI(4,5)P2, in turn, regulates mDia1 activity. Whether such a mechanism is in play at the myoblast fusion site needs to be further investigated (Deng, 2015).

A working model is proposed for the interplay between the actin regulators during myoblast fusion. Dia is recruited to the fusion site upon engagement of the recognition and adhesion receptors by a yet-to-be determined mechanism. It is proposed that PI(4,5)P2 signaling at the fusion site regulates the localization and activation of downstream targets such as Rho-family of small GTPases. These small GTPases lead to the activation of Dia. Activated Dia, in turn, polymerizes linear actin filaments and, in combination with the recognition and adhesion receptors and PI(4,5)P2, recruits the Arp2/3 NPFs, SCAR and WASp. Activation of these Arp2/3 NPFs at the fusion site would be accomplished by small GTPases such as Rac. These, in turn, would activate Arp2/3, leading to branched actin and formation of the F-actin focus and the invasive podosome. Whether the Arp2/3 NPFs such as SCAR/WAVE would negatively regulate Dia to downregulate linear actin polymerization, as suggested for mDia2 in cell culture, or whether Dia competes with WASp for barbed end binding remains to be investigated. However, these mechanisms would underscore a switch from linear F-actin filopodium formation to the linear and branched F-actin invasive podosome-like structure that is necessary for fusion (Deng, 2015).

The actin focus formed at the fusion site is an F-actin rich, invasive podosome-like structure that has been suggested to provide a mechanical force for FCMs to invade the FC/Myotube. Similar invasive actin structures named invadosomes have been seen in different cell types, such as podosomes in macrophages and invadopodia in cancer cell. Arp2/3 is known to play a key role in invadosome formation, and recent studies have revealed the involvement of formins in developing invadosomes. The current data indicate that specific temporal and spatial interactions between the formin Dia and Arp2/3 are required for the actin focus and invasive podosome formation. The data thus provide new mechanistic insights for the interplay of Arp2/3 and Formins during invadosome formation in these contexts (Deng, 2015).

Motoneurons regulate myoblast proliferation and patterning in Drosophila

Motoneurons directly influence the differentiation of muscle fibers, regulating features such as muscle fiber type and receptor development. Less well understood is whether motoneurons direct earlier events, such as the patterning of the musculature. In Drosophila, the denervation of indirect flight muscles results in a diminished myoblast population and smaller or missing muscle fibers. Whether the neuron-dependent control of myoblast number is due to regulation of cell division, motoneuron-dependent apoptosis, or nerve-dependent localization and migration of myoblasts, was examined. Denervation results in a reduced rate of cell division, as revealed by BrDU incorporation. There is no change in the frequency of apoptotic myoblasts following denervation. Using time lapse imaging of GFP-expressing myoblasts in vivo in pupae, it was observed that despite denervation, the migration and localization of myoblasts remains unchanged. In addition to reducing myoblast proliferation, denervation also alters the segregation of myoblasts into the de novo arising dorso-ventral muscles (DVMs). To address this effect on muscle patterning, the expression of the founder-cell marker Dumbfounded/Kirre (Duf) in imaginal pioneer cells was examined. There is a strong correspondence between cells that express Dumbfounded/Kirre and the number of DVM fibers, consistent with a role for these cells in establishing adult muscles. In the absence of innervation the Duf-positive cells are no longer detected, and muscle patterning is severely disrupted. These results support a model where specialized founder cells prefigure the adult muscle fibers under the control of the nervous system (Fernandes, 2005).

The motoneuron exerts a mitogenic influence on IFM myoblasts. Following unilateral denervation, the BrDU birthdating experiments revealed a significant decline in the rate of proliferation. This decline is likely sufficient to account for the reduced myoblast population observed in denervated hemisegments (previously quantified by morphometry; Fernandes, 1998). The smaller muscles that result are thus likely due to the smaller number of available myoblasts, and possibly to the absence of neuromuscular excitation following denervation (Fernandes, 2005).

Two alternate mechanisms were ruled out: there was no evidence for a change in the rate of myoblast cell death following denervation, indicating that the motoneuron does not provide an essential survival factor for the cells. Also, no significant change was observed in the migratory behavior of myoblasts following unilateral denervation, when examined at either the single cell or population level. This indicates that the reduced population size was not the result of myoblast emigration from denervated sites (Fernandes, 2005).

The second major effect of denervation was the gross disruption of normal muscle formation in the de novo arising DVMs. Normally, myoblasts coalesced into discrete primordia that prefigure the three sets of DVM muscle fibers. When denervated, the DVM myoblasts remain unpatterned, and the muscles fail to form. This may be due to a direct effect of the motoneuron on the myoblasts. However, denervation also disrupts the behavior of a potential intermediary player, the imaginal pioneer cells, that are thought to prefigure the DVM fibers as myoblast fusion targets (Fernandes, 2005).

BrDU birthdating experiments reveal a significant rise in the rate of DLM myoblast proliferation that normally occurs at 18-24 APF. In denervated regions, myoblast proliferation remains unchanged, holding steady at the earlier, basal level seen prior to the onset of myoblast fusion (at 12-16 h APF). Thus, DLM myoblast proliferation involves two components: a basal and nerve-independent phase (at 12-16 h APF) and a later incremental nerve-dependent phase (at 18-24 h APF). It is proposed that the nerve-dependent increase in myoblast proliferation regulates the number of myoblasts available for fusion, and thus is a way for motoneurons to control muscle size (Fernandes, 2005).

The nerve-dependent rise in DLM myoblast proliferation correlates with the expansion of motoneuronal terminal arbors on the muscle fiber surface. While it is possible that the expansion of motoneuron terminals and the change in myoblast proliferation are independent responses to exogenous hormone signals, the data argue that motoneurons strongly influence myoblast cell division, since proliferation is reduced following denervation. It is proposed that the growing motoneuron terminal either releases a factor that influences myoblast cell division, or alternatively potentiates myoblast responsiveness to available growth factors and mitogens. In either case, the nerve-dependent control of myoblast proliferation would in turn influence the growth of the muscle fiber (Fernandes, 2005).

The rise in myoblast proliferation observed during the nerve-dependent phase of DLM development resembles a feature seen during photoreceptor development in the Drosophila eye. During differentiation of the neuroepithelium, there is a rise in cell proliferation, referred to as the second mitotic wave (SMW), which produces additional precursors that are recruited to eventually form a complete ommatidium. It is likely that the rise in DLM myoblast proliferation similarly serves to maintain the size of the myoblast pool, so that cells can be continuously drawn from the pool until the desired muscle size is achieved (Fernandes, 2005).

That the two phases of DLM myogenesis differ in their nerve-dependence resembles events associated with vertebrate myogenesis. Mammalian skeletal muscles form in two waves: primary myotubes form first, and serve as scaffolds for secondary myotube formation. Primary myogenesis is independent of innervation, while secondary myogenesis is nerve dependent. This is due to the presence of a nerve-dependent population of myoblasts essential for secondary myotube formation (Fernandes, 2005).

The DVMs develop from the de novo fusion of myoblasts, and critically depend on the motoneuron for muscle fiber formation (Fernandes, 1998 and Fernandes, 1999). Denervation also results in a failure of myoblasts to segregate into distinct DVM primordia. Myoblast patterning and fiber development for the DVMs have been proposed to depend on specialized imaginal pioneer cells. The 'imaginal pioneer' (IP) cells lie in close association with motoneuron arbors, as demonstrated by EM analysis, and are thus potentially dependent on neurons for their normal function or survival. The IP cells are thought to serve as myoblast fusion targets, and thus to prefigure the mature muscle fibers. There have been, however, no reported molecular markers for these cells (Fernandes, 2005).

In the Drosophila embryo the mesodermal cells that generate the somatic muscles are critically dependent on specialized founder cells. Each embryonic founder cell is the precursor of a specific muscle fiber, and is the target of fusion-competent myoblasts. The founder cells each express Dumbfounded/Kirre, a key component of the cell fusion machinery. In loss of function duf mutations myoblast fusion is disrupted (Fernandes, 2005).

Intriguingly, it was found that there are Duf-positive cells within the DVM myoblast pool. Their location, number, and size indicate that they are likely to be the IP cells previously described. The Duf-positive cells are present in the DVM I and II primordia in direct correspondence to the final number of DVM fibers, as is the case for the IP cells. Duf-positive cells have also been reported for other pupal muscles, and a correlation exists between Duf-positive cells and the numbers of both IFM and abdominal muscle fibers. Significantly, it was found that denervation affects the Duf cells of the DVM primordia. Although Duf-positive cells are initially present in the denervated hemisegments in the normal pattern and number (at 12 h APF), following denervation they are no longer reliably observed by 18-20 h APF. By 24 h APF, when control hemisegments possess well patterned Duf-positive DVMs, Duf-positive cells on the denervated side are rarely observed (Fernandes, 2005).

These observations support a model where Duf-positive IP cells in the pupa serve as fusion targets of myoblasts, as is the case for the Duf-positive founder cells in the embryo. Since the Duf molecule is an essential component of the cell fusion machinery, its disappearance following denervation suggests that fusion events are severely disrupted and may explain the associated muscle patterning defects. It cannot as yet be determine whether the loss of Duf labeling is due to a loss of Duf expression in the IP cells, or due to apoptosis. Distinguishing between these scenarios will require in situ time lapse imaging of vitally labeled IP cells in denervated hemisegments (Fernandes, 2005).

DLM fibers arise from larval muscles that persist into the pupal stage. Like all embryonically established somatic muscle fibers, the persistent larval fibers also do not depend on the motoneuron for their formation or maintenance. When denervated, the larval fibers persist and DLM fibers still form, albeit at a slower rate (Fernandes, 1998; Fernandes. 1999). Duf expression is also detected in the persistent larval fibers, consistent with the fact that they function as myoblast fusion targets. However, unlike the DVMs, denervation does not result in a loss of Duf expression in the developing DLMs. The reason for this independence remains uncharacterized, but likely reflects the distinct origin of these cells from larval precursor muscles (Fernandes, 2005).

A dependence on motoneurons for the regulation of muscle size and patterning has been observed for several insect systems. When abdominal Drosophila muscles are denervated, the adult fibers are significantly reduced in mass. The most prominent effect involves the male-specific muscle (MSM) of the fifth abdominal segment of the adult. This muscle is larger than other body wall muscle fibers, a difference attributed to the enhanced recruitment of myoblasts from a common myoblast pool. When the abdominal myoblast pool is reduced experimentally through hydroxyurea treatment, a smaller muscle is present at the MSM location in segment A5. A BrDU labeling analysis remains to be performed to confirm the role of myoblast proliferation on MSM development (Fernandes, 2005).

Denervation studies in Manduca have similarly shown that proliferation of myonuclei is reduced in leg, abdominal, and DLM muscles. At the onset of metamorphosis, muscle precursors appear in the region of the future adult muscles and become associated with tendons (leg muscles) or persistent larval muscles (DLM). This accumulation is then followed by the appearance of proliferating 'myonuclei' within the developing primordia. By contrast, in the case of Drosophila DLMs, BrDU incorporation is restricted to myoblasts present outside the primordia, and there is no evidence of nuclear division within the muscle fibers (Fernandes, 2005).

In conclusion, it is proposed that the motoneuron critically influences the size of the myoblast pool through a direct effect on myoblast cell division, and that this helps regulate the final size of adult muscle fibers. The motoneuron has a second role in regulating the development of de novo forming fibers, where it is essential for the partitioning of myoblasts into muscle primordia. Moreover, continued Duf labeling within the primordia depends on the motoneuron's presence. Thus, the motoneuron influences both the number of cells available for fusion, as well as potentially regulates the fusion events themselves. This is an elegant mechanism for controlling muscle fiber differentiation during myogenesis, and may have evolved as a way to ensure that muscle primordia develop into muscles that meet the diverse demands placed on them by the nervous system (Fernandes, 2005).

A non-signaling role of Robo2 in tendons is essential for Slit processing and muscle patterning

Coordinated locomotion of an organism relies on the development of proper musculoskeletal connections. In Drosophila, the Slit-Robo signaling pathway guides muscles to tendons. This study shows that the Slit receptor Roundabout 2 (Robo2) plays a non-cell-autonomous role in directing muscles to their corresponding tendons. Robo2 is expressed by tendons, and its non signaling activity in these cells promotes Slit cleavage producing a cleaved Slit-N-terminal guiding signal, which provides short-range signaling into muscles. Consistently, robo2 mutant embryos exhibited a muscle phenotype similar to that of slit, which could not be rescued by a muscle-specific Robo2 expression but rather by an ectodermally derived Robo2. Alternatively this muscle phenotype could be induced by tendon-specific robo2 RNAi. It was further shown that membrane immobilization of Slit, or its N-terminal cleaved form on tendons bypasses the functional requirement for Robo2 in tendons, verifying that the major role of Robo2 is to promote the association of Slit with the tendon cell membrane. Cleaved Slit (Slit-N) tends to oligomerize whereas full-length uncleavable Slit does not. It is therefore proposed that Slit-N oligomers produced at the tendon membrane by Robo2 signal to the approaching muscle by combined Robo;Robo3 activity. These findings establish a Robo2-mediated mechanism, independent of signaling essential to limiting Slit distribution, which might be relevant to the regulation of Slit-mediated short-range signaling in additional systems (Ordan, 2015).

To determine the individual contribution of Robo family receptors to the embryonic muscle pattern, the orientation of the three lateral transverse muscles (LT1-3) and of the dorsal acute muscle 3 (DA3) was analyzed in Drosophila embryos at stage 16 lacking distinct Robo receptors, and it was compared to that of slit. In slit mutant embryos the LT muscles are closer to the posterior segment border and are not spaced correctly, and the DA3 muscle does not extend its leading edge at orthogonal fashion. The orientation of the LT and the DA3 muscles in robo or in robo3 mutants, exhibited almost normal positioning relative to their wild-type (WT) counterparts. Quantification of the ratio between the distance of LT3 muscle and the posterior segment border, divided to overall segmental width (DLT3/Ds) revealed no significant difference between robo and WT, or robo3 and WT. However, in the double robo;robo3 mutant embryos the orientation of these muscles resembled that of slit. robo2 mutants exhibited a phenotype that was comparable to that of slit mutants; the LT muscles were not equally spaced and were closer to the posterior segmental border, a phenotype observed in 85% of the segments. Quantification of DLT3/Ds revealed a smaller value in robo2 mutant. In addition, in 33% of the robo2 mutant segments, the DA3 muscle lost its diagonal orientation and often migrated in a straight ventral-dorsal orientation (Ordan, 2015).

To reveal the dynamic nature of the process of muscle elongation in robo2 mutants, individual muscles were followed in live robo2 mutant and control embryos using specific GFP-marked LT, or DA3 muscles in stage 13-16 throughout muscle elongation. This analysis showed that the polarity of the mutant muscles was similar to that of WT control muscles; however, the LT muscles tended to elongate closer to the segment border and were not evenly spaced. The DA3 muscle often did not turn towards the posterior segment, and both muscles reached their target tendons more slowly. These results suggested differential contributions of the Robo receptors to the directed elongation of LT and DA3 muscles (Ordan, 2015).

To reveal the relative distribution and expression levels of the Robo receptors in muscles, advantage was taken of flies carrying HA tag that was knocked-in within the genomic region of either of the Robo receptors, and embryos were labeled with anti-HA. Robo-HA accumulated at the muscle-tendon junction, and was observed at low levels on the muscle surfaces. Robo2 levels were prominent along ectodermal stripes, but not on muscles, and Robo3 labeling was indistinguishable from background. Double labeling with anti-Robo2 and Stripe or with CD8-GFP driven by the stripe-GAL4 driver verified the ectodermal expression of Robo2 at the surfaces of the tendon cells. The relatively high ectodermal expression of Robo2-HA and its lack of staining in the affected LT and DA3 muscles were consistent with a muscle non-autonomous function of Robo2 in mediating LT and DA3 muscle-patterning (Ordan, 2015).

To address whether undetectable Robo2 protein functioning in the muscles, the ability of Robo2 to rescue the robo2 mutant muscle phenotype was tested when expressed in muscles. Driving Robo2 expression in muscles of robo2 mutant embryos using the mef2-GAL4 driver did not rescue the muscle phenotype, but rather worsen the muscle pattern in all embryos tested compared to wild type embryos overexpressing Robo2. In contrast, knockdown of Robo2 in tendons of robo2 -/+ heterozygous embryos (using sr-GAL4>robo2 RNAi) led to a phenotype similar to that of robo2 mutant; the LT muscles were mis-patterned in 84% of the segments, and the DA3 muscle was aberrant in 31% of the segments. Importantly, partial rescue of the LT muscle pattern was achieved by expressing Robo2 in the ectoderm using the 69-GAL4 driver. Moreover, embryos in which Robo has been inserted into the robo2 locus, rescued the robo2 muscle phenotype, supporting the critical contribution of Robo2 unique ectodermal distribution rather than its signaling activity. In summary, the ectodermal expression of Robo2, its knockdown in tendons, and the ectodermal rescue imply that Robo2 functions in the ectoderm to induce the muscle pattern (Ordan, 2015).

Next, the requirement was addressed for Slit in the ectodermal activity of Robo2, associated with guiding the LT and DA3 muscles. Strong genetic interaction was found between robo2 and slit in inducing the LT and DA3 muscle pattern. Trans-heterozygous embryos for both robo2 and slit exhibited an aberrant pattern of the LT muscles in 82% of the segments relative to 0 or 10% in robo2 /+ or slit/+ embryos, respectively. In the DA3 muscle, 10% of the segments of the trans-heterozygous slit/robo2 embryos exhibited aberrant muscle orientation, relative to 0% in either of the single heterozygous mutants. These results imply that Robo2 activity is mediated through its ligand Slit (Ordan, 2015).

Next, whether Robo2 affects Slit cleavage, which is critical to short-range Slit signaling, was tested. To this end, Robo2 was overexpressed in the entire embryonic ectoderm using the 69B-GAL4 driver and followed endogenous Slit cleavage by western blotting using anti-Slit antibody, reactive with the C-terminal of Slit; this antibody recognizes both full-length Slit (~170kDa) and the C-terminal cleaved polypeptide, Slit-C, (~80kDa). Strikingly, overexpression of Robo2 in the ectoderm led to an average 9.7-fold increase in the ratio of Slit-C relative to Slit-FL levels, implicating Robo2 in promoting Slit cleavage in the ectoderm. In contrast, overexpression of Robo2 in the muscles (using the mef2-GAL4 driver) did not have this effect. Remarkably, similar enhanced Slit cleavage was observed following overexpression of truncated Robo2 lacking the cytoplasmic domain (Evans and Bashaw, 2010). Notably, overexpression of Robo in the ectoderm similarly enhanced Slit cleavage (data not shown). These results implicated a non-signaling function for Robo2 in promoting Slit cleavage (Ordan, 2015).

It was reasoned that covalently bound Slit oligomers, shown to form by previous crystallographic studies, would enable Slit to bind both juxtaposing Robo receptors, on the muscle side (Robo and/or Robo3) and on the tendon side (Robo2). Therefore Slit oligomerization was tested for in embryos. Overexpression of either GFP-tagged cleaved Slit-N (Slit-N-GFP), or uncleavable full length Slit-UC-myc, was induced in the embryonic ectoderm with 69B-GAL4 driver. The embryo extracts were then boiled under reducing (with β- mercaptoethanol, BME) or non-reducing conditions (without BME), separated on SDS-PAGE, and further reacted by western, either with anti-GFP, or anti-Myc. Under non-reducing conditions, Slit-N-GFP exhibited slower electrophoretic mobility relative to its mobility in reduced conditions (expected to be 124kDa), supporting the formation of Slit-N-GFP oligomers by disulfide bonds. In contrast, uncleavable Slit (Slit-UC-myc) did not exhibit differential electrophoretic mobility in natured versus denatured conditions. This result is consistent with the unique ability of Slit-N to oligomerize (Ordan, 2015).

Taken together, these results suggest that Robo2 in tendons enhances Slit cleavage, producing Slit-N oligomers at the tendon-cell membrane that are potentially capable of binding both Robo2 in cis, and Robo/Robo3 in trans, leading to spatially restricted Slit signaling to the elongating muscle. Knocked-in membrane-bound full length Slit bypasses the requirement for Robo2. It was reasoned that if the primary function of Robo2 is to promote localized Slit-N oligomers, the knocked-in membrane-bound uncleavable Slit, (Slit- uncleavable(UC)-CD8), would rescue the phenotype of robo2. Knocked-in Slit-UC-CD8 was recombined with robo2 and the phenotype of the DA3 and LT muscles was analyzed in homozygous embryos. Whereas in robo2 mutants, the orientation of the LT muscles was defective in 85% of the segments, the addition of knocked-in Slit-UC-CD8 showed only 37% defective segments. Likewise the DLT3/Ds ratio was rescued in the robo2;Slit-UC-CD8 embryos. Given that Slit-UC-CD8 by itself produced a moderate muscle phenotype, it is concluded that Slit-UC- CD8 is capable of rescuing the robo2 phenotype of the LT muscles. For the DA3 muscle, whereas 32% of the segments showed a phenotype in robo2 mutants, robo2 mutant embryos recombined with Slit-UC-CD8 showed a muscle phenotype in only 13% of the segments, and Slit-UC-CD8 mutants showed only 5% defective segments (Ordan, 2015).

Moreover, consistent with a major function of Robo2 in promoting Slit cleavage, overexpression of the active cleaved Slit-N-GFP in tendon cells rescued the robo2 mutant LT muscle phenotype in 73% of the segments, whereas expression of Slit-UC-myc alone did not. These results are consistent with a major function for Robo2 in promoting Slit-N accumulation at the tendon cell- membrane, presumably by retaining Slit-FL and enhancing its availability proteolytic cleavage. Slit-N oligomers potentially bind to both Robo2 at the tendon side as well as to Robo and/or Robo3 at the muscle side to promote unidirectional short-range Slit-repulsive signal in muscles. In this context Robo2 cytoplasmic signaling domain is dispensable, consistent with Robo2 function in chordotonal organs and with Robo2 non autonomous function in the CNS (Ordan, 2015).

In conclusion, the findings of this study demonstrate a novel, non-signaling role for Robo2 in tendons in regulating the local distribution of active cleaved Slit oligomers at the tendon cell-membrane. This signal is critical in promoting Slit short-range signaling in muscles essential for directional muscle elongation. Such mechanism might be significant in other setups where Slit promotes signaling between neighboring cells (Ordan, 2015).

Genetic control of the distinction between fat body and gonadal mesoderm

The somatic muscles, the heart, the fat body, the somatic part of the gonad and most of the visceral muscles are derived from a series of segmentally repeated primordia in the Drosophila mesoderm. This work describes the early development of the fat body and its relationship to the gonadal mesoderm, as well as the genetic control of the development of these tissues. The first sign of fat body development is the expression of serpent in segmentally repeated clusters within the trunk mesoderm in parasegments 4-9. Segmentation and dorsoventral patterning genes define three regions in each parasegment in which fat body precursors can develop. The primary and secondary dorsolateral fat body primordia are formed ventral to the visceral muscle primoridium in each parasegment. The ventral secondary cluster forms more ventrally in the posterior portion of each parasegment. Fat body progenitors in these regions are specified by different genetic pathways. Two dorsolateral regions require engrailed and hedgehog (within the even-skipped domain) for their development while the ventral secondary cluster is controlled by wingless. Ubiquitous mesodermal en expression leads to an expansion of the primary clusters into the sloppy-paired domain, resulting in a continuous band of serpent-expressing cells in parasegments 4-9. The observed effect of en on fat body development is seen not only on mesodermal overexpression but also when en is overexpressed in the ectoderm. Loss of wingless leads to an expansion of the dorsolateral fat body primordium. decapentaplegic and one or more unknown genes determine the dorsoventral extent of these regions. High levels of Dpp repress serpent, resulting in the formation of visceral musculature, an alternative cell fate (Reichmann, 1998).

In each of parasegments 10-12 one of these primary dorsolateral regions generates somatic gonadal precursors instead of fat body. The balance between fat body and somatic gonadal fate in these serially homologous cell clusters is controlled by at least five genes. A model is suggested in which tinman, engrailed and wingless are necessary to permit somatic gonadal develoment, while serpent counteracts the effects of these genes and promotes fat body development. In wg mutant embryos, all dorsolateral mesodermal cells, including those in parasegments 10-12, acquire fat body fate. This phenotype can be interpreted as the combined effects of two separate functions of wg: (1) wg is necessary to repress fat body development in the dorsolateral mesoderm underlying the wg domain in all parasegments; (2) wg is required in the primary cluster to permit somatic gonadal precursor instead of fat body development in parasegments 10-12. Loss of engrailed results in the absence of demonstrable somatic gonadal precursors, similar to the situation in tinman mutants. Ubiquitous mesodermal en expression leads to the formation of additional somatic gonadal precursor cells in parasegments 10-12. The homeotic gene abdominalA limits the region of serpent activity by interfering in a mutually repressive feed back loop between gonadal and fat body development. It is unlikely that abdA represses srp directly, since srp can be expressed in cells in which abdA is active. abdA might prevent srp from inhibition of a somatic gonadal precursor competence factor (Riechmann, 1998).

A nutrient sensor mechanism controls Drosophila growth

Organisms modulate their growth according to nutrient availability. Although individual cells in a multicellular animal may respond directly to nutrient levels, growth of the entire organism needs to be coordinated. This study provides evidence that in Drosophila, coordination of organismal growth originates from the fat body, an insect organ that retains endocrine and storage functions of the vertebrate liver. A genetic screen for growth modifiers discovered slimfast, a gene that encodes an amino acid transporter. Remarkably, downregulation of slimfast specifically within the fat body causes a global growth defect similar to that seen in Drosophila raised under poor nutritional conditions. This involves TSC/TOR signaling in the fat body, and a remote inhibition of organismal growth via local repression of PI3-kinase signaling in peripheral tissues. These results demonstrate that the fat body functions as a nutrient sensor that restricts global growth through a humoral mechanism (Colombani, 2003).

In multicellular organisms, the control of growth depends on the integration of various genetic and environmental cues. Nutrient availability is one of the major environmental signals influencing growth and, as such, has dictated adaptative responses during evolution toward multicellularity. In particular, complex humoral responses ensure that growth and development are properly coordinated with nutritional conditions (Colombani, 2003).

In isolated cells, amino acid withdrawal leads to an immediate suppression of protein synthesis, suggesting that cells are protected by active sensing mechanims that block translation prior to depletion of internal amino acid stores. In many mammalian cell types, changes in amino acid diet affect the binding of the translation repressor 4EBP1 to initiation factor eIF4E and the activity of ribosomal protein S6 kinase (S6K). These two signaling events require the activity of TOR (target of rapamycin), a conserved kinase recently shown to participate in a nutrient-sensitive complex both in mammalian cells and in yeast. Mutations in the Drosophila TOR homolog (dTOR) results in cellular and physiological responses characteristic of amino acid deprivation and establish that TOR is cell autonomously required for growth in a multicellular organism. Furthermore, the TSC (tuberous sclerosis complex) tumor suppressor, consisting of a TSC1 and TSC2 heterodimer (TSC1/2), as well as the small GTPase Rheb participate to the regulation of TOR function. Overall, these data suggest that TSC, Rheb, TOR, and S6K participate in a conserved pathway that coordinates growth with nutrition in a cell-intrinsic manner (Colombani, 2003).

In multicellular organisms, humoral controls are believed to buffer variations in nutrient levels. However, little is known about how growth of individual cells is coordinated. In vertebrates, growth-promoting action of the growth hormone (GH) is mostly relayed to peripheral tissues through the production of IGF-I. Binding of IGF-I to its cognate receptor tyrosine kinase (IGF-IR) induces phosphorylation of insulin receptor substrates (IRS), which in turn activate a cascade of downstream effectors. These include phospho-inositide 3-kinase (PI3K), which generates the second messenger phosphatidylinositol-3,4,5-P3 (PIP3), and thereby activates the AKT/PKB kinase. Genetic manipulation of IGF-I, IGF-IR, PI3K, and AKT in mice modulates tissue growth in vivo thus demonstrating a requirement of the IGF pathway for growth. In Drosophila, both loss- and gain-of function studies have also exemplified the role of a conserved insulin/IGF signaling pathway in the control of growth. Ligands for the unique insulin receptor (Inr) constitute a family of seven peptides related to insulin, the Drosophila insulin-like peptides (Dilps). Remarkably, three dilp genes (dilp2, dilp3, and dilp5) are expressed in a cluster of seven median neurosecretory cells (m-NSCs) in the larval brain, suggesting that they have an endocrine function. Indeed, ablation of the seven dilp-expressing mNSCs in larvae induces a systemic growth defect (Colombani, 2003).

Both in flies and mice, mutations in IRS provoke growth retardation as well as female sterility similar to what is observed in starved animals. Moreover, PI3K activity in Drosophila larvae depends on the availability of proteins in the food. Overall, this supports the notion that the insulin/IGF pathway might coordinate tissue growth with nutritional conditions. However, upon amino acid withdrawal, neither PI3K nor AKT/PKB activities are downregulated in mammalian or insect cells in culture, suggesting that this pathway does not directly respond to nutrient shortage. Hence, an intermediate sensor mechanism must link nutrient availability to insulin/IGF signaling (Colombani, 2003).

An intriguing possibility is that specific organs could function as nutrient sensors and induce a nonautonomous modulation of insulin/IGF growth signaling in response to changes in nutrient levels. This study used a genetic approach in Drosophila to assess both the cellular and humoral responses to amino acid deprivation in the context of a developing organism. The insect fat body (FB) has important storage and humoral functions associated with nutrition, comparable to vertebrate liver and adipose tissue. During larval stages, the FB accumulates large stores of proteins, lipids, and carbohydrates, which are normally degraded by autophagy during metamorphosis in order to supply the developing tissues but can also be remobilized during larval life to compensate transitory nutrient shortage. In addition to its storage function, the FB also has endocrine activity and supports growth of imaginal disc explants and DNA replication of larval brains in coculture. This study demonstrates that the FB operates as a sensor for variations in nutrient levels and coordinates growth of peripheral tissues accordingly via a humoral mechanism (Colombani, 2003).

In the course of a P[UAS]-based overexpression screen for growth modifiers, a P[UAS]-insertion line (UY681) was found to cause growth retardation upon ectopic activation. Sequence analysis revealed that P(UY)681 is inserted in a predicted gene (CG11128) that encodes a putative protein showing strong homology with amino acid permeases of the cationic amino acid transporter (CAT) family. The P[UAS] element is inserted in the first intron of the CG11128 gene, potentially driving transcription of an antisense RNA in a GAL4-dependent manner. To assess the function of this transporter, 3H-arginine uptake was measured in S2 cells. Results indicate that amino acid uptake is either enhanced by transfection of a CG11128 cDNA or suppressed by RNAi, indicating that the encoded protein presents CAT activity. In situ hybridization revealed basal levels of CG11128 expression in most larval tissues but much higher levels in the FB and the gut, two tissues involved in amino acid processing (Colombani, 2003).

By P element remobilization, an imprecise excision was obtained that deletes the sequences encoding the N-terminal half of the protein. 87% of homozygous mutant animals die during larval stages. The few viable adults emerged after a 2 day delay and were smaller and markedly slimmer than control animals. The associated gene was named slimfast (slif) and the excision allele slif1. Weight measurement indicated that homozygous slif1 adult males displayed a 16% mass reduction compared to control. Accordingly, adult wing size was reduced by 8% due to a reduction of both cell size and cell number. When the slif1 allele was in trans to Df(3L)Δ1AK, a deficiency covering the locus, larval lethality was slightly enhanced, suggesting that slif1 corresponds to a strong hypomorphic allele. The amino acid transporter function of slif, as well as the phenotypes observed upon reduction of slif function suggest that slif mutant animals might suffer amino acid deprivation. A major consequence of amino acid deprivation in larvae is the remobilization of nutrient stores in the FB, which typically results in aggregation of storage vesicles. Consistently, fusion of storage vesicles was observed in the FB of slif1 larvae and was indistinguishable from that observed in animals fed on protein-free media (Colombani, 2003).

GAL4 induction of P(UY)681 resulted in a growth-deficient phenotype similar to that of slif1 loss of function. The antisense orientation of P(UY)681 suggested that the growth defect following GAL4 induction was due to an RNAi effect. Indeed, Northern blot analysis revealed that ubiquitous GAL4-dependent activation of P(UY)681 using the daughterless-GAL4 (da-GAL4) driver strongly reduced slif mRNA levels. Only two of the three alternative first exons are potentially affected by the antisense RNA, possibly explaining the residual accumulation of slif mRNAs in da-GAL4; P(UY)681 animals. Most of these animals died at larval stage, similar to what was observed for slif1 mutants. Specific induction of P(UY)681in the wing disc using the MS1096-GAL4 driver provoked a reduction of the adult wing size, which could be either rescued by coactivation of a UAS-slif transgene or enhanced by reducing slif gene dosage with the heterozygous Df(3L)Δ1AK deficiency. Thus, GAL4-dependent activation of P(UY)681 reduces slif function and defines a conditional loss-of-function allele hereafter termed slifAnti (Colombani, 2003).

As expected, loss of slif function using the slifAnti allele also mimicked amino acid deprivation. Accordingly, ubiquitous slifAnti induction in growing larvae resulted in storage vesicle aggregation and strong reduction of global S6 kinase activity, similar to what was reported in animals raised on protein-free diet. Additionally, an increase in PEPCK1 gene transcription was observed, similar to the effect of amino acid withdrawal. In summary, this study has identified two loss-of-function alleles of the slif gene whose defects mimic physiological aspects of amino acid deprivation. Importantly, the conditional slifAnti allele provides a unique tool to mimic an amino acid deprivation in a tissue-specific manner (Colombani, 2003).

This study established that the FB is a sensor tissue for amino acid levels, as downregulation of the Slif amino acid transporter within the FB is sufficient to induce a general reduction in the rate of larval growth. In contrast, specific disruption of slif in imaginal discs, larval gut, or salivary glands did not induce a nonautonomous growth response, suggesting that these tissues do not participate in the systemic control of growth. The dilp-expressing median neurosecretory cells (m-NSCs) also affect growth control, since selective ablation of these cells in the larval brain induces an overall reduction of animal size. In response to complete sugar and protein starvation, the m-NSCs stop expressing dilp3 and dilp5 genes, suggesting that these neurons also sense nutrient levels. This study shows that the selective reduction of slif function in these cells has no obvious effect on tissue growth and animal development. This indicates that the seven dilp-expressing m-NSCs do not constitute a general amino acid sensor. In contrast, the role of m-NSCs in carbohydrate homeostasis and the observation that they stop expressing certain dilp genes when larvae are deprived of sugar rather suggests that these cells have a role in sensing carbohydrate levels (Colombani, 2003 and references therein).

This analysis also provides a framework in which to understand the phenotype of minidisc, a mutation in an amino acid transporter gene that exhibits nonautonomous growth defects in imaginal discs (Colombani, 2003).

In a number of model systems, both PI3K and TOR have been implicated in linking growth to nutritional status and, until recently, were considered as intermediates of a common regulatory pathway. In yeast, the TOR kinase is part of a cell-autonomous nutrient sensor, which controls protein synthesis, ribosome biogenesis, nutrient import, and autophagy. Genetic analysis in Drosophila indicates that dTOR is required for cell-intrinsic growth control. The results obtained using the slifAnti allele in the wing disc indicate that individual tissues have indeed the potential to respond to amino acid deprivation in a cell-autonomous manner. Nonetheless, this study also demonstrates that the TOR nutritional checkpoint participates in a systemic control of larval growth emanating from the FB. Within a developing organism, each cell may integrate these two distinct inputs regarding nutritional status, one originating from a systemically-acting FB sensor, and the other from TOR-dependent signaling in individual cells. One can further speculate that depending on the strength and duration of starvation, different in vivo nutritional checkpoints will be hierarchically recruited to protect the animal and that the systemic control might, in most physiological situations, override the cell-autonomous control. Indeed, as the data demonstrate, the FB sensor is sufficient to induce a general and coordinated response to starvation without calling individual cell-autonomous mechanisms into play (Colombani, 2003).

Several lines of evidence indicate that the PI3K pathway is not part of the sensor mechanism in FB cells. First, a sensor for PI3K activity in the FB is only marginally affected by amino acid deprivation in that tissue, indicating that the cell-autonomous response to amino acid starvation does not directly influence PI3K signaling. This is reminiscent of previous observations in mammalian cultured cells, showing that PI3K activity does not respond to variations in amino acid levels. Moreover, inhibition of PI3K signaling by dPTEN expression in the FB is not sufficient to trigger the sensing mechanism. Although, dPTEN overexpression causes a complete disappearance of the PI3K sensor accompanied by growth suppression of FB cells, the FB maintains a critical mass that allows for normal larval growth. In contrast, the regulatory subunit p60 whose overexpression potently inhibits PI3-kinase in flies has been shown to induce a systemic effect on larval growth when overexpressed in the FB using an Adh-Gal4 driver. This study found that a pumpless ppl-GAL4-directed expression of p60 also provokes a strong suppression of larval growth and a dramatic inhibition of FB development in young larvae. Thus, the systemic effect on growth observed upon p60 overexpression most likely results from a drastic reduction of FB mass, which then fails to support normal larval growth (Colombani, 2003).

These results further indicate that PI3K signaling is a remote target of the humoral message that originates from the FB in response to amino acid deprivation. This is in agreement with previous data showing that PI3K activity is downregulated by dietary amino acid deprivation and explains why global PI3-kinase inhibition mimics cellular and organismal effects of starvation. The existence of a humoral relay reconciles these in vivo studies with the absence of direct PI3K responsiveness to amino acid levels (Colombani, 2003).

The relative resistance of imaginal disc growth to the systemic control exerted by the FB correlates with maintenance of PI3K activity in these tissues. This is in agreement with previous observations that cells in the larval brain and in imaginal discs maintain a slow rate of proliferation under protein starvation, while larval endoreduplicating tissues (ERTs) arrest. This difference might be attributed to the basal levels of dilp2 expression observed in imaginal discs, allowing a moderate growth rate of these tissues through an autocrine/paracrine mechanism. It was recently shown that clonal induction of PI3K potently induces cell-autonomous growth response even in fasting larvae, indicating that some nutrients are still accessible to support cell growth within a fasted larva. The main function of a general sensor could be to preserve these limited nutrients for use by high priority tissues. In this context, local PI3K activation through an autocrine loop in imaginal tissues could favor the growth of prospective adult structures in adverse food conditions. Thus, the FB would have an active role in controlling the allocation of resources depending on nutritional status. In this respect, it is noteworthy that FB cells are relatively resistant to the FB-derived humoral signal, since the PI3K sensor is not drastically affected in the FB of ppl>slifAnti animals. Thereby, essential regulatory functions of the FB could be preserved even in severely restricted nutritional conditions (Colombani, 2003).

How does the FB signal to other tissues? This study suggests that a humoral signal relays information from the FB amino acid sensor and systemically inhibits PI3K signaling. In addition, this downregulation is not due to a direct inhibition of dilp expression by neurosecretory cells in the brain. Nevertheless, it cannot be ruled out that the secretion of these molecules is subjected to regulation in the mNSCs. Both in vivo and in insect cell culture, several imaginal discs growth factors (IDGF) secreted by the FB have been proposed to function synergistically with Dilp signaling to promote growth. However, this study did not find any modification of IDGF expression in the FB of larvae raised on water- or sugar-only diet, or upon FB induction of slifanti. In vertebrates, the different functions of the circulating IGF-I are modulated through its association with IGF-BPs and acid labile subunit (ALS). In particular, the formation of a ternary complex with ALS leads to a considerable extension of IGF-I half-life. The finding that a Drosophila ALS ortholog is expressed within the FB in an amino acid-dependent manner provides a new avenue to study the molecular mechanisms of nonautonomous growth control mediated by the FB (Colombani, 2003).

This study highlights the contribution that genetics can provide to unravel the mechanisms of physiological control. Using a genetic tool to mimic amino acid deprivation, it was demonstrated that nutrition systemically controls body size through an amino acid sensor operating in the FB. It is proposed that (1) in metazoans, a systemic nutritional sensor modulates the conserved TOR-signaling pathway, and (2) the response to sensor activation is relayed by a hormonal mechanism, which triggers an Inr/PI3K-dependent response in peripheral tissues (Colombani, 2003).

Role and regulation of starvation-induced autophagy in the Drosophila fat body

In response to starvation, eukaryotic cells recover nutrients through autophagy, a lysosomal-mediated process of cytoplasmic degradation. Autophagy is known to be inhibited by TOR signaling, but the mechanisms of autophagy regulation and its role in TOR-mediated cell growth are unclear. Signaling through TOR and its upstream regulators PI3K and Rheb is necessary and sufficient to suppress starvation-induced autophagy in the Drosophila fat body. In contrast, TOR's downstream effector S6K promotes rather than suppresses autophagy, suggesting S6K downregulation may limit autophagy during extended starvation. Despite the catabolic potential of autophagy, disruption of conserved components of the autophagic machinery, including ATG1 and ATG5, does not restore growth to TOR mutant cells. Instead, inhibition of autophagy enhances TOR mutant phenotypes, including reduced cell size, growth rate, and survival. Thus, in cells lacking TOR, autophagy plays a protective role that is dominant over its potential role as a growth suppressor (Scott, 2004).

Autophagy likely evolved in single-cell eukaryotes to provide an energy and nutrient source allowing temporary survival of starvation. In yeast, Tor1 and Tor2 act as direct links between nutrient conditions and cell metabolism. These proteins sense nutritional status by an unknown mechanism, and effect a variety of starvation responses including changes in transcriptional and translational programs, nutrient import, protein and mRNA stability, cell cycle arrest, and induction of autophagy. Autophagy thus occurs in the context of a comprehensive reorganization of cellular activities aimed at surviving low nutrient levels (Scott, 2004).

In multicellular organisms, TOR is thought to have retained its role as a nutrient sensor but has also adopted new functions in regulating and responding to growth factor signaling pathways and developmental programs. Thus in a variety of signaling, developmental, and disease contexts, TOR activity can be regulated independently of nutritional conditions. In these cases, autophagy may be induced in response to downregulation of TOR despite the presence of abundant nutrients and may potentially play an important role in suppressing cell growth rather than promoting survival. Identification of the tumor suppressors PTEN, and TSC1 and TSC2 as positive regulators of autophagy provides correlative evidence supporting such a role for autophagy in growth control. Alternatively, since TOR activity is required for proper expression and localization of a number of nutrient transporters, inactivation of TOR may lead to reduced intracellular nutrient levels, and autophagy may therefore be required under these conditions to provide the nutrients and energy necessary for normal cell metabolism and survival (Scott, 2004).

The results presented here provide genetic evidence that under conditions of low TOR signaling, autophagy functions primarily to promote normal cell function and survival, rather than to suppress cell growth. This conclusion is based on the finding that genetic disruption of autophagy does not restore growth to cells lacking TOR, but instead exacerbates multiple TOR mutant phenotypes. It is important to note that mutations in TOR do not disrupt larval feeding, and thus disruption of autophagy is detrimental in TOR mutants despite the presence of ample extracellular nutrients. The finding that autophagy is critical in cells lacking TOR further supports earlier studies suggesting that inactivation of TOR causes defects in nutrient import, resulting in an intracellular state of pseudo-starvation (Scott, 2004).

Can the further reduction in growth of TOR mutant cells upon disruption of autophagy be reconciled with the potential catabolic effects of autophagy? TOR regulates the bidirectional flow of nutrients between protein synthesis and degradation through effects on nutrient import, autophagy, and ribosome biogenesis. When TOR is inactivated, rates of nutrient import and protein synthesis decrease, resulting in a commensurate reduction in mass accumulation and cell growth. In addition, autophagy is induced to maintain intracellular nutrient and energy levels sufficient for normal cell metabolism. When autophagy is experimentally inhibited in cells lacking TOR, this reserve source of nutrients is blocked, leading to a further decrease in energy levels, protein synthesis, and growth. It is noted that autophagy may have additional functions in cells with depressed TOR signaling, including recycling of organelles damaged by the absence of TOR activity, or selective degradation of cell growth regulators, analogous to the regulatory roles of ubiquitin-mediated degradation (Scott, 2004).

Autophagy is required for normal developmental responses to inactivation of insulin/PI3K signaling in the nematode C. elegans. In response to starvation or disruption of insulin/PI3K signaling, C. elegans larvae enter a dormant state called the dauer. Autophagy has been observed in C. elegans larvae undergoing dauer formation: disruption of a number of ATG homologs interfers with normal dauer morphogenesis. Importantly, simultaneous disruption of insulin/PI3K signaling and autophagy genes results in lethality, similar to the results presented in this study. Thus despite significant differences in developmental strategies for surviving nutrient deprivation, autophagy plays an essential role in the starvation responses of yeast, flies, and worms (Scott, 2004).

The prevailing view that S6K acts to suppress autophagy was founded on correlations between induction of autophagy and dephosphorylation of rpS6 in response to amino acid deprivation or rapamycin treatment. However, the genetic data presented in this study argue strongly against a role for S6K in suppressing autophagy: unlike other positive components of the TOR pathway, null mutations in S6K do not induce autophagy in fed animals. It is suggested that the observed correlation between S6K activity and suppression of autophagy is due to common but independent regulation of S6K and autophagy by TOR. Thus, autophagy suppression and S6K-dependent functions such as ribosome biogenesis represent distinct outputs of TOR signaling (Scott, 2004).

How might TOR signal to the autophagic machinery, if not through S6K? In yeast, this is accomplished in part through regulation of Atg1 kinase activity and Atg8a gene expression (Kamada, 2000 and Kirisako, 1999). The demonstration of a role for Drosophila ATG1 and ATG8 homologs [see TG8a (CG32672) and ATG8b (CG12334)] in starvation-induced autophagy, and the genetic interaction observed between ATG1 and TOR, are consistent with a related mode of regulation in higher eukaryotes. However, it is noted that other components of the yeast Atg1 complex such as Atg17 and Atg13, whose phosphorylation state is rapamycin sensitive, do not have clear homologs in metazoans, indicating that differences in regulation of autophagy by TOR are likely (Scott, 2004).

In addition to excluding a role for S6K in suppression of autophagy, these results reveal a positive role for S6K in induction of autophagy. S6K may promote autophagy directly, through activation of the autophagy machinery, or indirectly through its effects on protein synthesis. The latter possibility is consistent with previous reports that protein synthesis is required for expansion and maturation of autophagosomes. Interestingly, despite being required for autophagy, S6K is downregulated under conditions that induce it, including chronic starvation and TOR inactivation. Consistent with this, it was found that lysotracker staining is significantly weaker in chronically starved animals or in TOR mutants than in wild-type animals starved 3-4 hr. Furthermore, expression of constitutively activated S6K has no effect in wild-type, but restores lysotracker staining in TOR mutants to levels similar to those of acutely starved wild-type animals. It is suggested that downregulation of S6K may limit rates of autophagy under conditions of extended starvation or TOR inactivation and that this may protect cells from the potentially damaging effects of unrestrained autophagy (Scott, 2004).

Co-culture and conditioned media experiments have shown that the Drosophila fat body is a source of diffusible mitogens. The fat body has also been shown to act as a nutrient sensor through a TOR-dependent mechanism and to regulate organismal growth through effects on insulin/PI3K signaling. The results in this study extend these findings by showing that this endocrine response is accompanied by the regulated release of nutrients through autophagic degradation of fat body cytoplasm. Preventing this reallocation of resources, either through constitutive activation of PI3K or through inactivation of ATG genes, results in profound nutrient sensitivity. Thus, in response to nutrient limitation, the fat body is capable of simultaneously restricting growth of peripheral tissues through downregulation of insulin/PI3K signaling and providing these tissues with a buffering source of nutrients necessary for survival through autophagy (Scott, 2004).

The two origins of hemocytes in Drosophila

As in many other organisms, the blood of Drosophila consists of several types of hemocytes, which originate from the mesoderm. By lineage analyses of transplanted cells, two separate anlagen have been defined that give rise to different populations of hemocytes: embryonic hemocytes and lymph gland hemocytes. The anlage of the embryonic hemocytes is restricted to a region within the head mesoderm between 70% and 80% egg length. In contrast to all other mesodermal cells, the cells of this anlage are already determined as hemocytes at the blastoderm stage. Unexpectedly, these hemocytes do not degenerate during late larval stages, but have the capacity to persist through metamorphosis and are still detectable in the adult fly. A second anlage, which gives rise to additional hemocytes at the onset of metamorphosis, is located within the thoracic mesoderm at 50% to 53% egg length. After transplantation within this region, clones were detected in the larval lymph glands. Labeled hemocytes are released by the lymph glands not before the late third larval instar. The anlage of these lymph gland-derived hemocytes is not determined at the blastoderm stage, as indicated by the overlap of clones with other tissues. These analyses reveal that the hemocytes of pupae and adult flies consist of a mixture of embryonic hemocytes and lymph gland-derived hemocytes, originating from two distinct anlagen that are determined at different stages of development (Holz, 2003).

The origin of the embryonic hemocytes (EH) can be traced back to the head mesoderm of late stage 11 embryos by morphological criteria. Owing to the fact that srp is expressed in a narrow stripe within the cephalic mesoderm at the blastoderm stage and that a loss of srp function leads to a complete loss of embryonic hemocytes, this domain is considered to be the primordium of the EH. By homotopic single-cell transplantations it was possible to restrict the anlage to a sharply delimitated region located at 70% to 80% EL within the mesoderm, exactly corresponding to the cephalic expression domain of srp. The fact that none of the EH clones overlapped with other tissues indicates that the hemocytes are already determined at the blastoderm stage. This was confirmed by heterotopic transplantations from the EH anlage into the abdominal mesoderm; these transplanted cells give rise to hemocytes. Since mesodermal cells transplanted into the EH anlage do not transform into embryonic hemocytes, the determining factor is not able to induce a hemocyte fate within these cells and seems to function cell-autonomously. A good candidate for such a factor is Srp. However, since srp is also expressed in many other tissues that do not give rise to hemocytes, there must be additional genes that lead to a determination of the EH at the blastoderm stage. The early determination of the EH is quite unusual, since all other mesodermal tissues analyzed to date -- including the anlage of the lymph gland-derived hemocytes -- are not restricted to a tissue-specific fate prior to the second postblastodermal mitoses. This might be a developmental adaptation of the EH, which at stage 12 are already differentiated into functional macrophages and are responsible for the removal of apoptotic cells within developing tissues (Holz, 2003).

It is commonly believed that in Drosophila during larval development the EH population is entirely replaced by hemocytes that have been released by the larval lymph glands. However, it is possible to trace hemocytes originating from the head mesoderm through all stages of development until 14-day-old adult flies. The number of hemocytes progressively rises during larval life, from less than 200 to more than 5000 per individual. Cell lineage analyses unambiguously demonstrate that this increase is due to postembryonic proliferation of the EH. The contribution of the lymph glands to the hemocyte population was determined by means of cell lineage analyses. These studies reveal that the lymph glands do not release blood cells into the hemocoel during all larval stages but exclusively at the end of the third larval instar (Holz, 2003).

With the onset of metamorphosis, additional hemocytes are released from the lymph glands. Although the lymph glands do not persist through metamorphosis, the marked hemocytes released by the labeled lymph glands are still detectable in adult flies. Hence, all hemocytes found throughout larval life originate solely from the EH anlage, whereas the pupal and imaginal blood is made up of two different populations: EH and LGH (Holz, 2003).

The two populations of hemocytes share many functional, morphological and genetic similarities. In both cases, the determination of hemocytes depends on srp, while the specification towards the distinct blood cell types is induced by the expression of lozenge (lz) glia cells missing (gcm) and the gcm homolog gcm2. Both EH and LGH differentiate into podocytes, crystal cells and plasmatocytes. Hemocytes of both populations have the capability to adopt macrophage characteristics. However, despite all similarities, the history of the two populations is quite different, since they originate from two different mesodermal regions and are determined at different developmental stages. In view of the fact that the lymph glands do not release hemocytes before the onset of metamorphosis under nonimmune conditions, all hemocytes found in the larval hemocoel represent EH (Holz, 2003).

The many similarities between EG and LGH raise the question why there are two populations at all. A massive release of hemocytes by the lymph glands is seen just at the onset of pupation. The lymph glands additionally have the capacity to differentiate and release a special type of hemocytes, the lamellocytes, under immune conditions even before the onset of metamorphosis. Thus, because under nonimmune conditions the lymph glands do not release any cells before the onset of pupation, it might be their primary role to provide a reservoir of immune defensive hemocytes. The massive apoptosis and accumulation of cell debris might be a secondary trigger to stimulate proliferation and release of the lymph gland hemocytes (Holz, 2003).

The Drosophila lymph gland as a developmental model of hematopoiesis

Drosophila hematopoiesis occurs in a specialized organ called the lymph gland. In this systematic analysis of lymph gland structure and gene expression, the developmental steps in the maturation of blood cells (hemocytes) from their precursors are defined. In particular, distinct zones of hemocyte maturation, signaling and proliferation in the lymph gland during hematopoietic progression are described. Different stages of hemocyte development have been classified according to marker expression and placed within developmental niches: a medullary zone for quiescent prohemocytes, a cortical zone for maturing hemocytes and a zone called the posterior signaling center for specialized signaling hemocytes. This establishes a framework for the identification of Drosophila blood cells, at various stages of maturation, and provides a genetic basis for spatial and temporal events that govern hemocyte development. The cellular events identified in this analysis further establish Drosophila as a model system for hematopoiesis (Jung, 2005).

In the late embryo, the lymph gland consists of a single pair of lobes containing ~20 cells each. These express the transcription factors Srp and Odd skipped (Odd), and each cluster of hemocyte precursors is followed by a string of Odd-expressing pericardial cells that are proposed to have nephrocyte function. These lymph gland lobes are arranged bilaterally such that they flank the dorsal vessel, the simple aorta/heart tube of the open circulatory system, at the midline. By the second larval instar, lymph gland morphology is distinctly different in that two or three new pairs of posterior lobes have formed and the primary lobes have increased in size approximately tenfold (to ~200 cells. By the late third instar, the lymph gland has grown significantly in size (approximately another tenfold) but the arrangement of the lobes and pericardial cells has remained the same. The cells of the third instar lymph gland continue to express Srp (Jung, 2005).

The third instar lymph gland also exhibits a strong, branching network of extracellular matrix (ECM) throughout the primary lobe. This network was visualized using several GFP-trap lines in which GFP is fused to endogenous proteins. For example, line G454 represents an insertion into the viking locus, which encodes a Collagen IV component of the extracellular matrix. The hemocytes in the primary lobes of G454 (expressing Viking-GFP) appear to be clustered into small populations within pockets or chambers bounded by GFP-labeled branches of various sizes. Other lines, such as the uncharacterized GFP-trap line ZCL2867, also highlight this branching pattern. What role this intricate ECM network plays in hematopoiesis, as well as why multiple cells cluster within these ECM chambers, remains to be determined (Jung, 2005).

Careful examination of dissected, late third-instar lymph glands by differential interference contrast (DIC) microscopy revealed the presence of two structurally distinct regions within the primary lymph gland lobes that have not been previously described. The periphery of the primary lobe generally exhibits a granular appearance, whereas the medial region looks smooth and compact. These characteristics were examined further with confocal microscopy using a GFP-trap line G147, in which GFP is fused to a microtubule-associated protein. The G147 line is expressed throughout the lymph gland but, in contrast to nuclear markers such as Srp and Odd, distinguishes morphological differences among cells because the GFP-fusion protein is expressed in the cytoplasm in association with the microtubule network. Cells in the periphery of the lymph gland make relatively few cell-cell contacts, thereby giving rise to gaps and voids among the cells within this region. This cellular individualization is consistent with the granularity of the peripheral region observed by DIC microscopy. By contrast, cells in the medial region were relatively compact with minimal intercellular space, which is also consistent with the smoother appearance of this region by DIC microscopy. Thus, in the late third instar, the lymph gland primary lobes consist of two physically distinct regions: a medial region consisting of compactly arranged cells, which was termed the medullary zone; and a peripheral region of loosely arranged cells, termed the cortical zone (Jung, 2005).

Mature hemocytes have been shown to express several markers, including collagens, Hemolectin, Lozenge, Peroxidasin and P1 antigen. The expression of the reporter Collagen-gal4 (Cg-gal4), which is expressed by both plasmatocytes and crystal cells, is restricted to the periphery of the primary lymph gland lobe. Comparison of Cg-gal4 expression in G147 lymph glands, in which the medullary zone and cortical zone can be distinguished, reveals that maturing hemocytes are restricted to the cortical zone. In fact, the expression of each of the maturation markers mentioned above is found to be restricted to the cortical zone. The reporter hml-gal4 and Pxn, which are expressed by the plasmatocyte and crystal cell lineages, are extensively expressed in this region. Likewise, the expression of the crystal cell lineage marker Lozenge is restricted in this manner. The spatial restriction of maturing crystal cells to the cortical zone was verified by several means, including the distribution of melanized lymph gland crystal cells in the Black cells background and analysis of the terminal marker ProPOA1. The cortical zone is also the site of P1 antigen expression, a marker of the plasmatocyte lineage. The uncharacterized GFP fusion line ZCL2826 also exhibits preferential expression in the cortical zone. Last, it was found that the homeobox transcription factor Cut is preferentially expressed in the cortical zone of the primary lobe. Although the role of Cut in Drosophila hematopoiesis is currently unknown, homologs of Cut are known to be regulators of the myeloid hematopoietic lineage in both mice and humans. Cells of the rare third cell type, lamellocytes, are also restricted to the cortical zone, based upon cell morphology and the expression of a msn-lacZ reporter (msn06946). In summary, based on the expression patterns of several genetic markers that identify the three major blood cell lineages, it is proposed that the cortical zone is a specific site for hemocyte maturation (Jung, 2005).

The medullary zone was initially defined by structural characteristics and subsequently by the lack of expression of mature hemocyte markers. However, several markers have been identified that are exclusively expressed in the medullary zone at high levels but not the cortical zone. Consistent with the compact arrangement of cells in the medullary zone, it was found that Drosophila E-cadherin (DE-cadherin or Shotgun) is highly expressed in this region. No significant expression of DE-cadherin was observed among maturing cells in the cortical zone. E-cadherin, in both vertebrates and Drosophila, is a Ca2+-dependent, homotypic adhesion molecule often expressed by epithelial cells and is a crucial component of adherens junctions. Attempts to study DE-cadherin mutant clones in the medullary zone where the protein is expressed were unsuccessful since no clones were recoverable. The reporter lines domeless-gal4 and unpaired3-gal4 are preferentially expressed in the medullary zone. The gene domeless (dome) encodes a receptor molecule known to mediate the activation of the JAK/STAT pathway upon binding of the ligand Unpaired. The unpaired3 (upd3) gene encodes a protein with homology to Unpaired and has been associated with innate immune function. These gal4 lines are in this study only as markers that correlate with the medullary zone and, at the present time, there is no evidence that their associated proteins have a role in lymph gland hematopoiesis. Other markers of interest with preferential expression in the medullary zone include the molecularly uncharacterized GFP-trap line ZCL2897 and actin5C-GFP. Cells expressing hemocyte maturation markers are not seen in the medullary zone. It is therefore reasonable to propose that this zone is largely populated by prohemocytes that will later mature in the cortical zone. Prohemocytes are characterized by their lack of maturation markers, as well as their expression of several markers described as expressed in the medullary zone (Jung, 2005).

The posterior signaling center (PSC), a small cluster of cells at the posterior tip of each of the primary (anterior-most) lymph gland lobes, is defined by its expression of the Notch ligand Serrate and the transcription factor Collier. During this analysis, several additional markers were identified that exhibit specific or preferential expression in the PSC region. For example, it was found that the reporter Dorothy-gal4 is strongly expressed in this zone. The Dorothy gene encodes a UDP-glycosyltransferase, which belongs to a class of enzymes that function in the detoxification of metabolites. The upd3-gal4 reporter, which has preferential expression in the medullary zone, is also strongly expressed among cells of the PSC. Last, three uncharacterized GFP-gene trap lines, ZCL2375, ZCL2856 and ZCL0611 were found, that are preferentially expressed in the PSC. This analysis has made it clear that the PSC is a distinct zone of cells that can be defined by the expression of multiple gene products (Jung, 2005).

The PSC can be defined just as definitively by the characteristic absence of several markers. For example, the RTK receptor Pvr, which is expressed throughout the lymph gland, is notably absent from the PSC. Likewise, dome-gal4 is not expressed in the PSC, further suggesting that this population of cells is biased toward the production of ligands rather than receptor proteins. Maturation markers such as Cg-gal4, which are expressed throughout the cortical zone, are not expressed by PSC cells. Additionally, the expression levels of the hemocyte marker Hemese and the Friend-of-GATA protein U-shaped are dramatically reduced in the PSC when compared with other hemocytes of the lymph gland. Taken together, both the expression and lack of expression of a number of genetic markers defines the cells of the PSC as a unique hemocyte population (Jung, 2005).

In contrast to primary lobes of the third instar, maturing hemocytes are generally not seen in the secondary lobes. Correspondingly, secondary lobes often have a smooth and compact appearance, much like the medullary zone of the primary lobe. Consistent with this appearance, secondary lymph gland lobes also express high levels of DE-cadherin. The size of the secondary lobe, however, varies from animal to animal and this correlates with the presence or absence of maturation markers. Smaller secondary lobes contain a few or no cells expressing maturation markers, whereas larger secondary lobes usually exhibit groups of differentiating cells. Direct comparison of DE-cadherin expression in secondary lobes with that of Cg-gal4, hml-gal4 or Lz revealed that the expression of these maturation markers occurs only in areas in which DE-cadherin is downregulated. Therefore, although there is no apparent distinction between cortical and medullary zones in differentiating secondary lobes, there is a significant correlation between the expression of maturation markers and the downregulation of DE-cadherin, as is observed in primary lobes (Jung, 2005).

The relatively late 'snapshot' of lymph gland development in the third larval instar establishes the existence of spatial zones within the lymph gland that are characterized by differences in structure as well as gene expression. In order to understand how these zones form over time, lymph glands of second instar larvae, the earliest time at which it was possible to dissect and stain, were examined for the expression of hematopoietic markers. As expected, Srp and Odd are expressed throughout the lymph gland during the second instar since they are in the late embryo and third instar lymph gland. Likewise, the hemocyte-specific marker Hemese is expressed throughout the lymph gland at this stage, although it is not present in the embryonic lymph gland (Jung, 2005).

To determine whether the cortical zone is already formed or forming in second instar lymph glands, the expression of various maturation markers were examined in a pair-wise manner to establish their temporal order. Of the markers examined, hml-gal4 and Pxn are the earliest to be expressed. The majority of maturing cells were found to be double-positive for hml-gal4 and Pxn expression, although a few cells were found to express either hml-gal4 or Pxn alone. This indicates that the expression of these markers is initiated at approximately the same time, although probably independently, during lymph gland development. The marker Cg-gal4 is next to be expressed since it was found among a subpopulation of Pxn-expressing cells. Finally, P1 antigen expression is initiated late, usually in the early third instar. Interestingly, the early expression of each of these maturation markers is restricted to the periphery of the primary lymph gland lobe, indicating that the cortical zone begins to form in this position in the second instar. Whenever possible, each genetic marker was directly compared with other pertinent markers in double-labeling experiments, except in cases such as the comparison of two different gal4 reporter lines or when available antibodies were generated in the same animal. In such cases, the relationship between the two markers, for example dome-gal4 and hml-gal4, was inferred from independent comparison with a third marker such as Pxn (Jung, 2005).

By studying the temporal sequence of expression of hemocyte-specific markers, one can describe stages in the maturation of a hemocyte. It should be noted, however, that not all hemocytes of a particular lineage are identical. For example, in the late third instar lymph gland, the large majority of mature plasmatocytes (~80%) expresses both Pxn and hml-gal4, but the remainder express only Pxn (~15%) or hml-gal4 (~5%) alone. Thus, while plasmatocytes as a group can be characterized by the expression of representative markers, populations expressing subsets of these markers indeed exist. It remains unclear at this time whether this heterogeneity in the hemocyte population is reflective of specific functional differences (Jung, 2005).

In the third instar, Pxn is a prototypical hemocyte maturation marker, while immature cells of the medullary zone express dome-gal4. Comparing the expression of these two markers in the second instar reveals an interesting developmental progression. A group of cells along the peripheral edge of these early lymph glands already express Pxn. These developing hemocytes downregulate the expression of dome-gal4, as they do in the third instar. Next to these developing hemocytes is a group of cells that expresses dome-gal4 but not Pxn; these cells are most similar to medullary zone cells of the third instar and are therefore prohemocytes. Interestingly, there also exists a group of cells in the second instar that expresses neither Pxn nor dome-gal4. This population is most easily seen in the medial parts of the gland, close to the centrally placed dorsal. These cells resemble earlier precursors in the embryo, except they express the marker Hemese. These cells are called pre-prohemocytes. Interpretation of the expression data is that pre-prohemocytes upregulate dome-gal4 to become prohemocytes. As prohemocytes begin to mature into hemocytes, dome-gal4 expression is downregulated, while the expression of maturation markers is initiated. The prohemocyte and hemocyte populations continue to be represented in the third instar as components of the medullary and cortical zones, respectively (Jung, 2005).

The cells of the PSC are already distinguishable in the late embryo by their expression of collier. It was found that the canonical PSC marker Ser-lacZ is not expressed in the embryonic lymph gland and is only expressed in a small number of cells in the second instar. This relatively late onset of expression is consistent with collier acting genetically upstream of Ser. Another finding was that the earliest expression of upd3-gal4 parallels the expression of Ser-lacZ and is restricted to the PSC region. Finally, Pvr and dome-gal4 are excluded from the PSC in the second instar, similar to what is seen in the third instar (Jung, 2005).

To determine whether maturing cortical zone cells are indeed derived from medullary zone prohemocytes, a lineage-tracing experiment was performed in which dome-gal4 was used to initiate the permanent marking of all daughter cell lineages. In this system, the dome-gal4 reporter expresses both UAS-GFP and UAS-FLP. The FLP recombinase excises an intervening FRT-flanked 'STOP cassette', allowing constitutive expression of lacZ under the control of the actin5C promoter. At any developmental time point, GFP is expressed in cells where dome-gal4 is active, while lacZ is expressed in all subsequent daughter cells regardless of whether they continue to express dome-gal4. In this experiment, cortical zone cells are permanently marked with ß-galactosidase despite not expressing dome-gal4 (as assessed by GFP), indicating that these cells are derived from a dome-gal4-positive precursor. This result is consistent with and further supports independent marker analysis that shows that dome-gal4-positive prohemocytes downregulate dome-gal4 expression as they initiate expression of maturation markers representative of cortical zone cells. As controls to the above experiment, the expression patterns of two other gal4 lines, twist-gal4 and Serrate-gal4 were determined. The reporter twist-gal4 is expressed throughout the embryonic mesoderm from which the lymph gland is derived. Accordingly, the entire lymph gland is permanently marked by ß-galactosidase despite a lack of twist-gal4 expression (GFP) in the third instar lymph gland. Analysis of Ser-gal4 reveals that PSC cells remain a distinct population of signaling cells that do not contribute to the cortical zone (Jung, 2005).

Genetic manipulation of Pvr function provides valuable insight into its involvement in the regulation of temporal events of lymph gland development. To analyze Pvr function, FLP/FRT-based Pvr-mutant clones were generated in the lymph gland early in the first instar and then examined during the third instar for the expression of maturation markers. It was found that loss of Pvr function abolishes P1 antigen and Pxn expression, but not Hemese expression. The crystal cell markers Lz and ProPOA1 are also expressed normally in Pvr-mutant clones, consistent with the observation that mature crystal cells lack or downregulate Pvr. The fact that Pvr-mutant cells express Hemese and can differentiate into crystal cells suggests that Pvr specifically controls plasmatocyte differentiation. Pvr-mutant cells do not become TUNEL positive but do express the hemocyte marker Hemese and can differentiate into crystal cells, all suggesting that the observed block in plasmatocyte differentiation within the mutant clone is not due to cell death. Additionally, Pvr-mutant clones were large and not significantly different in size from their wild-type twin spots. Thus, the primary role of Pvr is not in the control of cell proliferation. Targeting Pvr by RNA interference (RNAi) revealed the same phenotypic features, confirming that Pvr controls the transition of Hemese-positive cells to plasmatocyte fate (Jung, 2005).

Entry into S phase was monitored using BrdU incorporation and distinct proliferative phases were identified that occur during lymph gland hematopoiesis. In the second instar, proliferating cells are evenly distributed throughout the lymph gland. By the third instar, however, the distribution of proliferating cells is no longer uniform; S-phase cells are largely restricted to the cortical zone. This is particularly evident when BrdU-labeled lymph glands are co-stained with Pxn. Medullary zone cells, which can be identified by the expression of dome-gal4, rarely incorporate BrdU. Therefore, the rapidly cycling prohemocytes of the second instar lymph gland quiesce as they populate the medullary zone of the third instar. As prohemocytes transition into hemocyte fates in the cortical zone, they once again begin to expand in number. This is supported by the observation that the medullary zone in white pre-pupae does not appear diminished in size, suggesting that the primary mechanism for the expansion of the cortical zone prior to this stage is through cell division within the zone. Proliferating cells in the secondary lobes continue to be distributed uniformly in the third instar, suggesting that secondary-lobe prohemocytes do not reach a state of quiescence as do the cells of the medullary zone. These results indicate that cells of the lymph gland go through distinct proliferative phases as hematopoietic development proceeds (Jung, 2005).

This analysis of the lymph gland revealed three key features that arise during development. The first feature is the presence of three distinct zones in the primary lymph gland lobe of third instar larvae. Two of these zones, termed the cortical and medullary zones, exhibit structural characteristics that make them morphologically distinct. These zones, as well as the third zone, the PSC, are also distinguishable by the expression of specific markers. The second key feature is that cells expressing maturation markers such as Lz, ProPOA1, Pxn, hml-gal4 and Cg-gal4 are restricted to the cortical zone. The medullary zone is consistently devoid of maturation marker expression and is therefore defined as a region composed of immature hemocytes (prohemocytes). The finding of different developmental populations within the lymph gland (prohemoctyes and their derived hemocytes) is similar to the situation in vertebrates where it is known that hematopoietic stem cells and other blood precursors give rise to various mature cell types. Additionally, Drosophila hemocyte maturation is akin to the progressive maturation of myeloid and lymphoid lineages in vertebrate hematopoiesis. The third key feature of lymph gland hematopoiesis is the dynamic pattern of cellular proliferation observed in the third instar. At this stage, the vast majority of S-phase cells in the primary lobe are located in the cortical zone, suggesting a strong correlation between proliferation and hemocyte differentiation. Compared with earlier developmental stages, cell proliferation in the medullary zone actually decreases by the late third instar, suggesting that these cells have entered a quiescent state. Thus, proliferation in the lymph gland appears to be regulated such that growth, quiescence and expansion phases are evident throughout its development (Jung, 2005).

Drosophila blood cell precursors, prohemocytes and maturing hemocytes each exhibit extensive phases of proliferation. The competence of these cells to proliferate seems to be a distinct cellular characteristic that is superimposed upon the intrinsic maturation program. Based on the patterns of BrdU incorporation in developing primary and secondary lymph gland lobes, it is possible to envision at least two levels of proliferation control during hematopoiesis. It is proposed that the widespread cell proliferation observed in second instar lymph glands and in secondary lobes of third instar lymph glands occurs in response to a growth requirement that provides a sufficient number of prohemocytes for subsequent differentiation. The mechanisms promoting differentiation in the cortical zone also trigger cell proliferation, which accounts for the observed BrdU incorporation in this zone and serves to expand the effector hemocyte population. The quiescent cells of the medullary zone represent a pluripotent precursor population because they, similar to vertebrate hematopoietic precursors, rarely divide and give rise to multiple lineages and cell types (Jung, 2005).

Based on this analysis a model is proposed by which hemocytes mature in the lymph gland. Hematopoietic precursors that populate the early lymph gland are first distinguishable as Srp+, Odd+ (S+O+) cells. These will eventually give rise to a primary lymph gland lobe where the steps of hemocyte maturation are most apparent. During the first or early second instar, these S+O+ cells begin to express the hemocyte-specific marker Hemese (He) and the tyrosine kinase receptor Pvr. Such cells can be called pre-prohemocytes and, in the second instar, cells expressing only these markers occupy a narrow region near the dorsal vessel. Subsequently, a subset of these Srp+, Odd+, He+, Pvr+ (S+O+H+Pv+) pre-prohemocytes initiate the expression of dome-gal4 (dg4), thereby maturing into prohemocytes. The prohemocyte population (S+O+H+Pv+dg4+) can be subdivided into two developmental stages. Stage 1 prohemocytes, which are abundantly seen in the second instar, are proliferative, whereas stage 2 prohemocytes, exemplified by the cells of the medullary zone, are quiescent. As development continues, prohemocytes begin to downregulate dome-gal4 and express maturation markers (M; becoming S+O+H+Pv+dg4lowM+). Eventually, dome-gal4 expression is lost entirely in these cells (becoming S+O+H+Pv+dg4-M+), found generally in the cortical zone. Thus, the maturing hemocytes of the cortical zone are derived from prohemocytes previously belonging to the medullary zone. This is supported by lineage-tracing experiments that show cells expressing medullary zone markers can indeed give rise to cells of the cortical zone. In turn, the medullary zone is derived from the earlier, pre-prohemocytes. Early cortical zone cells continue to express successive maturation markers (M) as they proceed towards terminal differentiation. Depending on the hemocyte type, examples of expressed maturation markers are Pxn, P1, Lz, L1, msn-lacZ, etc. These studies have shown that differentiation of the plasmatocyte lineage requires Pvr, while previous work has shown that the Notch pathway is crucial for the crystal cell fate. Both the JAK/STAT and Notch pathways have been implicated in lamellocyte production (Jung, 2005).

Previous investigations have demonstrated that similar transcription factors and signal transduction pathways are used in the specification of blood lineages in both vertebrates and Drosophila. Given this relationship, Drosophila represents a powerful system for identifying genes crucial to the hematopoietic process that are conserved in the vertebrate system. The work presented here provides an analysis of hematopoietic development in the Drosophila lymph gland that not only identifies stage-specific markers, but also reveals developmental mechanisms underlying hemocyte specification and maturation. The prohemocyte population in Drosophila becomes mitotically quiescent, much as their multipotent precursor counterparts in mammalian systems. These conserved mechanisms further establish Drosophila as an excellent genetic model for the study of hematopoiesis (Jung, 2005).

Subdivision and developmental fate of the head mesoderm in Drosophila

This paper defines temporal and spatial subdivisions of the embryonic head mesoderm and describes the fate of the main lineages derived from this tissue. During gastrulation, only a fraction of the head mesoderm (primary head mesoderm; PHM) invaginates as the anterior part of the ventral furrow. The PHM can be subdivided into four linearly arranged domains, based on the expression of different combinations of genetic markers (tinman, heartless, snail, serpent, mef-2, zfh-1). The anterior domain (PHMA) produces a variety of cell types, among them the neuroendocrine gland (corpus cardiacum). PHMB, forming much of the'T-bar' of the ventral furrow, migrates anteriorly and dorsally and gives rise to the dorsal pharyngeal musculature. PHMC is located behind the T-bar and forms part of the anterior endoderm, besides contributing to hemocytes. The most posterior domain, PHMD, belongs to the anterior gnathal segments and gives rise to a few somatic muscles, but also to hemocytes. The procephalic region flanking the ventral furrow also contributes to head mesoderm (secondary head mesoderm, SHM) that segregates from the surface after the ventral furrow has invaginated, indicating that gastrulation in the procephalon is much more protracted than in the trunk. This study distinguishes between an early SHM (eSHM) that is located on either side of the anterior endoderm and is the major source of hemocytes, including crystal cells. The eSHM is followed by the late SHM (lSHM), which consists of an anterior and posterior component (lSHMa, lSHMp). The lSHMa, flanking the stomodeum anteriorly and laterally, produces the visceral musculature of the esophagus, as well as a population of tinman-positive cells that is interpreted as a rudimentary cephalic aorta ('cephalic vascular rudiment'). The lSHM contributes hemocytes, as well as the nephrocytes forming the subesophageal body, also called garland cells (de Velasco, 2005).

The mesoderm is a morphologically distinct cell layer that can be recognized in early embryos of most bilaterian phyla and that gives rise to tissues interposed between ectodermal and endodermal epithelia, including muscle, connective, blood, vascular, and excretory tissue. Besides the differentiative fate of tissues derived from it, the mesoderm shares several common properties in regard to its formation during gastrulation. The anlage of the mesoderm is sandwiched in between the anlage of the endoderm and the neurectoderm. This has been documented in most detail in anamniote vertebrates, where signals from the vegetal blastomeres (the anlage of the endoderm) act on the adjacent marginal zone of the future ectoderm to induce mesoderm. Although gastrulation proceeds quite differently in arthropods from the way it does in chordates, the proximity of the mesodermal anlage to future endoderm and neurectoderm is conserved, and numerous signaling pathways and transcriptional regulators that share similar function and expression patterns in arthropods and chordates have been identified (de Velasco, 2005 and references therein).

Following gastrulation, the mesoderm is subdivided along the dorso-ventral axis into several subdivisions laid out in a distinct dorso-ventral order. In vertebrates, cells located in the dorsal part of the mesoderm anlage give rise to notochord and somites, which in turn produce muscular, skeletal, and connective tissue. Next to the somitic mesoderm is the intermediate mesoderm that will form the excretory and reproductive system. The ventral mesoderm (lateral plate) gives rise to blood, vascular system, visceral musculature, and coelomic cavity. In arthropods, fundamentally similar mesodermal subdivisions can be recognized, and similarities extend to the relative positions these domains obtain relative to each other and relative to the adjacent neurectoderm. For example, precursors of visceral muscles, vascular system, and blood are at the edge of the mesoderm facing away from the neural primordium (ventral in vertebrates, dorsal in arthropods (de Velasco, 2005 and references therein).

The subdivision of the vertebrate mesoderm into distinct longitudinal tissue columns with different fates is seen throughout the trunk and head of the embryo. However, several significant differences between the head and the trunk are immediately apparent. For example, cells derived from the anterior neurectoderm form the neural crest that migrates laterally and gives rise to many of the tissues that are produced by mesoderm in the trunk. As a result, the fates taken over by the head mesoderm are more limited than those of the trunk mesoderm. In contrast, the head mesoderm produces several unique lineages, such as the heart (cardiac mesoderm) and a population of early differentiating macrophages. Moreover, some of the signaling pathways responsible for inducing different mesodermal fates in the trunk appear to operate in a different manner in the head. A recently described example is the Wnt signal that induces somatic musculature in the trunk, but inhibits the same fate in the head (de Velasco, 2005 and references therein).

The head mesoderm of arthropods, like that of vertebrates, also appears to deviate in many ways from the trunk mesoderm. For example, specialized lineages like embryonic blood cells and nephrocytes forming the subesophageal body (also called garland cells) arise exclusively in the head. That being said, very little is known about how the arthropod head mesoderm arises and what types of tissues derive from it. The existing literature mainly uses histology, which severely limits the possibilities of following different cell types forward or backward in time. In this paper, several molecular markers have been used to initiate more detailed studies of the head mesoderm in Drosophila. The goal was to establish temporal and spatial subdivisions of the head mesoderm and, using molecular markers expressed from early stages onward, to follow the fate of the lineages derived from this embryonic tissue. Besides hemocytes and pharyngeal muscles described earlier, the head mesoderm also gives rise to several other lineages, including visceral muscle, putative vascular cells, nephrocytes, and neuroendocrine cells. The development of the head mesoderm is discussed in comparison with the trunk mesoderm and in the broader context of insect embryology (de Velasco, 2005).

The Drosophila head mesoderm, as traditionally defined, includes all mesoderm cells originating anterior to the cephalic furrow. The formation of the head mesoderm is complicated by the fact that (unlike the mesoderm of the trunk) only part of it invaginates with the ventral furrow; by far, the majority of head mesoderm cells, recognizable in a stage 10 or 11 embryo, segregate from the surface epithelium of the head after the ventral furrow has formed. Another complicating factor is that head mesoderm cells derived from different antero-posterior levels adopt very different fates, unlike the situation in the trunk where mesodermal fates within different segments along the AP axis are fairly homogenous, with obvious exceptions such as the gonadal mesoderm that is derived exclusively from a subset of abdominal segments. Using several different markers, this study has followed the origin, migration pathways, and later, fates of head mesoderm cells (de Velasco, 2005).

The anterior part of the ventral furrow, called primary head mesoderm (PHM) in the following, includes cells that will contribute to diverse tissues, including muscle, hemocytes, endoderm, and several ill-defined cell populations closely associated with the brain and neuroendocrine system. For clarification, the anterior ventral furrow will be divided into the following domains:

The anterior lip of the T-bar (PHMA) is the source of the corpus cardiacum, as well as other gt-positive cells that at least in part end up as nerve cells flanking the frontal connective and frontal ganglion. These cells continue the expression of giant throughout late embryonic development; they represent a hitherto unknown class of nonneuroblast-derived neurons (de Velasco, 2005).

The posterior lip of the T-bar (PHMB) can be followed towards later stages by its continued expression of htl. These cells, called the procephalic somatic mesoderm, form a bilateral cluster that moves dorso-anteriorly into the labrum and becomes the dorsal pharyngeal musculature. Htl expression almost disappears in these cells around late stage 11, but is reinitiated at stage 12 and stays strong until stage 14, when the dorsal pharyngeal muscles differentiate. Many of the genes expressed in the somatic musculature of the trunk and its precursors (Dmef2, beta-3-tubulin) are also expressed in the procephalic somatic mesoderm (de Velasco, 2005).

The part of the ventral furrow posteriorly adjacent to the T-bar (PHMC) expresses srp, forkhead (fkh), and other endoderm/hemocyte markers. After the ventral furrow closes in the ventral midline (stage 7/8), these cells form a compact median mass, most of which represents part of the anterior endoderm that gives rise to the midgut epithelium. Starting at around this stage, the lateral part of the hemocyte-forming 'secondary head mesoderm' ingresses in between the endoderm and the surface ectoderm. It is likely that some of the PHMC cells invaginating already with the ventral furrow, along with the cells that form the anterior endoderm, also give rise to hemocytes. Precursors of hemocytes and midgut are difficult to distinguish during and shortly after ventral furrow invagination since both express srp and other markers shared between hemocytes and midgut precursors. At around stage 9, the two populations of precursors disengage. The endoderm remains a compact mesenchyme attached to the invaginating stomodeum; hemocyte precursors move dorsally and take on the shape of expanding vertical plates interposed in between endoderm and ectoderm (de Velasco, 2005).

Domain PHMD, the short portion of the ventral furrow situated posterior to the endoderm, along with a considerable portion of the mesoderm behind the cephalic furrow, forms the mesoderm of the three gnathal segments (mandible, maxilla, labium). The gnathal mesoderm in many ways behaves like the mesoderm of thoracic and abdominal segments. It gives rise to somatic muscle (the lateral pharyngeal muscles), visceral muscle, and fat body. Unlike trunk mesoderm, gnathal mesoderm does not produce cardioblasts and pericardial cells. Instead, a large proportion of gnathal mesoderm cells, joining the anteriorly adjacent secondary procephalic mesoderm, adopt the fate of hemocytes (de Velasco, 2005).

Besides the ventral furrow, other parts of the ventral procephalon produce head mesoderm in a complex succession of delamination and ingression events. The head mesoderm that forms from outside the ventral furrow will be called 'secondary mesoderm' (SHM) in the following. Based on the time of formation and the position relative to the stomodeum, the following phases and domains of secondary head mesoderm development can be distinguished.

Following the obliteration of the ventral furrow at stage 8, the eSHM delaminates from the ventral surface 'meso-ectoderm' (considering that this epithelium still contains mesodermal progenitors!) flanking the endodermal mass. The eSHM forms two monolayered sheets that gradually move dorsally and posteriorly; by stage 9, the eSHM cells line the basal surface of the emerging head neuroblasts. An undefined number of primary head mesoderm cells derived from domain PHMC of the ventral furrow are mingled together with the eSHM cells. The ultimate fate of the eSHM is that of hemocytes: they express srp, followed slightly later by other blood cell markers (e.g., peroxidasin and asrij). A subset of hemocytes, called crystal cells, derive from precursors that form a morphologically conspicuous cluster at the dorsal edge of the eSHM, identifiable from early stage 10 onward by the expression of lz. The mechanism by which at least part of the eSHM delaminates is unique. Thus, it is formed by the vertically oriented division of the surface epithelium, whereby the inner daughters will become eSHMe and the outer ones ectoderm. The focus of vertical mitosis has named the procephalic domain in which it occurs 'mitotic domain #9' (de Velasco, 2005).

From late stage 9 onward, the early SHMs are followed inside the embryo by the closely adjacent posterior late SHMs. One cluster of posterior late secondary head mesoderm (lSHMp) cells delaminates from the surface epithelium flanking the posterior lip of the stomodeum; a second lSHMp cluster appears at the same stage at a slightly more posterior level. The first cluster seems to contribute to the hemocyte population; the posterior cluster gives rise to the nephrocytes forming the subesophageal body (also called garland cells; labeled by CG32094). Garland cell precursors are initially arranged as a paired cluster latero-ventrally of the esophagus primordium; subsequently, the clusters fuse in the midline and form a crescent underneath the esophagus. Garland cells are distinguished from crystal cells by their size, location, and arrangement: crystal cells are large, round cells grouped in an oblong cloud dorso-anterior to the proventriculus. Garland cells are smaller, closely attached to each other, and lie ventral of the esophagus (de Velasco, 2005).

During stages 10 and 11, cells delaminate beside and anterior to the stomodeum, originating from the anlage of the esophagus and the epipharynx (labrum). These cells, called anterior late secondary head mesoderm cells (lSHMa), can be followed by their expression of tin. Two groups can be distinguished. The tin-positive cells delaminating from the esophageal anlage (es) give rise to the visceral musculature (vm) surrounding the esophagus. These cells lose tin expression soon after their segregation, but can be recognized by other visceral mesoderm markers such as anti-Connectin. More dorsally, in the anlage of the clypeolabrum (cl) delaminate, the dorsal subpopulation of the lSHMas, which rapidly migrates posteriorly on either side and slightly dorsal of the esophagus, can be found. These cells retain expression of tin into the late embryo. They assemble into two longitudinal rows stretching alongside the roof of the esophagus primordium. During late embryogenesis, they move posteriorly along with the esophagus towards a position behind the brain commissure. Many of the tin-positive SHMs apparently undergo apoptosis: initially counting approximately 25 on either side, they decrease to 12-15 at stage 14 to finally form a single, irregular row of about 15 cells total in the late embryo. These cells come into contact with the anterior tip of the dorsal vessel. This formation of previously undescribed cells, for which the term 'procephalic vascular cells', is proposed, is interpreted as a rudiment of the head aorta, which forms a prominent part of the dorsal vessel in many insect groups (de Velasco, 2005).

On the basis of additional molecular markers, the tin-positive procephalic vascular cells are further subdivided into two populations. The first subpopulation expresses the muscle and cardioblast-specific marker Dmef2; the second type is Dmef2-negative. In the dorsal vessel of the trunk, tin-positive cells also fall into a Dmef2-positive and a Dmef2-negative population. Dmef2-positive cells of the trunk represent the cardioblasts, myoendothelial cells lining the lumen of the dorsal vessel. Dmef2-negative/tin-positive cells form a somewhat irregular double row of cells attached to the ventral wall of the dorsal vessel. The ultimate fate of these cells has not been explored yet. However, preliminary data suggest that they develop into a muscle band that runs alongside the larval dorsal vessel. This would correspond to the situation in other insects in which such a ventral cardiac muscle band has been described (de Velasco, 2005).

The role of tinman in the formation of the procephalic vascular rudiment was investigated by assaying tin-mutant embryos for the expression of Dmef2. Similar to the cardioblasts of the trunk, the Dmef2-positive cells of the procephalic vascular rudiment are absent in tin mutants. It is quite likely that the (Dmef2-negative) remainder of the procephalic vascular rudiment is affected as well by loss of tin, but in the absence of appropriate markers (besides tin itself, which is not expressed in the mutant), it was not possible to substantiate this proposal (de Velasco, 2005).

At the time of appearance of the ventral furrow, segmental markers such as hh do not allow the distinction between distinct 'preoral' segments. Thus, hh is expressed in a wide procephalic stripe in front of the regularly sized mandibular stripe. During stage 7, the procephalic hh stripe splits into an anterior, antennal stripe and a posterior, short, intercalary stripe. The anterior lip of the ventral furrow (domain PHMA) coincides with the anterior boundary of the antenno-intercalary stripe. Thus, the primary head mesoderm and endoderm originating from within the anterior ventral furrow can be considered a derivative of the antennal and intercalary segments. This interpretation is supported by the expression of the homeobox gene labial (lab) found in the intercalary segment. The labial domain covers much of the anterior ventral furrow, including domains PHMB-C (de Velasco, 2005).

Morphogenetic movements in the ventral head, associated with the closure of the ventral furrow, the formation of the stomodeal placode, and the subsequent invagination of the stomodeum result in a shift of head segmental boundaries. The antennal segment tilts backward, as can be seen from the orientation of the antennal hh stripe that from stage 8 onward forms an almost horizontal line, connecting the cephalic furrow with the sides of the stomodeal invagination (which falls within the ventral realm of the antennal segment, in Drosophila as well as other insects). Since the expression of hh, like that of engrailed (en), coincides with the posterior boundary of a segment, the territory located ventral to the antennal hh stripe falls within the intercalary segment. This implies that most, if not all, of the posterior late SHM, is intercalary in origin. It is further plausible to consider that the anterior lSHM belongs to the intercalary and antennal segment. The vascular cells of the head, a conspicuous derivative of the anterior lSHM in Drosophila, are derived from the antennal mesoderm in other insects. The labrum, with which much of the anterior lSHM is associated, represents a structure that has always been difficult to integrate in the segmental organization of the head. Most likely the labrum represents part of the intercalary segment; this would help explain some of the unusual characteristics of the head mesoderm (de Velasco, 2005).

In conclusion, several fundamental similarities are found between the mesoderm of the head and that of the trunk regarding the tissues they give rise to, and possibly the signaling pathways deciding over these fates. After an initial phase of structural and molecular homogeneity, the trunk mesoderm becomes subdivided into a dorsal and a ventral domain by a Dpp-signaling event that emanates from the dorsal ectoderm. The dorsal domain, characterized by the Dpp-dependent continued expression of tinman, becomes the source of visceral and cardiogenic mesoderm, among other cell types. A role of Dpp/BMP signaling in cardiogenesis seems to be conserved among insects and vertebrates. Subsequent signaling steps, involving both Wingless and Notch/Delta, separate between these two fates and further subdivide the cardiogenic mesoderm into several distinct lineages, such as cardioblast, pericardial cells, and secondary hemocyte precursors (lymph gland). As a result of these signaling events, Tinman and several other fate-determining transcription factors become restricted to their respective lineages: tin to the cardioblasts, odd to pericardial cells and hemocyte precursors, zfh1 and srp to hemocyte precursors and fat body. Dmef2 and several other transcription factors become restricted to various combinations of muscle types (somatic, visceral, cardiac) (de Velasco, 2005).

In the head mesoderm, the above genes are associated with similar fates. Tin and Dmef2 appear widely in the procephalic ventral furrow and the anterior lSHM before getting restricted to the procephalic vascular rudiment and/or the pharyngeal musculature, respectively. In contrast with the initially ubiquitous expression of Tin and Dmef2 in the trunk mesoderm, those parts of the head mesoderm giving rise to hemocytes (PHMC, posterior lSHM) never express these mesodermal genes. Previous work has shown that the head gap gene buttonhead (btd) is responsible for the early repression of tin in the above mentioned domains of the head mesoderm. The early absence of Tin and Dmef2 in the head mesodermal hemocyte precursors is paralleled by the presence of Srp and Zfh1 in these cells. Interestingly, Srp/Zfh-positive cells of the head produce only hemocytes and no fat body, suggesting that an as-yet-uncharacterized signaling step prevents the formation of fat body in the head. It is tempting to speculate that there exists within the mesoderm a 'blood/fat body equivalence group'. Blood cells and fat body share not only the expression of fate-determining genes such as srp and zfh1, but also, later, functional properties that have to do with immunity. In the trunk, the blood/fat body equivalence group gives rise mostly to fat body, producing only a limited number of hemocyte precursors in the dorsal mesoderm of the thoracic segments. In the head, on the other hand, all cells of the equivalence group become hemocytes (de Velasco, 2005).

Attention is drawn to another mesodermal lineage that produces related, yet not identical, cell types in the trunk and the head: the nephrocytes. Nephrocytes are defined by their characteristic ultrastructure (membrane invaginations sealed off by junctions) that attests to their excretory function. In the trunk, nephrocytes are represented by the pericardial cells that settle beside the cardioblasts; a newly discovered nephrocyte population ('star cells') invading the Malpighian tubules is derived from the mesoderm of the tail segments. In the head, nephrocytes aggregate near the junction between esophagus and proventriculus as the subesophageal body, also called garland cells. The fact that from the early stages of development onward different transcription factors are expressed in garland cells and pericardial cells suggests that these cells perform similar, yet not fully overlapping, functions (de Velasco, 2005).

Mononuclear muscle cells in Drosophila ovaries revealed by GFP protein traps

Genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into human muscle physiology and disease. Studies in Drosophila have been particularly useful for discovering key genes involved in muscle specification, myoblast fusion, and sarcomere organization. The muscles of the Drosophila female reproductive system have received little attention despite extensive work on oogenesis. This study used newly available GFP protein trap lines to characterize of ovarian muscle morphology and sarcomere organization. The muscle cells surrounding the oviducts are multinuclear with highly organized sarcomeres typical of somatic muscles. In contrast, the two muscle layers of the ovary, which are derived from gonadal mesoderm, have a mesh-like morphology similar to gut visceral muscle. Protein traps in the Fasciclin 3 gene produced Fas3::GFP that localized in dots around the periphery of epithelial sheath cells, the muscle surrounding ovarioles. Surprisingly, the epithelial sheath cells each contain a single nucleus, indicating these cells do not undergo myoblast fusion during development. Consistent with this observation, the Flp/FRT system was used to efficiently generate genetic mosaics in the epithelial sheath, suggesting these cells provide a new opportunity for clonal analysis of adult striated muscle (Hudson, 2008).

Two protein trap lines showed a striped pattern in ovarian muscle. The GFP transposon in these lines was inserted into the genes for two uncharacterized proteins, CG30084 and CG6416, both of which contain a PDZ domain and a ZASP motif (ZM). CG30084 also contains four LIM domains in its C-terminus. Both CG30084 and CG6416 proteins show homology to the human Z-disc Alternatively Spliced Protein (ZASP) that localizes to the Z-disc in skeletal muscle. Like the ZASP gene, both CG30084 and CG6416 encode a number of alternative splice forms, almost all of which could be tagged based on the locations of the protein trap insertions. An additional isoform of CG30084 has been annotated that encodes only LIM domains and cannot be tagged. Due to the homology of these genes to ZASP, CG30084 was designated as Zasp52, and CG6416 as Zasp66 based on the cytological location of the genes (Hudson, 2008).

The availability of protein trap lines was especially useful for describing intercellular contacts (e.g., Fas3::GFP, Ilk::GFP inserted into Integrin linked kinase) and for showing the location of 'new' sarcomere components (e.g., Zasp52::GFP, Zasp66::GFP). The muscle cells of the oviducts, which are derived from genital disks, have highly organized sarcomeres and a myotube organization typical of somatic muscles in insects, and likely arise by myoblast fusion during development. In contrast, the visceral muscle layers surrounding ovarioles and ovaries have a mesh-like shape similar to the visceral muscles of the gut. Surprisingly, it was found that the epithelial sheath muscles contain one nucleus rather than two as in gut visceral muscles, making them the only known adult somatic or visceral muscle that does not arise by myoblast fusion. It remains to be determined whether the peritoneal sheath cells are mononuclear as well (Hudson, 2008).

The nature of the epithelial sheath muscle makes it ideally suited for analysis of muscle function. The morphology of the cells can be viewed in great detail with an array of available sarcomere and cellular markers. Importantly, the ability to produce cells with a defined genotype by mitotic clone induction provides a new opportunity for studying the phenotypes in adult muscles caused by homozygous mutations in muscle genes, even if the mutations are lethal during early development. Finally, it is straightforward to do short-term in vitro culturing of ovarioles with the epithelial sheath intact and contracting; thus, the behavior of cells bearing mutations can be studied in live tissue under controlled conditions (Hudson, 2008).

Variation in mesoderm specification across drosophilids is compensated by different rates of myoblast fusion during body wall musculature development

It has been shown that species separated by relatively short evolutionary distances may have extreme variations in egg size and shape. Those variations are expected to modify the polarized morphogenetic gradients that pattern the dorso-ventral axis of embryos. Currently, little is known about the effects of scaling over the embryonic architecture of organisms. This problem was examined by asking if changes in embryo size in closely related species of Drosophila modify all three dorso-ventral germ layers or only particular layers, and whether or not tissue patterning would be affected at later stages. This paper reports that changes in scale affect predominantly the mesodermal layer at early stages, while the neuroectoderm remains constant across the species studied. Next, the fate of somatic myoblast precursor cells that derive from the mesoderm was examined to test whether the assembly of the larval body wall musculature would be affected by the variation in mesoderm specification. The results show that in all four species analyzed, the stereotyped organization of the body wall musculature is not disrupted and remains the same as in D. melanogaster. Instead, the excess or shortage of myoblast precursors is compensated by the formation of individual muscle fibers containing more or less fused myoblasts. These data suggest that changes in embryonic scaling often lead to expansions or retractions of the mesodermal domain across Drosophila species. At later stages, two compensatory cellular mechanisms assure the formation of a highly stereotyped larval somatic musculature: an invariable selection of 30 muscle founder cells per hemisegment, which seed the formation of a complete array of muscle fibers, and a variable rate in myoblast fusion that modifies the number of myoblasts that fuse to individual muscle fibers (Belu, 2011).

The data shows that within evolutionary distances as short as 5 mya of divergence time, there can be extreme variations in mesoderm specification. In most species analyzed, the width of mesodermal domain decreases or increases according to embryo size. However, this is not an absolute rule as D. simulans has a larger mesodermal domain than D. melanogaster, despite the fact that those two species vary less in their DV axis (Belu, 2011).

In contrast to the plasticity seen in the mesoderm specification, the width of the neuroectoderm remains constant across species. This latter result is in agreement with two important findings regarding the development of the ventral nerve cord. First, neuroblast maps are nearly identical in a broad range of insect species, sharing similarities even with the crustacean phylum. Second, experiments of genetic manipulation that altered the width of D/V expression domains within the neuroectoderm resulted in the duplication or elimination of neuroblasts of particular identity. Thus, the stable width in the neuroectoderm appears to be essential for the generation of correct neural lineages and axonal scaffolds within Drosophila species. However, the mechanisms that protect the neuroectoderm from scaling effects remain elusive (Belu, 2011).

Based on the stereotyped arrays of muscle fibers and innervation patterns observed in the different species, the data indicate that the mesodermal alterations can be compensated later in development. These corrections would involve an invariable selection of 30 FCs per hemisegment, and a variable rate of myoblast fusion that allows more cells to be incorporated to each muscle fiber. These two cellular mechanisms cooperate and prevent supernumerary or lack of muscle fibers (Belu, 2011).

What protects the development of the somatic body wall musculature from variations in the mesoderm size? This study highlights some key differences between the myogenesis and neurogenesis that may explain why the assembly of the somatic body wall has more alternate ways to cope with the early variations in mesodermal specification than does the ventral nerve cord (Belu, 2011).

The initial steps of both myogenesis and neurogenesis are similar and rely on the formation of groups of equivalent cells, the promuscular and proneural groups, from which a single progenitor cell is selected through lateral inhibition. In the case of the neural progenitor cell, or neuroblast, its identity is determined once it delaminates from the proneural group, when it initiates stereotyped divisions giving rise to a defined number and types of neurons/glial cells. In contrast, the progenitor of somatic muscles undergoes additional asymmetric cell divisions before it gives rise to FCs and adult muscle progenitor cells. Thus, modifications in the specification of muscle progenitor cells and/or their asymmetric cell divisions could generate an identical outcome of 30 embryonic FCs in the different Drosophila species (Belu, 2011).

Another difference between mesodermal and neural tissue specification is the fact that the entire neuroectodermal domain contributes to the formation of a stereotyped tissue, whereas the mesodermal domain is further subdivided and gives rise to non-stereotyped tissues as well, such as the fat body, hematopoietic system and visceral musculature. Therefore, species with reduced mesodermal domain might still be able to assemble the same numbers of promuscular groups at the expense of other mesodermal precursor cells that form non-stereotyped tissues (Belu, 2011).

Finally, the present study reveals that the myoblast fusion step, which is unique to myogenesis, is an important compensatory mechanism for the formation of the somatic body wall musculature (Belu, 2011).

The data shows that during myogenesis of D. busckii and D. pseudoobscura, fewer myoblasts are fused together to form slender muscle fibers in comparison to D. melanogaster. In contrast, more myoblasts fuse into single fibers in D. simulans and D. sechellia, resulting in fibers of increased size. The differential regulation of fusion events appears to be the only characteristic of FC identity that is unique to each species (Belu, 2011).

One of the main regulators of myoblast fusion is the adhesion molecule Kin of Irre/Dumbfounded (Kirre/Duf), which is expressed exclusively by FCs and functions as an attractant to FCMs. The expression of kirre/duf is down-regulated once the correct number of fused FCMs is achieved for a given muscle fiber. If this down-regulation of kirre/duf is modulated by the number of FCM that are aggregated, then there are two ways of increasing myoblast fusion. One would be if inhibitory signals released from fused FCMs are weaker in strength and the other would be if the sensitivity of kirre/duf to these signals is lower. In either case, more myoblasts would be added to the fiber. Recently, the cis-regulatory region of kirre/duf gene was identified in a group of Drosophila species, including D. pseudoobscura and D. simulans, and was found to have stretches of sequence divergence. These results support the view that modifications in the cis-regulatory sequence of kirre/duf could be responsible for different rates of myoblast fusion observed in these Drosophila species. However, further tests would be needed to determine whether constructs with kirre/duf from D. simulans and D. pseudoobscura inserted in D. melanogaster respond as expected by creating fibers with more or less myoblasts, respectively (Belu, 2011).

Variations in embryo size impose challenges to developing organisms, which must be overcome to ensure viability. In all species investigated in this study, some separated by several million years and others by only several thousand years, it was noted that alterations in mesodermal size were resolved by a common mechanism that increases or decreases the rate of myoblast fusion to generate the same stereotyped array of muscles. Since the variation in mesodermal domain and myoblast fusion rates occurred within very short evolutionary distances, these are fast evolving traits. Consistent with this view, there is evidence from the literature that genes belonging to the Toll and Dorsal/NFkappaB pathway, which participate in both immune response and D/V patterning, are fast evolving within twelve Drosophila species. This finding can be explained as adaptation to new pathogens found in the particular niches these species occupy. However, a recent comparison of the genomes of three melanogaster sister species identified components of the Dorsal/NFkappaB pathway that diverged the most in D. melanogaster, but the least in the pair D. simulans/D. sechellia, despite the fact that the latter two species do occupy completely different niches (i.e. one is cosmopolitan and the other is restricted to the plant Morinda, respectively). These data provide further evidence that D/V patterning itself, and not only immunity, evolves fast and point out to specific candidates in the Dorsal/NFkappaB pathway undergoing those changes (Belu, 2011).

Glycolysis supports embryonic muscle growth by promoting myoblast fusion

Muscles ensure locomotion behavior of invertebrate and vertebrate organisms. They are highly specialized and form using conserved developmental programs. To identify new players in muscle development, Drosophila and zebrafish gene expression databases were screened for orthologous genes expressed in embryonic muscles. More than 100 candidates were selected. Among them is the glycolysis gene Pglym78/pgam2, the attenuated expression of which results in the formation of thinner muscles in Drosophila embryos. This phenotype is also observed in fast muscle fibers of pgam2 zebrafish morphants, suggesting affected myoblast fusion. Indeed, a detailed analysis of developing muscles in Pglym78 RNAi embryos reveals loss of fusion-associated actin foci and an inefficient Notch decay in fusion competent myoblasts, both known to be required for fusion. In addition to Pglym78, the screen identifies six other genes involved in glycolysis or in pyruvate metabolism (Pfk, Tpi, Gapdh, Pgk, Pyk, and Impl3). They are synchronously activated in embryonic muscles and attenuation of their expression leads to similar muscle phenotypes, which are characterized by fibers with reduced size and the presence of unfused myoblasts. The data also show that the cell size triggering insulin pathway positively regulates glycolysis in developing muscles and that blocking the insulin or target of rapamycin pathways phenocopies the loss of function phenotypes of glycolytic genes, leading to myoblast fusion arrest and reduced muscle size. Collectively, these data suggest that setting metabolism to glycolysis-stimulated biomass production is part of a core myogenic program that operates in both invertebrate and vertebrate embryos and promotes formation of syncytial muscles (Tixier, 2013).

Founder cells regulate fiber number but not fiber formation during adult myogenesis in Drosophila

During insect myogenesis, myoblasts are organized into a pre-pattern by specialized organizer cells. In the Drosophila embryo, these cells have been termed founder cells and play important roles in specifying muscle identity and in serving as targets for myoblast fusion. A group of adult muscles, the dorsal longitudinal (flight) muscles, DLMs, is patterned by persistent larval scaffolds; the second set, the dorso-ventral muscles, DVMs is patterned by mono-nucleate founder cells (FCs) that are much larger than the surrounding myoblasts. Both types of organizer cells express Dumbfounded, which is known to regulate fusion during embryonic myogenesis. The role of DVM founder cells as well as the DLM scaffolds was tested in genetic ablation studies using the UAS/Gal4 system of targeted transgene expression. In both cases, removal of organizer cells prior to fusion, causes formation of supernumerary fibers, suggesting that cells in the myoblast pool have the capacity to initiate fiber formation, which is normally inhibited by the organizers. In addition to the large DVM FCs, some (smaller) cells in the myoblast pool also express Dumbfounded. It is proposed that these cells are responsible for seeding supernumerary fibers, when DVM FCs are eliminated prior to fusion. When these cells are also eliminated, myogenesis fails to occur. In the second set of studies, targeted expression of constitutively active RasV12 also resulted in the appearance of supernumerary fibers. In this case, the original DVM FCs are present, suggesting alterations in cell fate. Taken together, these data suggest that DVM myoblasts are able to respond to cues other than the original founder cell, to initiate fusion and fiber formation. Thus, the role of the large DVM founder cells is to generate the correct number of fibers, but they are not required for fiber formation itself. Evidence is also presented that the DVM FCs may arise from the leg imaginal disc (Atreya, 2008).

Founder cells (FCs) play an important role during myogenesis as they usually represent a pre-pattern prior to the onset of myoblast fusion. In the Drosophila embryo, each of the 30 muscle fibers in a hemisegment is seeded by a single founder cell. A group of adult flight muscles in Drosophila, the dorso-ventral muscles (DVMs) is patterned by founder cells which unlike the embryo are much larger than surrounding myoblasts. These cells have been referred to as imaginal pioneers and express the embryonic founder cell marker Dumbfounded, as detected by reporter activity. Duf is known to serve as an attractant for myoblasts and to thereby promote fusion. This study examined a role for the DVM FCs in organizing fiber formation. First the FCs were eliminated prior to the onset of fusion through targeted expression of the cell death gene reaper. Removal of these cells does not abolish fiber formation, but rather, excessive numbers of thinner fibers are formed. This outcome resembles what is observed when larval scaffolds for a related group of muscles, the dorsal longitudinal muscles (DLMs), are ablated. These scaffolds also express Duf, and the outcomes of their genetic ablation served as a useful comparison of muscle patterning in a set of functionally related fibers. In examining DVM myogenesis closely, it was found that in addition to the large Duf-expressing FCs, some (smaller) cells in the myoblast pool also express Duf, and it is proposed that these are the alternate/replacement founder cells that can seed fiber formation in absence of the large FCs. Interestingly, supernumerary fibers do not develop when reaper expression is maintained through the period of myogenesis, indicating that Duf expression is necessary in the alternate/replacement founder cells for fiber formation. In a second set of experiments, the Ras-signaling pathway was constitutively activated in using the rP298-Gal4 driver, and this manipulation also resulted in an increase in fiber number. The increase was not due to proliferation of pre-existing founder cells, and the supernumerary fibers develop in presence of the existing FCs. These outcomes for ablations of DVM organizers suggest that (1) DVM fibers can arise in the absence of the large elongate FCs; (2) a subset of cells in the myoblast pool have the potential to initiate fiber formation; (3) signals from the FC normally suppress this capability, so that the correct pattern of muscle fibers can be generated; and (4) interfering with the signaling results in the formation of supernumerary fibers. Thus, the most critical role of the DVM FCs is to regulate fiber number (Atreya, 2008).

An important consequence of eliminating the DVM FCs is that multiple fibers are formed, suggesting that cells from the myoblast pool are recruited to initiate fusion. Formation of supernumerary fibers is also observed when larval scaffolds that pattern the functionally opposing muscle group, the DLMs are ablated, or eliminated using reaper. Thus, although the two muscle groups develop using different modes of development, the larval scaffolds and the single celled FCs serve a similar function-specification of the number of adult fibers. These organizers serve as a pre-pattern, partitioning myoblasts through attractive cues, and subsequently when fusion begins, the final muscle pattern begins to emerge. The size of the myoblast pool remains unaffected by the manipulation (targeted reaper expression) and this is borne out by the fact that muscle volume in the adult thorax is not altered. These outcomes are similar to what is observed after DLM scaffolds are genetically ablated or laser ablated (Fernandes and Keshishian, 1996).

If a role for the large DVM founder cells is to initiate fusion, how does it occur in their absence? Two possibilities are suggested: that myoblasts randomly fuse with each other, or that 'replacement' founder cells begin to seed fiber formation. If the first scenario were true, many more than the 6-7 supernumerary fibers would be expected. Also, it would be a departure from the well understood separation of 'founder' and 'fusion-competent' fates (reviewed in Taylor, 2003) and the normal tendency of myoblasts to only fuse with a founder/scaffold. The current results do suggest the recruitment of 'alternate/replacement' founder cells. The rP298 promoter is active in a small subset of cells in the myoblast pool as early as 6 h APF, as detected by using the rP298-Gal4/UAS mCD8GFP. These cells are the same size as other cells in the myoblast pool and thus much smaller than the DVM FCs. It is proposed that these smaller Duf-expressing cells are recruited to initiate fusion when FCs are eliminated. They are capable of sustaining fusion, as suggested by multiple EWG-positive nuclei within a supernumerary fiber. It was also observed that fiber formation is disrupted upon prolonged exposure to reaper, which is most likely due to elimination of the smaller Duf-expressing cells as well. Thus, it appears that Duf expression is necessary in these cells to generate supernumerary fibers (Atreya, 2008).

It is useful to compare another manipulation which also eliminates Duf-lacZ expressing FCs, but which has different outcomes with respect to fiber formation. When the mesothorax is denervated at the third larval instar, the expression of Dumbfounded in DVM FCs is gradually abolished during the period of fusion, 12-24 h APF. Although the size of the myoblast pool is unaffected by the denervation, and Duf-expressing FCs are present prior to fusion (0-12 h APF), DVM fibers fail to form. In light of the reaper studies, it is reasonable to propose that during the 0-12 h period, DVM FCs engage in a 'lateral inhibition' process that is sufficient to prevent cells of the myoblast pool from seeding fibers during the subsequent fusion phase. Moreover, just as innervation is needed to maintain Duf expression in the FCs during the fusion phase, it may also be necessary to maintain expression in the smaller 'replacement/alternate' founder cells (Atreya, 2008).

In considering a role for the smaller Duf-positive cells during normal development, it is proposed that a subset of cells in the myoblast pool have the capability to initiate fiber formation, and that interactions with the founder cell prior to fusion are responsible for active suppression of this 'organizer' competency. When the FCs are eliminated, it is conceivable that the suppression is incomplete and they can go on to seed fibers. Thus, the smaller Duf-expressing cells serve as a reserve pool of founder cells to initiate fusion when necessary. The suppression could involve signaling mechanisms such as those that have been described for the embryonic mesoderm, wherein an equivalence group is first defined, from which a single founder cell is then selected, and the potential of the remaining cells is inhibited (Atreya, 2008).

Supernumerary fibers can arise despite the presence of pre-existing rP298-lacZ positive FCs Overexpression of RasV12 with the rP298-Gal4 driver prior to the onset of fusion also results in the development of supernumerary fibers. Two additional phenotypes are seen compared to what is seen under conditions of reaper expression (1) the large rP298-lacZ expressing FCs are still present and (2) many more rP298-lacZ expressing nuclei are seen in the myoblast pool than in controls. The formation of additional rP298-lacZ expressing cells is considered first. The BrDU studies demonstrate that they do not arise by proliferation. Thus the cells are being generated from the myoblast pool due to a change in cell fate. Two possibilities are suggested: one that activation of the Ras/Map kinase pathway in FCs signals cells in the myoblast pool to turn on rP298, and the second and more intriguing possibility considers the small subset of cells that express the rP298-reporter at low levels. There may be additional cells that are below the level of detection with the lacZ reporter; if the Ras/Map kinase pathway is activated in several of these cells, it would similarly result in activation of the rP298 promoter, giving rise to the increased number of rP298-positive cells. The levels of RasV12 activated in these unfused myoblasts may be just enough to alter cell fate, conferring founder cell like properties. In the Drosophila embryo, when RasV12 is targeted to the embryonic mesoderm an overproduction of progenitor cells is seen, which is brought about as a result of a change in cell fate of mesodermal cells. Although RasV12 is also being targeted to the large DVM FC, it does not alter cell fate there since that cell is already expressing high levels of rP298 and it continues to seed fiber formation, as in controls (Atreya, 2008).

Ras regulates intracellular signaling through the ERK/MAP kinase pathway, and RasV12 is known to increase levels of activated MAPK within cells. A recent report on founder cell specification during adult abdominal myogenesis in Drosophila has shown that restricted activation of the Ras/Map kinase pathway by the FGF receptor Htl, in a few myoblasts reinforces founder cell properties in them, leading to the upregulation of rP298-lacZ levels, and the eventual loss of reporter expression in the other myoblasts. While expression of Htl has not been reported in IFM myoblasts, a similar mechanism that uses the ERK/MAPK pathway might be at play. It is also considered that overexpression of RasV12 causes death of the founder cells. But this is clearly not the case, since TUNEL labeling does not show any cell death, and the original founder cells are present and seed fiber formation (Atreya, 2008).

Expressing RasV12 in DLM scaffolds has two prominent outcomes. First, there is an increase in the number of rows of nuclei within the developing fibers. The increase in number of nuclei is suggestive of rapid fusion, and an outcome of enhanced Ras signaling. Another interesting aspect is in regard to muscle splitting. The six DLM fibers are generated as a collective outcome of the longitudinal splitting of the three larval scaffolds and fusion of myoblasts with these scaffolds. It is thought that as myoblasts fuse with the scaffolds there is an increase in surface area, which is manifested as splitting. Under conditions of RasV12 expression, there is an increase in the number of nuclei within each DLM fiber, indicative of rapid fusion. However, in 45% of animals, splitting does not proceed normally. It follows therefore, that fusion occurs too rapidly, and becomes uncoupled from a sustained incorporation of new membrane, which is then manifested as a lag in splitting. Under conditions of nerve ablation, a lag in muscle splitting is observed, but in that case, a reduced pool of myoblasts and slower fusions are responsible (Atreya, 2008).

Which of the known organizer cells do they resemble — grasshopper pioneer, larval scaffolds, or embryonic FCs? The cells that organize the DVM myoblasts share features that are common to embryonic founder cells and to grasshopper pioneers. Like the embryonic FCs and grasshopper pioneer cells, they serve as fusion targets and prefigure a muscle fiber. They bear a closer resemblance to grasshopper pioneers with respect to their large size including cellular expanse. However, they are unique with respect to the manner in which they interact with surrounding myoblasts, a feature that has been revealed as a result of manipulations carried out in the present study. They are not necessary for fiber formation, per se, as in their absence, a reserve pool of 'alternate/replacement' FCs is recruited. When these cells are removed as well, fiber formation fails and this scenario is the most similar to the outcome of targeting reaper to embryonic founder cells (Atreya, 2008).

A distinction between adult and embryonic myogenesis is exemplified by the manner in which founder cells are selected during abdominal myogenesis. rP298-lacZ expressing founder cells in the adult abdomen are specified by signaling through the FGF receptor, Heartless, which is under positive as well as negative regulation through Sprouty and Heartbroken. This is different from founder cell specification in the embryo which uses Notch-mediated lateral inhibition. This difference between embryonic and adult abdominal muscles is particularly striking since the two muscle groups are more similar to each other with respect to the occurrence of segmentally repeated patterns, a feature that is not present in the adult thorax. The manner in which founder cells of the adult thoracic muscles are specified is not known, but it does not include a Notch-mediated lateral inhibition (Atreya, 2008).

In considering how a myoblast pool is patterned, these studies thus far have shown that an important property of organizer cells that regulate DVM patterning, is the regulation of fiber number. This property is related to the expression of Duf, which is known to serve as an attractant for myoblasts. Thus, the pre-pattern of large, elongate rP298-lacZ positive cells enables myoblast segregation in a manner that the fidelity of fiber number is ensured. There is a scattering of smaller rP298-lacZ expressing adult myoblasts in the pool, and these have the potential to seed fiber formation. The manipulations have shown that the DVM FCs suppress these smaller myoblasts from seeding fibers, and that interfering with this capacity can result in the formation of supernumerary fibers being generated. The interference can take the form of eliminating FCs or by activating the Ras/Map kinase pathway. Interestingly, cues from the epidermis are able to guide the nascent supernumerary fibers to attach appropriately. The organizer cells are contacted by neurons prior to the onset of fusion and this is the basis for another property that is distinct from embryonic founder cells -- the nerve dependence of rP298-lacZ expression. The innervating neurons continue to exert an influence on myogenesis during the stages of fusion and proliferation as well (Atreya, 2008).

Future work will be aimed at examining the mechanisms by which the correct number of FCs is established, the signaling mechanisms underlying founder cell-myoblast interactions, and how supernumerary founder cells are normally prevented from arising in the myoblast pool (Atreya, 2008).

The bHLH transcription factor Hand is required for proper wing heart formation in Drosophila

The Hand basic helix-loop-helix transcription factors play an important role in the specification and patterning of various tissues in vertebrates and invertebrates. This study has investigated the function of Hand in the development of the Drosophila wing hearts which consist of somatic muscle cells as well as a mesodermally derived epithelium. Hand was found to be essential in both tissues for proper organ formation. Loss of Hand leads to a reduced number of cells in the mature organ and loss of wing heart functionality. In wing heart muscles Hand is required for the correct positioning of attachment sites, the parallel alignment of muscle cells, and the proper orientation of myofibrils. At the protein level, α-Spectrin and Dystroglycan are misdistributed suggesting a defect in the costameric network. Hand is also required for proper differentiation of the wing heart epithelium. Additionally, the handC-GFP reporter line is not active in the mutant suggesting an autoregulatory role of Hand in wing hearts. Finally, in a candidate-based RNAi mediated knock-down approach Daughterless and Nautilus were identified as potential dimerization partners of Hand in wing hearts (Togel, 2013).

In hand null mutants, wing hearts are formed but exhibit severe morphological defects resulting in loss of wing heart function. Consequently, almost all individuals display opaque wings and are unable to fly. Moreover, over time many of the mutant flies accumulate hemolymph in their wings. This long term effect occurs also very frequently in flies that completely lack wing hearts. During wing inflation, hemolymph is forced into the wings by elevated hemolymph pressure in the thorax which is effectuated by rhythmic contractions of the abdomen. However, this does not result in uncontrolled hemolymph accumulation as observed in animals lacking wing heart function since the epidermal cells of the wings still interconnect the opposing wing surfaces at this stage. Only after their delamination, more hemolymph may accumulate resulting in balloon-like wings which explains the long term character of this phenotype. However, since a rather large amount of hemolymph may accumulate in the wings some mechanism must exist that prevents backflow into the body cavity. In the tubular connection between wing and wing heart, a back-flow valve exists that prohibits hemolymph flow from the body cavity into the wing and thus is unsuitable to maintain a large amount of hemolymph inside the wing. In the region of the hinge, no valves are present and hemolymph may freely enter or leave the wings. It is therefore assumed that the apoptotic epidermal cells that remain in the wings due to loss of wing heart function form clots in the inflow and outflow tracts and thereby block hemolymph passage. Animals exhibiting these long term effects are probably affected in various ways. Most obviously, flies with filled wings have difficulties moving around and tend to fall during climbing. However, there are probably also physiological effects since the amount of hemolymph trapped in the wings must be lacking in the body cavity and should therefore affect internal hemolymph pressure as well as tissue homeostasis. Thus, it is proposed that the long term effects on wing morphology may contribute to the observed shortened life span of adult hand mutants (Togel, 2013).

Based on handC-GFP reporter activity, wing hearts express hand throughout their entire development and probably also during their mature state. However, the requirement for Hand seems only critical during early pupal stages at the time when the wing hearts are formed. Similarly, hand mutants display a phenotype in the adult only with regard to the heart and the midgut indicating that Hand is likewise required only during metamorphosis in these organs. In the adult heart, loss of hand leads to disorganized myofibrils, a phenotype that was also observed in mature wing heart muscles. Additionally, it was found that the attachment sites are less regular leading to a disruption of the dorso-ventral order of the muscle cells and loss of their parallel alignment. In many cases, muscle cells even form ectopic attachment sites in an area where they never occur in the wild-type. In an attempt to characterize the phenotype at the protein level, it was found that α-Spectrin and Dystroglycan are not properly distributed. Both proteins constitute components of the costameric network and are enriched at the membrane overlying Z-discs in the wild-type. In hand mutants, however, their pattern is altered to a more or less homogenous distribution at the membrane. In knock-downs of α- or β-Spectrin in the postsynaptic neuromuscular junction (NMJ), it was shown that spectrins are required for normal growth of NMJs and normal distribution of Dlg at the junctions. The enlarged NMJs visible in the Dlg staining, support the observation that α-Spectrin is misdistributed in hand mutants. The similar localization of α-Spectrin and Dystroglycan at the membrane raises the question whether their misdistribution in the mutant is somehow interconnected or independent of each other. Spectrins are organized in tetramers, consisting of two α/β-Spectrin heterodimers, which bind actin and are connected to the plasma membrane via Ankyrins. Ankyrins, in turn, have binding sites for Dystroglycan and E-Cadherin and together Ankyrin and the actin/spectrin network are thought to stabilize cell-cell and cell-matrix attachments. A hint that the misdistribution of α-Spectrin and Dystroglycan may be interconnected comes from observations in Dystrophin deficient mdx mice. There, Dystroglycan and β-Spectrin are both irregularly distributed but always co-localize. This let the authors conclude that their organization is coordinated. A possible explanation for the general loss of costamere organization may be that the costameric γ-Actin, although expressed normally, does not form a stable link between Z-discs and the membrane in mxd mice. However, loss of Dystrophin results mostly in the disruption of the linear arrangement of the proteins at the Z- and M-lines and does not lead to their homogenous distribution as observed in hand mutants. Nevertheless, the data obtained in this study and the phenotypic analysis of loss of function studies strongly suggest that the Spectrin and Dystroglycan phenotypes in hand mutants are interconnected caused by a, yet unknown, defect in the costameric network. Moreover, the misdistribution of these two proteins suggests that other proteins might be affected in a similar way including receptors required for directed outgrowth of muscle cells and proper targeting of tendon cells. This would explain why many muscle cells attach at improper positions or are misaligned. However, the cytoskeleton is not affected as a whole since the muscle cells still attain an elongated shape with attachment sites at their ends and a wild-typic βPS-Integrin pattern (Togel, 2013).

In the epithelium, loss of hand results in the failure of cells to integrate into the developing epithelium leading to gaps and the loss of cells. In mature organs, cells are predominantly missing in the area that dorsally extends the muscle cells suggesting that epithelial cells have greater difficulties attaching to their own type than to the muscle cells. On the protein level, it was found that Arm does not localize to the periphery of the cells except for small dot-like areas in the remaining filopodia-like cellular interconnections. Arm (β-Catenin) constitutes an intracellular adapter protein that links the transmembrane receptor E-Cadherin to actin filaments in adherens junctions. Adherens junctions are predominantly found between cells of the same type whereas Integrin based hemiadherens junctions connect to the ECM and additionally form specialized junctions between different cell types (e.g. myotendinous junctions). Based on the correct distribution of βPS-Integrin and the absence of Arm at the cell borders in hand mutants, it could be that the epithelial cells are able to form hemiadherens junctions towards the muscle cells but fail to establish a sufficient number of adherens junctions towards other epithelial cells. However, an alternative explanation would be that the formation of hemiadherens junctions is not affected and the remaining cells are simply too far apart to establish proper cell-cell contacts. Further experiments are needed to clarify this point (Togel, 2013).

It has been suggested that Hand proteins are involved in the inhibition of apoptosis based on the observation that loss of Hand function leads to hypoplasia and that block of apoptosis in the mutant background, at least partially, rescues the hand phenotype. This study observed a similar effect with respect to wing heart cell number. However, live cell imaging showed that also in the wild-type muscle cells are removed by apoptosis suggesting that this is a normal process during regulation of muscle cell number. Consequently, block of apoptosis in the controls led to an increase in muscle cell number. This suggests further that wing hearts in general have the potential to form more functional muscle cells. In hand mutants, the same removal of muscle cells occurs indicating that hand does not in general block apoptosis in wing hearts. Moreover, since the inhibition of apoptosis by P35 also affects the apoptosis involved in regulation of muscle cell number it cannot be excluded that the observed effect is actually induced hyperplasia in the mutant background mimicking a rescue instead of a real rescue of the hand phenotype. Additionally, live cell imaging showed that the cells of the wing heart epithelium forming the dorsal extension arise at their correct position in a sufficient number so that no gaps are visible. Only after they fail to establish proper cell–cell contacts they are removed from the wing hearts. It is therefore proposed that loss of cells by apoptosis in hand mutants is only a secondary effect caused by the inability of cells to integrate into the forming wing hearts (Togel, 2013).

It has been shown that Hand proteins can function as transcriptional activators in vertebrates and invertebrates. However, no direct targets have been identified in Drosophila so far. This study reports that the handC-GFP reporter line shows almost no activity in wing heart progenitors during postembryonic development suggesting that hand itself is a direct target of Hand. Remarkably, in some individuals a few nuclei of the wing hearts still show reporter activity indicating that hand is not the only transcription factor involved in postembryonic activation of the reporter. Moreover, the fact that the hand null mutant is not always a null with respect to reporter activation makes it a variable phenotype. Similarly, the severity of the phenotype observed in individual wing hearts (e.g. left and right side of the same animal) may differ considerably. So, how can a null mutation cause variable phenotypes? The answer may lie in the fact that all bHLH transcription factors need to form homo- or heterodimers for DNA binding. It was therefore proposed that the absence of a bHLH transcription factor not only affects its direct downstream targets but also the entire bHLH factor stoichiometry within the cell suggesting that the pool of bHLH dimers might be dynamically balanced. In the absence of Hand, new and presumably also artificial bHLH dimers are formed which consequently can cause a variety of delicate differentiation defects. The scenario is becoming even more complicated by the observation that the dimerization property of Hand is modulated by its phosphorylation state as well as by the finding that Hand can inhibit the dimerization of other transcription factors by blocking their protein interaction sites (Togel, 2013).

A crucial prerequisite for understanding the bHLH network in wing hearts is therefore the identification of dimerization partners. In a candidate based RNAi approach, two bHLH proteins, Da and Nau, were identified which evoke a phenotype very similar to the hand mutant. In order to verify the indication that these factors are interacting with Hand, Y2H analysis was applied and and an interaction between both Hand and Da as well as Hand and Nau was confirmed at the protein level. Furthermore, in vertebrates it was shown that these proteins are also able to form heterodimers with each other and that Hand is able to compete for heterodimer formation and DNA binding. Thus, based on Y2H interaction as well as phenotype similarity, the potential bHLH network in wing hearts likely includes Hand/Da and Hand/Nau heterodimers which activate different sets of downstream genes. In hand null mutants, the balance may be shifted to Da/Nau heterodimers or even Da/Da or Nau/Nau homodimers which may be able to activate some of Hand's target genes but with lower or higher efficiency. The competition of all these dimers with different transcriptional activation efficiency for the hand targets might explain the variations observed in the hand mutants (Togel, 2013).

The Dmca1D channel mediates Ca inward currents in Drosophila embryonic muscles
The channel mechanisms underlying membrane excitation in the Drosophila embryonic body wall muscle, whose biophysical properties have been poorly characterized. The inward current underlying the action potential was solely mediated by a high-threshold class of voltage-gated Ca2+ channels, which exhibited slow inactivation, Ca2+-permeability with saturation at high [Ca2+]OUT, and sensitivity to a Ca2+ channel blocker, Cd2+. The Ca2+ current in the embryonic muscle was completely eliminated in Dmca1D mutants, indicating that the Dmca1D-encoded Ca2+-channel is the major mediator of inward currents in the body wall muscles throughout the embryonic and larval stages.

Noncanonical roles for Tropomyosin during myogenesis
For skeletal muscle to produce movement, individual myofibers must form stable contacts with tendon cells and then assemble sarcomeres. The myofiber precursor is the nascent myotube, and during myogenesis the myotube completes guided elongation to reach its target tendons. Unlike the well-studied events of myogenesis, such as myoblast specification and myoblast fusion, the molecules that regulate myotube elongation are largely unknown. In Drosophila, hoi polloi (hoip) encodes a highly-conserved RNA binding protein and hoip mutant embryos are largely paralytic due to defects in myotube elongation and sarcomeric protein expression. The hoip mutant background was used as a platform to identify novel regulators of myogenesis, and surprising developmental functions were uncovered for the sarcomeric protein Tropomyosin 2 (Tm2). Hoip responsive sequences were identified in the coding region of the Tm2 mRNA that are essential for Tm2 protein expression in developing myotubes. Tm2 overexpression rescued the hoip myogenic phenotype by promoting F-actin assembly at the myotube leading edge, by restoring the expression of additional sarcomeric RNAs, and by promoting myoblast fusion. Embryos that lack Tm2 also showed reduced sarcomeric protein expression, and embryos that expressed a gain-of-function Tm2 allele showed both fusion and elongation defects. Tropomyosin therefore dictates fundamental steps of myogenesis prior to regulating contraction in the sarcomere (Williams, 2015).

Surface apposition and multiple cell contacts promote myoblast fusion in Drosophila flight muscles

Fusion of individual myoblasts to form multinucleated myofibers constitutes a widely conserved program for growth of the somatic musculature. This study used electron microscopy methods to study this key form of cell-cell fusion during development of the indirect flight muscles (IFMs) of Drosophila melanogaster. It was found that IFM myoblast-myotube fusion proceeds in a stepwise fashion and is governed by apparent cross talk between transmembrane and cytoskeletal elements. Cell adhesion was found to be necessary for bringing myoblasts to within a minimal distance from the myotubes. The branched actin polymerization machinery acts subsequently to promote tight apposition between the surfaces of the two cell types and formation of multiple sites of cell-cell contact, giving rise to nascent fusion pores whose expansion establishes full cytoplasmic continuity. Given the conserved features of IFM myogenesis, this sequence of cell interactions and membrane events and the mechanistic significance of cell adhesion elements and the actin-based cytoskeleton are likely to represent general principles of the myoblast fusion process (Dhanyasi, 2015).

A model of muscle atrophy based on live microscopy of muscle remodelling in Drosophila metamorphosis

Genes controlling muscle size and survival play important roles in muscle wasting diseases. In Drosophila melanogaster metamorphosis, larval abdominal muscles undergo two developmental fates. While a doomed population is eliminated by cell death, another persistent group is remodelled and survives into adulthood. To identify and characterize genes involved in the development of remodelled muscles, a workflow was developed consisting of in vivo imaging, targeted gene perturbation and quantitative image analysis. Inhibition of TOR signalling and activation of autophagy promote developmental muscle atrophy in early, while TOR and yorkie activation are required for muscle growth in late pupation. Changes were discovered in the localization of myonuclei during remodelling that involve anti-polar migration leading to central clustering followed by polar migration resulting in localization along the midline. The Cathepsin L orthologue Cp1 was demonstrated to be required for myonuclear clustering in mid, while autophagy contributes to central positioning of nuclei in late metamorphosis. In conclusion, studying muscle remodelling in metamorphosis can provide new insights into the cell biology of muscle wasting (Kuleesha, 2016).

Transcriptome analysis of IFM-specific actin and myosin nulls in Drosophila melanogaster unravels lesion-specific expression blueprints across muscle mutations

Muscle contraction is a highly fine-tuned process that requires the precise and timely construction of large protein sub-assemblies to form sarcomeres. Mutations in many genes encoding constituent proteins of this macromolecular machine result in defective functioning of the muscle tissue. However, the pathways underlying muscle degeneration, and manifestation of myopathy phenotypes are not well understood. This study explored transcriptional alterations that ensue from the absence of the two major muscle proteins - myosin and actin - using the Drosophila indirect flight muscles. The aim of this study was to understand how the muscle tissue responds as a whole to the absence of either of the major scaffold proteins, whether the responses are generic to the tissue; or unique to the thick versus thin filament systems. The results indicated that muscles respond by altering gene transcriptional levels in multiple systems active in muscle remodelling, protein degradation and heat shock responses. However, there were some responses that were filament-specific signatures of muscle degeneration, like immune responses, metabolic alterations and alterations in expression of muscle structural genes and mitochondrial ribosomal genes. These general and filament-specific changes in gene expression may be of relevance to human myopathies (Madan, 2017).

Distortion of the Actin A-triad results in contractile disinhibition and cardiomyopathy
Striated muscle contraction is regulated by the movement of tropomyosin over the thin filament surface, which blocks or exposes myosin binding sites on actin. Findings suggest that electrostatic contacts, particularly those between K326, K328, and R147 on actin and tropomyosin, establish an energetically favorable F-actin-tropomyosin configuration, with tropomyosin positioned in a location that impedes actomyosin associations and promotes relaxation. This study provides data that directly support a vital role for these actin residues, termed the A-triad, in tropomyosin positioning in intact functioning muscle. By examining the effects of an A295S alpha-cardiac actin hypertrophic cardiomyopathy-causing mutation, over a range of increasingly complex in silico, in vitro, and in vivo Drosophila muscle models, it is proposed that subtle A-triad-tropomyosin perturbation can destabilize thin filament regulation, which leads to hypercontractility and triggers disease. These efforts increase understanding of basic thin filament biology and help unravel the mechanistic basis of a complex cardiac disorder. ators, the type I and type II receptors. Endocytosis orchestrates the assembly of signaling complexes by coordinating the entry of receptors with their downstream signaling mediators. Recently, it was shown that the C. elegans type I bone morphogenetic protein (BMP) receptor SMA-6 (see Drosophila Thickveins), part of the TGFbeta family, is recycled through the retromer complex while the type II receptor, DAF-4 (see Drosophila Punt) is recycled in a retromer-independent, ARF-6 dependent manner. From genetic screens in C. elegans aimed at identifying new modifiers of BMP signaling, this study reports on SMA-10, a conserved LRIG (leucine-rich and immunoglobulin-like domains) transmembrane protein. It is a positive regulator of BMP signaling that binds to the SMA-6 receptor. This study shows that the loss of sma-10 leads to aberrant endocytic trafficking of SMA-6, resulting in its accumulation in distinct intracellular endosomes including the early endosome, multivesicular bodies (MVB), and the late endosome with a reduction in signaling strength. These studies show that trafficking defects caused by the loss of sma-10 are not universal, but affect only a limited set of receptors. Likewise, in Drosophila, it was found that the fly homolog of sma-10, lambik (lbk), reduces signaling strength of the BMP pathway, consistent with its function in C. elegans and suggesting evolutionary conservation of function. Loss of sma-10 results in reduced ubiquitination of the type I receptor SMA-6, suggesting a possible mechanism for its regulation of BMP signaling (Viswanathan, 2017).

Live imaging and analysis of muscle contractions in Drosophila embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. Previously work has shown that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. This study presents a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching; this method was used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. Body posture was also examined and advantage was taken of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching (Chandel, 2019).

Drosophila Nedd4-long reduces Amphiphysin levels in muscles and leads to impaired T-tubule formation

Drosophila Nedd4 (dNedd4) is a HECT ubiquitin ligase with two main splice isoforms: dNedd4 short (dNedd4S) and long (dNedd4Lo). DNedd4Lo has a unique N-terminus containing a Pro-rich region. While dNedd4S promotes neuromuscular synaptogenesis, dNedd4Lo inhibits it and impairs larval locomotion. To delineate the cause of the impaired locomotion, binding partners to the N-terminal unique region of dNedd4Lo were sought in larval lysates. Mass-spectrometry identified Amphiphysin (dAmph). dAmph is a postsynaptic protein containing SH3-BAR domains, which regulates muscle transverse tubule (T-tubule) formation in flies. The interaction was validated by coimmunoprecipitation, and direct binding between dAmph-SH3 domain and dNedd4Lo-N-terminus was demonstrated. Accordingly, dNedd4Lo was colocalized with dAmph postsynaptically and at muscle T-tubules. Moreover, expression of dNedd4Lo in muscle during embryonic development led to disappearance of dAmph and to impaired T-tubule formation, phenocopying amph null mutants. This effect was not seen in muscles expressing dNedd4S or a catalytically-inactive dNedd4Lo(C->A). It is proposed that dNedd4Lo destabilizes dAmph in muscles, leading to impaired T-tubule formation and muscle function (Safi, 2016).

The Drosophila melanogaster larval body wall muscles are established during embryogenesis beginning with the invagination of the mesoderm, which spreads along the ectoderm and then forms numerous mesodermal derivatives. Somatic mesodermal specification produces three different types of myoblasts. Fusion of muscle founder cells and fusion-competent myoblasts form the syncytial myotube, which develops into the embryonic and larval body wall muscles. After myoblast fusion, nuclei are positioned correctly throughout the myotube and form connections to surrounding tendon cells to establish the myotendinous junction, which is innervated by motorneurons in a process called neuromuscular (NM) synaptogenesis. The contractile apparatus is then assembled, and muscles begin to contract. During larval stages, the essential muscle pattern created in the embryo does not change, except that the muscles continue to expand along with the growth of the larva. In Drosophila larva, a repeated pattern of 30 unique muscle fibers is present in each abdominal hemisegment, which are innervated by 36 motor neurons. Each muscle fiber is distinguishable by size, shape, orientation, number of nuclei, innervation, and tendon attachment sites. Throughout development, internal and external cues guide muscles to adopt specific properties that allow them to perform particular functions (Safi, 2016).

Ubiquitination is the process of conjugating ubiquitin onto proteins, and it plays an important role in controlling protein degradation/stability, as well as in trafficking, sorting, and endocytosis of transmembrane proteins. The ubiquitination cascade involves three enzymes: E1, E2, and E3, with the last responsible for substrate recognition and ubiquitin transfer, either indirectly (e.g., RING E3 ligases) or directly (Safi, 2016).

Although many proteins are involved in the regulation of NM synaptogenesis in flies, the role of the ubiquitin system in this process is less well characterized. Studies have shown that the RING-family ubiquitin ligase complex Highwire inhibits synapse formation and function by inhibiting the kinase Wallenda/DLK1, the activator of JNK , whereas the deubiquitinating enzyme Fat Facet targets Liquid Facet/Epsin and promotes synaptic growth (Safi, 2016 and references therein).

Neuronal precursor cell expressed developmentally down-regulated 4 (Nedd4) family members belong to the HECT family of E3 ligases and contain a common C2-WW(n)-HECT domain architecture. Drosophila contains a single dNedd4 gene, which undergoes alternative splicing to produce several splice isoforms, including two prominent ones: dNedd4-short (dNedd4S) and dNedd4-long (dNedd4Lo). Differences between the two isoforms of dNedd4 include an alternate start codon site, resulting in a longer N-terminal region in dNedd4Lo, and an extra exon inserted between those encoding WW1 and WW2 domains (Zhong, 2011; Safi, 2016 and references therein).

Previous studies have shown that dNedd4S promotes NM synaptogenesis in flies by interacting and ubiquitinating Commissureless (Comm), which leads to endocytosis of Comm from the muscle surface, a step required for NM synaptogenesis. Whereas dNedd4S is essential for proper NM synaptogenesis, dNedd4Lo inhibits it (Zhong, 2011). Of importance, dNedd4Lo also inhibited normal larval locomotion (Zhong, 2011). These adverse effects of dNedd4Lo were caused by unique N-terminal and Middle regions found in dNedd4Lo (and absent from dNedd4S) and required a functional HECT domain (Zhong, 2011). Of interest, during embryonic muscle development, dNedd4Lo expression is dramatically decreased, whereas that of dNedd4S remains relatively high (Zhong, 2011; Safi, 2016 and references therein).

Because it was observed that the muscle and synaptogenesis defects of dNedd4Lo larvae were not caused by altered phosphorylation of dNedd4Lo, dNedd4Lo-mediated inhibition of catalytic activity of dNedd4S, or diminished effects of dNedd4Lo on Comm endocytosis (Zhong, 2011), it was suspected that dNedd4Lo might inhibit muscle development and/or function by interacting with other proteins via its unique regions (Safi, 2016).

This study identified, using mass spectrometry, Drosophila Amphiphysin (dAmph) as a binding partner of the unique N-terminal region of dNedd4Lo. Amphiphysins are members of the BAR-SH3 domain-containing family of proteins. Mammalian amphiphysin Amph I is involved in endocytosis and synaptic vesicle recycling during neurotransmission by interacting with clathrin/dynamin. In contrast, both mammalian Amph IIb (Bin1) and its fly orthologue, dAmph, are postsynaptic, lack binding sites for clathrin/dynamin, and are not involved in endocytosis. Instead, Bin1 and dAmph regulate transverse tubule (T-tubule) biogenesis in muscles (Safi, 2016).

This study demonstrates that the N-terminus of dNedd4Lo directly binds to dAmph-SH3 domain. In accord, dNedd4Lo and dAmph are colocalized postsynaptically at neuromuscular junctions (NMJs) and muscle transverse tubules (T-tubules). The data show that dNedd4Lo regulates the levels of dAmph postsynaptically and in muscles. Moreover, expression of dNedd4Lo in muscles results in impaired T-tubule formation, phenocopying amph-null mutants. These results demonstrate an important role of dNedd4Lo in regulating T-tubule organization of Drosophila muscles (Safi, 2016).

Previous work has shown that unlike dNedd4 (dNedd4S), muscle-specific overexpression of the dNedd4Lo isoform inhibits NM synaptogenesis and leads to impaired larval locomotion and lethality (Zhong, 2011). This effect required the catalytic activity of dNedd4Lo, since a mutant dNedd4Lo(C->A) with an inactivating mutation in the HECT domain (Cys -> Ala) was not inhibitory. This suggested that the same gene (dNedd4) can encode isoforms with opposite functions. In accord with this, it was observed that during stages of embryonic development when synaptogenesis takes place (14-24 h), dNedd4S expression remains relatively high, whereas that of the inhibitory dNedd4Lo is strongly reduced (Zhong, 2011), suggesting tight regulation of expression of these isoforms to promote muscle development at a very precise time. The inhibitory effect of dNedd4Lo is mediated by two regions unique to dNedd4Lo (N-terminus and Middle region), as deletion of these unique regions alleviated the synaptogenesis defects and increased viability. Therefore it is postulated that the unique regions of dNedd4Lo might negatively regulate NM synaptogenesis and muscle development by targeting specific substrate(s) (Safi, 2016).

This study identified dAmph as a binding partner for the unique N-terminal region of dNedd4Lo by mass spectrometry and validated the interaction in vitro and in vivo in flies. Transiently expressed dAmph coimmunoprecipitates with dNedd4Lo but not with dNedd4S in Drosophila S2 cells and demonstrated direct binding between dAmph-SH3 domain and the dNedd4Lo N-terminus. In vivo results showed that dAmph colocalizes with dNedd4Lo postsynaptically at neuromuscular junctions and muscle T-tubules, where their expression overlaps with the postsynaptic/T-tubule marker, Dlg. Of importance, it was demonstrated that dNedd4Lo expression significantly reduced the levels of dAmph in the postsynaptic region and muscles, an effect not observed in larvae expressing dNedd4S or dNedd4Lo(C->A). As expected, due to the disappearance of endogenous dAmph in larvae expressing dNedd4Lo (and the inability to 'treat' live larvae with proteasome inhibitors), it was not possible to detect ubiquitination of dAmph in these larvae. In addition to biochemical interactions, genetic interactions were also shown between dNedd4Lo and dAmph (Safi, 2016).

The reduction in dAmph levels in the dNedd4Lo-expressing muscles correlated with impaired T-tubule formation, mimicking the phenotype of the amph-null flies. These results could help explain (along with previously described NM synaptogenesis defects; Zhong, 2011) the observed locomotion defects in the dNedd4Lo-overexpressing larvae. At present, it is not possible to quantify the contribution of the T-tubule defects versus the NM synaptogenesis defects to the impaired muscle locomotion/function (Safi, 2016).

Interestingly, it was found that flies overexpressing the dAmph(ΔSH3) mutant in muscles showed reduced localization at the postsynaptic region and T-tubules and no longer colocalized with dNedd4. It is known that the SH3 domain of some membrane-associated proteins is important for their targeting to specific subcellular locations. Similarly, it was found that the SH3 domain of dAmph is also important for its location in the postsynaptic region and the muscle, since the ΔSH3 dAmph protein mislocalized to a region near the muscle plasma membrane. It is not known whether this SH3-dependent localization is related to the ability of this domain to bind dNedd4Lo or due to its interaction(s) with other molecules (Safi, 2016).

Amphiphysin has been implicated in T-tubule biogenesis due to its N-terminal amphipathic helix and BAR domain (N-BAR), which promotes membrane curvature. The BAR domain of the isoform of mammalian amphiphysin 2 (Bin1) is known to be associated with T-tubule formation in skeletal and cardiac muscles, where it induces tubular plasma membrane invaginations. Similar to Bin1, dAmph was shown to participate in plasma membrane remodeling during cleavage furrow ingression, which is required for de novo formation of cells in the Drosophila embryo; the BAR domain of dAmph is required for the formation of endocytic tubules that form at the cleavage furrow tips. This study shows that dNedd4Lo expression reduces the levels of dAmph in the muscle and significantly inhibits T-tubule formation. The degradation of dAmph by dNedd4Lo could impair T-tubule biogenesis by the BAR domain of dAmph, which could help to explain the larval locomotion defects that were observed (Safi, 2016).

Cardiac Bin1 has been implicated in calcium channel trafficking and formation of the inner membrane folds of the cardiac T-tubules. Bin1 localizes to cardiac T-tubules with the L-type calcium channel, Cav1.2, by tethering dynamic microtubules to membrane scaffolds, allowing targeted delivery of Cav1.2 to cardiac T-tubules. Knockdown of Bin1 reduces surface Cav1.2 and delays development of the calcium transient. In cardiomyopathy, decrease in Bin1 alters T-tubule morphology and can cause arrhythmia. Mice with cardiac Bin1 deletion show decreased T-tubule folding, which leads to free diffusion of local extracellular ions, prolonging action-potential duration and increasing susceptibility to arrhythmias. Bin1 is also important for maintenance of intact T-tubule structure and Ca2+ homeostasis in adult skeletal muscle. Adult mouse skeletal muscles with Bin1 knockdown display swollen T-tubule structures, alterations to intracellular Ca2+ release, and compromised coupling between the voltage-gated calcium channel, dihydropyridine receptor (DHPR), and the intracellular calcium channel, ryanodine receptor 1 (Safi, 2016).

Similar to Bin1, dAmph is also required for the organization of the excitation-contraction coupling machinery of muscle. Accordingly, dAmph mutant larvae and flies show defects in T-tubule formation, severe locomotor defects, and flight impairments, indicative of defects in muscle function. Therefore degradation of dAmph in the muscle and inhibition of T-tubule biogenesis by dNedd4Lo could have adverse effects on the localization of T-tubule-associated calcium channels and coupling between the DHPR and ryanodine receptor, as a result altering calcium signaling in muscles. Efficient intracellular Ca2+ homeostasis in skeletal muscle requires intact triad junctional complexes comprising T-tubule invaginations of plasma membrane and terminal cisternae of sarcoplasmic reticulum. Because dNedd4Lo expression significantly reduced T-tubule projections, this would likely impair intracellular Ca2+ homeostasis and result in locomotor defects (Safi, 2016).

Although there is no direct homologue of dNedd4Lo in species other than Drosophila, the mammalian Nedd4 relative Itch has a proline-rich N-terminal region that binds the SH3 domain of Sorting Nexin 9 . In yeast, Rsp5 (the yeast orthologue of Nedd4 proteins) regulates the Amphiphysin homologue Rvs167 by monoubiquitination of lysine in the SH3 domain of Amphiphysin, demonstrating that Nedd4 family members can interact with SH3 domains, including that of amphiphysin, in other species in addition to flies (Safi, 2016).

In addition to dAmph, this study identified several other interacting partners of the N-terminal and Middle regions of dNedd4Lo that could potentially be targeted by dNedd4Lo. Similar to dAmph, two of these proteins, Syndapin (which, like dAmph, bound the unique N-terminus region of dNedd4Lo), and Sorting Nexin 9 (SH3PX1, which bound the unique Middle region of dNedd4Lo), contain BAR and SH3 domains. The SH3 domain-containing protein Cindr/CG31012 (orthologue of the mammalian Cd2ap and Cin85) was also identified as a binding partner to the unique N-terminus of dNedd4Lo. It is not known whether these proteins are bone fide substrates of dNedd4Lo or contribute to the NMJ and T-tubule defects caused by overexpression of dNedd4Lo in the muscle during development (Safi, 2016).

In conclusion, the severely reduced locomotion activity of larvae overexpressing dNedd4Lo in the muscle may be explained by both impaired neuromuscular synaptogenesis, which has been demonstrated previously (Zhong, 2011), and by impaired T-tubule formation as a result of dAmph degradation by dNedd4Lo, which is shown in this study. The defective T-tubule branching would likely impair coupling between the DHPR and ryanodine receptor, possibly by affecting the localization of calcium channels to the T-tubule network. Reduced surface calcium channels on the T-tubule network would alter calcium homeostasis and compromise excitation and contraction coupling, causing larval locomotor defects (Safi, 2016).

Identification and functional characterization of muscle satellite cells in Drosophila

Work on genetic model systems such as Drosophila and mouse has shown that the fundamental mechanisms of myogenesis are remarkably similar in vertebrates and invertebrates. Strikingly, however, satellite cells, the adult muscle stem cells that are essential for the regeneration of damaged muscles in vertebrates, have not been reported in invertebrates. This study shows that lineal descendants of muscle stem cells are present in adult muscle of Drosophila as small, unfused cells observed at the surface and in close proximity to the mature muscle fibers. Normally quiescent, following muscle fiber injury, these cells express Zfh1 and engage in Notch-Delta-dependent proliferative activity and generate lineal descendant populations, which fuse with the injured muscle fiber. In view of strikingly similar morphological and functional features, these novel cells are considered to be the Drosophila equivalent of vertebrate muscle satellite cells (Chaturvedi, 2017).

Tet protein function during Drosophila development

The TET (Ten-eleven translocation) 1, 2 and 3 proteins have been shown to function as DNA hydroxymethylases in vertebrates and their requirements have been documented extensively. Recently, the Tet proteins have been shown to also hydroxylate 5-methylcytosine in RNA. 5-hydroxymethylcytosine (5hmrC) is enriched in messenger RNA but the function of this modification has yet to be elucidated. Because Cytosine methylation in DNA is barely detectable in Drosophila, it serves as an ideal model to study the biological function of 5hmrC. This study characterized the temporal and spatial expression and requirement of Tet throughout Drosophila development. Tet was shown to be essential for viability as Tet complete loss-of-function animals die at the late pupal stage. Tet is highly expressed in neuronal tissues and at more moderate levels in somatic muscle precursors in embryos and larvae. Depletion of Tet in muscle precursors at early embryonic stages leads to defects in larval locomotion and late pupal lethality. Although Tet knock-down in neuronal tissue does not cause lethality, it is essential for neuronal function during development through its affects upon locomotion in larvae and the circadian rhythm of adult flies. Further, the function of Tet in ovarian morphogenesis is reported. Together, these findings provide basic insights into the biological function of Tet in Drosophila, and may illuminate observed neuronal and muscle phenotypes observed in vertebrates (Wang, 2018).

The scaffolding protein Cnk binds to the receptor tyrosine kinase Alk to promote visceral founder cell specification in Drosophila

In Drosophila melanogaster, the receptor tyrosine kinase (RTK) Anaplastic lymphoma kinase (Alk) and its ligand Jelly belly (Jeb) are required to specify muscle founder cells in the visceral mesoderm. This study identified a critical role for the scaffolding protein Cnk (Connector enhancer of kinase suppressor of Ras) in this signaling pathway. Embryos that ectopically expressed the minimal Alk interaction region in the carboxyl terminus of Cnk or lacked maternal and zygotic cnk did not generate visceral founder cells or a functional gut musculature, phenotypes that resemble those of jeb and Alk mutants. Deletion of the entire Alk-interacting region in the cnk locus affected the Alk signaling pathway in the visceral mesoderm and not other RTK signaling pathways in other tissues. In addition, the Cnk-interacting protein Aveugle (Ave) was shown to be critical for Alk signaling in the developing visceral mesoderm. Alk signaling stimulates the MAPK/ERK pathway, but the scaffolding protein Ksr, which facilitates activation of this pathway, was not required to promote visceral founder cell specification. Thus, Cnk and Ave represent critical molecules downstream of Alk, and their loss genocopies the lack of visceral founder cell specification of Alk and jeb mutants, indicating their essential roles in Alk signaling (Wolfstetter, 2017).

Receptor tyrosine kinase (RTK) signaling plays an essential role in development by transducing external signals into the nucleus and other cellular compartments, thereby altering gene expression and promoting intracellular responses. The hallmarks of RTK signaling are conserved among eukaryotic organisms and involve ligand-dependent activation of a transmembrane receptor protein tyrosine kinase and the recruitment of canonical intracellular signaling modules and cascades, such as the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. Alk activation stimulates this pathway through the guanosine triphosphatase Ras and the serine-threonine kinases Raf [a MAPK kinase kinase (MAPKKK)], MEK (a MAPK kinase), and MAPK/ERK. Other factors that contribute to or modulate the activity of this pathway have been identified, such as the kinase suppressor of Ras (Ksr), which was identified by mutagenesis screens in Ras-sensitized genetic backgrounds in Drosophila melanogaster and Caenorhabditis elegans. Because of inconsistent findings regarding the catalytic activity of its kinase domain, the role of Ksr has remained controversial. Different models have proposed distinct roles for Ksr as an activator of Raf in parallel to or downstream of Ras or as a scaffolding protein for the assembly of Raf-MEK protein complexes. There is no evidence for a direct interaction between Ksr and Ras, but dimerization between Ksr and Raf can stimulate Raf activity in a manner that is independent of the kinase activity of Ksr, suggesting that Ksr may act as a scaffold in the context of RTK signaling (Wolfstetter, 2017).

A genetic modifier screen using ectopic expression of a dominant-negative, chimeric version of Ksr in the fly eye led to the identification of another critical factor for Ras-ERK signaling named Connector enhancer of kinase suppressor of Ras (Cnk). The cnk locus encodes a large protein of 1557 amino acids containing an N-terminal sterile α motif (SAM), followed by a conserved region in Cnk (CRIC), a PDZ domain, proline-rich motifs, and a pleckstrin homology (PH) domain. The protein structure suggests that Cnk acts as a multidomain protein scaffold. Like Ksr, Cnk functions downstream of various RTK signaling events including epidermal growth factor receptor (EGFR)-tribution of Cnk to embryonic Torso signaling, supporting the finding - and EGFR-dependent air sac development in the dorsal thorax. Ectopic expression of the Cnk N-terminal region enhances the effects of activated RasV12 independently of MAPK/ERK activation in the Drosophila eye. The C-terminal region contains a Raf inhibitory region (RIR) that binds to and represses Raf, which is released upon phosphorylation of Cnk by the Src family kinase Src42A. Thus, Cnk functions as a molecular scaffold to support Ksr-mediated Raf activation and to recruit and integrate additional signaling components such as Src42A (Wolfstetter, 2017).

During embryonic development in D. melanogaster, the visceral mesoderm (VM) gives rise to a lattice of midgut muscles that ensheaths the larval midgut. The VM consists of naïve myoblasts that become specified as either founder cells (FCs) or fusion competent myoblasts (FCMs). Subsequently, the FCs fuse one-to-one with FCMs and eventually form the binucleate visceral myotubes. Specification of VM cells requires the Drosophila ortholog of the receptor anaplastic lymphoma kinase (ALK), initially identified as part of a chimeric protein created by the 2;5 (p23:q35) translocation in human anaplastic large cell lymphoma cell lines. Drosophila Alk is expressed in the segmental clusters of the embryo that segregate from the dorsal trunk mesoderm to form the VM. Alk protein can be detected at the membrane of all VM cells, but only the distal arch within each cluster comes into direct contact with a secreted, small low-density lipoprotein domain ligand named Jelly belly (Jeb). Binding of Jeb to the extracellular part of Alk activates a downstream signaling cascade that results in ERK phosphorylation and triggers expression of an FC-specific subset of genes including Hand, optomotor-blind-related-gene-1 (org-1), and kin of irre (kirre; also referred to as dumbfounded or duf). Jeb-Alk signaling is crucial for visceral myoblasts to commit to the FC fate. In the absence of either ligand or receptor, neither ERK phosphorylation nor the expression of FC-specific marker genes in the VM occurs. Moreover, visceral cells fail to undergo myoblast fusion, and the VM subsequently disintegrates in jeb and Alk mutant embryos (Wolfstetter, 2017).

This study has identified the multidomain scaffolding protein Cnk as a potential Alk binding partner and essential component in the Alk signaling pathway. Cnk bound to the intracellular part of Alk by its C-terminal region. Loss of cnk function or expression of dominant-negative cnk constructs in Drosophila interfered with Alk signaling in multiple developmental contexts. Moreover, germline clone-derived embryos lacking maternal and zygotic Cnk failed to specify visceral FCs and did not develop a functional midgut. In agreement with its proposed function, epistasis experiments revealed that Cnk operates between Ras and Raf in the Alk signaling pathway. Further targeted deletion of a minimal Alk interaction region (AIR) in Cnk resulted in a specific decrease of Jeb-Alk-induced ERK phosphorylation within the visceral FC row. Deletion of the larger Alk interacting region blocked specification of visceral FCs in response to Alk activation. Although the SAM domain containing Cnk binding partner Aveugle (Ave) was essential for Alk signaling, it was found that Ksr is not essentially required to drive Alk signaling in the developing VM. Thus, Cnk and its binding partner Ave serve as critical components for Alk signaling in Drosophila (Wolfstetter, 2017).

This study uncovered an essential function for the protein scaffold Cnk in Alk signaling. The identification of multiple Cnk preys as AlkICD interactors in the Y2H analysis revealed a region in Cnk that likely mediates this interaction and allowed definition of a minimal AIR that was sufficient to bind Alk. The importance of Cnk in Alk signaling was supported by the loss of FCs in the VM of germline clone-derived cnk mutants [cnk (m-/z-)], which genocopied the embryonic Alk loss-of-function phenotype and the dominant-negative effect of ectopic CnkAIR expression on visceral FC specification. The tissue-specific decrease of ERK phosphorylation in the visceral FC row of cnkΔAIR mutants and the loss of visceral FCs upon deletion of the entire Alk interacting region identified by the Y2H approach further support a direct interaction between Cnk and Alk (Wolfstetter, 2017).

Various interactions between Cnk and membrane-associated factors (although not other RTKs) have been reported. In cultured mammalian cells, CNK1 promotes insulin signaling by binding to and localizing cytohesins at the plasma membrane, and binding of CNK1 to the transmembrane ligand EphrinB1 links fibronectin-mediated cell adhesion to EphrinB-associated JNK signaling. Moreover, mammalian CNK2 (also called MAGUIN), the mammalian CNK homolog most similar to Drosophila Cnk, binds to various members of the membrane-associated MAGUK family proteins and Densin-180. It will be interesting to determine whether the RTK binding capacities of Cnk are limited to Alk (Wolfstetter, 2017).

Cnk localizes close to the plasma membrane. Although the Alk-Cnk interaction appears to be important for ERK activation and visceral FC specification in the VM, Alk does not appear to be required for the subcellular localization of Cnk. Whether the Alk-Cnk interaction depends on Alk activity, potentially resulting in posttranslational modification of Cnk, will be interesting to pursue in further studies. Notably, Drosophila Cnk is tyrosine-phosphorylated upon coexpression with the activated form of the RTK Sevenless (SEVS11) in S2 cells, and activation of the platelet-derived growth factor receptor induces tyrosine phosphorylation of mammalian CNK1, leading to changes in CNK1 subcellular localization (Wolfstetter, 2017).

Cnk is generally abundant in the Drosophila embryo where its function is required in various RTK signaling pathways. However, the CnkAIR appears to be critical only for Alk signaling. Morphological analysis further reveals a minor contribution of Cnk to embryonic Torso signaling, supporting the finding that Torso signals are processed by three or more parallel branches. This finding also agrees with earlier analyses of embryonic Torso signaling in avem-/z- mutants, which form terminally derived structures. Notably, the differences in tracheal phenotypes caused by the cnk63F null allele and the strong, Raf-repressing cnksag alleles, which have C-terminal nonsense mutations and abrogate Alk signaling in the VM, indicate that distinct Cnk domains are involved in specific RTK-signaling pathways. This notion is also supported by the observation that the strong, Heartless-related phenotypes exhibited by cnkm-/z- null mutants were barely apparent in cnkΔY2H m-/z- embryos, which, on the other hand, displayed a complete loss of Alk-driven FC specification in the VM (Wolfstetter, 2017).

Selectivity for a requirement of CNK by different RTKs has also been observed in mammalian cells because CNK2 appears to be required for nerve growth factor, but not EGF-induced ERK activation in PC12 cells. Therefore, Cnk contributes to multiple signaling events, but its importance to different RTKs varies, perhaps reflecting differential wiring of downstream signaling in different developmental processes, an aspect that will be interesting to explore in future studies. Cnk has been described as a protein scaffold that facilitates Ras-Raf-MAPK signaling at the plasma membrane, allowing signal integration to enhance Raf and MAPK activation. The epistatic analysis presented in this study shows that Cnk is required downstream of activated Alk and RasV12 but upstream of activated Raf in the VM, which agrees with previous studies in Drosophila. Thus, activated Ras seems to require Cnk to transmit signaling to Raf in the VM (Wolfstetter, 2017).

Alk activation at the membrane of a prospective visceral FC by the ligand Jeb, which is secreted from the neighboring somatic mesoderm, induces the Raf/MAPK/ERK signaling cascade, eventually leading to the transcriptional activation of downstream targets including kirre, org-1, and Hand. Cnk and Ave are core components of the Alk signaling pathway that are required downstream of the receptor and upstream of Raf to mediate visceral FC specification. Although not essential for Jeb-Alk signaling, Ksr appears to be required for full activation of ERK (Wolfstetter, 2017).

Ave directly binds to Cnk through an interface formed by their SAM domains. This interaction is thought to be necessary to recruit Ksr to a complex that in turn promotes Raf activation in the presence of activated Ras. Although this study identified Ave as a critical component for Cnk function downstream of Alk, the single Ksr in Drosophila was not required for Alk-mediated FC specification. Cnk was originally identified in a Ksr-dependent genetic screen in Drosophila, and its function has been proposed to mediate the association between Ksr and Raf, suggesting that Ksr should also play an important role in Alk signaling. However, the role of Ksr is unclear, with early reports suggesting an inhibitory function rather than an activating potential in RTK signaling). Ksr requires the presence of additional factors such as 14-3-3 proteins or activated Ras , and loss of Ksr-1 suppresses RasE13-induced but not wild-type signaling during C. elegans vulva formation, suggesting altered affinities of Ksr for different variants of Ras. Because this analyses revealed a function for ksr in driving robust ERK phosphorylation, it is plausible that, although nonessential, Ksr might enhance Alk signaling by integrating signals from activated Ras to Cnk-associated Raf (Wolfstetter, 2017).

The importance of ERK activation in RTK-mediated signaling in the Drosophila embryo is difficult to address directly because the rolled locus (encoding the only MAPK/ERK1-2 ortholog in Drosophila) is located close to the centromere and therefore not accessible for standard germline clone analysis. The removal of the 42-amino acid AIR from Cnk by genomic editing of the cnk locus suggests that ERK phosphorylation, even at decreased amounts, is sufficient to promote Alk-induced specification of visceral FCs in vivo. Whereas the AIR is sufficient to mediate the Alk-Cnk interaction in vitro, the ability of the cnkΔAIR mutant to support Alk-driven FC specification in the developing embryo highlights additional requirements. The Y2H analysis supports a direct interaction between Alk and Cnk mediated by a more extensive binding interface within the Alk-Y2H binding region of Cnk. Accordingly, deletion of this region in the cnk locus (cnkΔY2H) blocks VM specification in vivo. However, the possibility cannot be excluded that the Y2H region of Cnk may form interactions with additional Alk binding proteins, which could mediate the Alk-Cnk interaction in an indirect manner (Wolfstetter, 2017).

In summary, this study has identified an interaction between Alk and Cnk mediated by an Alk binding region in Cnk. This region was specifically required for the activation of ERK and formation of FCs downstream of the activated Alk receptor in the Drosophila VM. Together with Ave, Cnk represents an important signaling module that is required for Alk-mediated signaling during embryogenesis. Cnk and Ave represent molecules identified downstream of Alk, whose loss genocopies the lack of visceral FC specification of Alk and jeb mutants. Further work should allow a better understanding of the importance of Cnk in Alk signaling and whether this is conserved in mammalian systems (Wolfstetter, 2017).

The cis-regulatory dynamics of embryonic development at single-cell resolution

This study investigated the dynamics of chromatin regulatory landscapes during embryogenesis at single-cell resolution. Using single-cell combinatorial indexing assay for transposase accessible chromatin with sequencing (sci-ATAC-seq), chromatin accessibility was profiled in over 20,000 single nuclei from fixed Drosophila embryos spanning three embryonic stages: stage 5 blastoderm nuclei; 6-8 h after egg laying, to capture a midpoint in embryonic development when major lineages in the mesoderm and ectoderm are specified; and 10-12 h after egg laying, when cells are undergoing terminal differentiation. The results show that there is spatial heterogeneity in the accessibility of the regulatory genome before gastrulation, a feature that aligns with future cell fate, and that nuclei can be temporally ordered along developmental trajectories. During mid-embryogenesis, tissue granularity emerges such that individual cell types can be inferred by their chromatin accessibility while maintaining a signature of their germ layer of origin. Analysis of the data reveals overlapping usage of regulatory elements between cells of the endoderm and non-myogenic mesoderm, suggesting a common developmental program that is reminiscent of the mesendoderm lineage in other species. 30,075 distal regulatory elements were identified that exhibit tissue-specific accessibility. The germ-layer specificity of a subset of these predicted enhancers was validated in transgenic embryos, achieving an accuracy of 90%. Overall, these results demonstrate the power of shotgun single-cell profiling of embryos to resolve dynamic changes in the chromatin landscape during development, and to uncover the cis-regulatory programs of metazoan germ layers and cell types (Cusanovich, 2018).

Drosophila Hsp67Bc hot-spot variants alter muscle structure and function

The Drosophila Hsp67Bc gene encodes a protein belonging to the small heat-shock protein (sHSP) family, identified as the nearest functional ortholog of human HSPB8. The most prominent activity of sHSPs is preventing the irreversible aggregation of various non-native polypeptides. Moreover, they are involved in processes such as development, aging, maintenance of the cytoskeletal architecture and autophagy. In larval muscles Hsp67Bc localizes to the Z- and A-bands, which suggests its role as part of the conserved chaperone complex required for Z-disk maintenance. In addition, Hsp67Bc is present at neuromuscular junctions (NMJs), which implies its involvement in the maintenance of NMJ structure. This study reports the effects of muscle-target overexpression of Drosophila Hsp67Bc hot-spot variants Hsp67BcR126E and Hsp67BcR126N mimicking pathogenic variants of human HSPB8. Depending on the substitutions, a different impact was observed on muscle structure and performance. Expression of Hsp67BcR126E affects larval motility, which may be caused by impairment of mitochondrial respiratory function and/or by NMJ abnormalities manifested by a decrease in the number of synaptic boutons. In contrast, Hsp67BcR126N appears to be an aggregate-prone variant, as reflected in excessive accumulation of mutant proteins and the formation of large aggregates with a lesser impact on muscle structure and performance compared to the Hsp67BcR126E variant (Jablonska, 2018).

Nanoscopy reveals the layered organization of the sarcomeric H-zone and I-band complexes

Sarcomeres are extremely highly ordered macromolecular assemblies where structural organization is intimately linked to their functionality as contractile units. Although the structural basis of actin and Myosin interaction is revealed at a quasiatomic resolution, much less is known about the molecular organization of the I-band and H-zone. This study reports the development of a powerful nanoscopic approach, combined with a structure-averaging algorithm, that allowed determination of the position of 27 sarcomeric proteins in Drosophila melanogaster flight muscles with a quasimolecular, approximately 5- to 10-nm localization precision. With this protein localization atlas and template-based protein structure modeling, refined I-band and H-zone models were assembled with unparalleled scope and resolution. In addition, it was found that actin regulatory proteins of the H-zone are organized into two distinct layers, suggesting that the major place of thin filament assembly is an M-line-centered narrow domain where short actin oligomers can form and subsequently anneal to the pointed end (Szikora, 2020)

Johnston, J. S., Zapalac, M. E. and Hjelmen, C. E. (2020). Flying high-muscle-specific underreplication in Drosophila. Genes (Basel) 11(3). PubMed ID: 32111003

Flying high-muscle-specific underreplication in Drosophila

Drosophila underreplicate the DNA of thoracic nuclei, stalling during S phase at a point that is proportional to the total genome size in each species. In polytene tissues, such as the Drosophila salivary glands, all of the nuclei initiate multiple rounds of DNA synthesis and underreplicate. Yet, only half of the nuclei isolated from the thorax stall; the other half do not initiate S phase. To address this problem, flow cytometry was used to compare underreplication phenotypes between thoracic tissues. When individual thoracic tissues are dissected and the proportion of stalled DNA synthesis is scored in each tissue type, it was found that underreplication occurs in the indirect flight muscle, with the majority of underreplicated nuclei in the dorsal longitudinal muscles (DLM). Half of the DNA in the DLM nuclei stall at S phase between the unreplicated G0 and fully replicated G1. The dorsal ventral flight muscle provides the other source of underreplication, and yet, there, the replication stall point is earlier (less DNA replicated), and the endocycle is initiated. The differences in underreplication and ploidy in the indirect flight muscles provide a new tool to study heterochromatin, underreplication and endocycle control (Johnston, 2020).

The glutamic acid-rich long C-terminal extension of troponin T has a critical role in insect muscle functions

The troponin complex regulates the Ca2+-activation of myofilaments during striated muscle contraction and relaxation. Troponin genes emerged 500-700 million years ago during early animal evolution. Troponin T (TnT) is the thin filament-anchoring subunit of troponin. Vertebrate and invertebrate TnTs have conserved core structures, reflecting conserved functions in regulating muscle contraction, and also contain significantly diverged structures, reflecting muscle type- and species-specific adaptations. TnT in insects contains a highly diverged structure consisting of a long glutamic acid-rich C-terminal extension of ~70 residues with unknown function. C-terminally truncated Drosophila TnT (TpnT-CD70) retains binding of tropomyosin, troponin I, and troponin C, indicating a preserved core structure of TnT. However, the mutant TpnTCD70 gene residing on the X chromosome resulted in lethality in male flies. This X-linked mutation produces dominant-negative phenotypes, including decreased flying and climbing abilities, in heterozygous female flies. Immunoblot quantification with a TpnT-specific monoclonal antibody indicated expression of TpnT-CD70 in vivo and normal stoichiometry of total TnT in myofilaments of heterozygous female flies. Light and electron microscopy examinations revealed primarily normal sarcomere structures in female heterozygous animals, whereas Z-band streaming could be observed in the jump muscle of these flies. Although TpnT-CD70-expressing flies exhibited lower resistance to cardiac stress, their hearts were significantly more tolerant to Ca2+ overloading induced by high-frequency electrical pacing. These findings suggest that the Glu-rich long C-terminal extension of insect TnT functions as a myofilament Ca2+buffer/reservoir, potentially critical to the high-frequency asynchronous contraction of flight muscles (Cao, 2020).

Transcriptional silencers in Drosophila serve a dual role as transcriptional enhancers in alternate cellular contexts

A major challenge in biology is to understand how complex gene expression patterns are encoded in the genome. While transcriptional enhancers have been studied extensively, few transcriptional silencers have been identified, and they remain poorly understood. This study used a novel strategy to screen hundreds of sequences for tissue-specific silencer activity in whole Drosophila embryos. Almost all of the transcriptional silencers that were identified were also active enhancers in other cellular contexts. These elements are bound by more transcription factors than non-silencers. A subset of these silencers forms long-range contacts with promoters. Deletion of a silencer caused derepression of its target gene. These results challenge the common practice of treating enhancers and silencers as separate classes of regulatory elements and suggest the possibility that thousands or more bifunctional CRMs remain to be discovered in Drosophila and in humans (Gisselbrecht, 2019).

This study has adapted an enhancer-fluorescence-activated cell sorting-sequencing (FACS-seq) technology for highly parallel screening of elements for enhancer activity in Drosophila embryos (Gisselbrecht, 2013) into silencer-FACS-seq (sFS) technology, which enriches for elements that tissue specifically silence reporter gene expression. Briefly, this study generated a reporter vector, pSFSdist, which drives GFP expression under the control of an element from a library of candidate silencers, positioned at least 100 bp upstream of a strong, ubiquitous enhancer (ChIPCRM2078; Gisselbrecht, 2013). This vector contains a target sequence for a site-specific recombinase, permitting assaying all of the tested elements in the same genomically integrated context. Flies carrying single insertions from the reporter library are crossed to a strain in which the expression of the exogenous marker protein rat CD2 is driven in a tissue or cell type of interest, and the resulting informative embryos are dissociated to produce a single-cell suspension. By sorting for CD2+ cells in which GFP expression is reduced from the level driven by the strong ubiquitous enhancer in the absence of silencing activity, cells were enriched for that contained silencers active in the cell type of interest, which were then recover and identify by high-throughput sequencing. Insertion of an element with known mesodermal silencing activity into this vector consistently yielded a larger fraction of CD2+GFPreduced cells) than were observed when a negative control sequence (derived from Escherichia coli genomic DNA) was used (Gisselbrecht, 2019).

A library of 591 genomic elements, chosen to represent a variety of chromatin states or enhancer activity patterns, was designed to test for silencer activity in the embryonic mesoderm at stages 11-12 (5.5-7.5 h after embryo deposition). Since general features of silencers are unknown, this library was designed to test 3 main hypotheses about what kinds of sequences act as silencers in this developmental context (Gisselbrecht, 2019).

First, it was noted that 2 bifunctional CRMs had been identified previously in Drosophila that function as enhancers in one context and as silencers in other contexts. As this phenomenon is known to occur in multiple eukaryotic systems from a small number of examples and could be important in understanding the architecture of regulatory DNA, it was desirable to assess the generality of this phenomenon. Therefore, CRMs were selected from the REDfly and CAD2 databases that exhibited no or highly restricted mesodermal expression at embryonic stage 11. Elements associated with genes that show widespread mesodermal expression at this stage were filtered out. Second, a potential mechanistic signature of transcriptional silencers is the binding of well-characterized transcriptional corepressors, by analogy to the prediction of enhancers by binding of the coactivator CBP. Therefore genomic elements identified by ChIP as binding sites for the co-repressors Groucho or CtBP were selected. As Groucho has canonically been associated with long-range repression and CtBP has been associated with short-range repression, it was predicted that Groucho binding sites would be a richer source of silencer activity in this assay, in which candidate silencers were placed >100 bp upstream of the enhancer driving reporter gene activity (Gisselbrecht, 2019).

Third, genomic regions were selected that were associated with the markers of both enhancers and repressed chromatin structure in whole-mesoderm or whole-embryo experiments. It was reasoned that silencers are active regulatory elements, distinct from the silenced chromatin that results from their activity, yet must recruit factors that exert repressive functions, and therefore may show association with both classes of chromatin marks. Moreover, these 'bivalent' chromatin states may represent sequences of the above-mentioned type, that act as enhancers in one cell type and as silencers in another. In this class, two sets of sequences were included: (1) DNase I hypersensitive sites (DHSs) that colocalize with ChIP signal for the well-studied repressive chromatin mark histone H3 trimethyllysine 27 (H3K27me3) in sorted mesoderm, and (2) coincident mesodermal peaks for H3K27me3, the canonical enhancer mark histone H3 monomethyllysine 4 (H3K4me1), and histone H3 acetyllysine 27 (H3K27ac), which has been associated with active enhancers and promoters. All of the sequences identified from genome-wide ChIP methods were filtered for the absence of widespread mesodermal expression of associated nearby genes (Gisselbrecht, 2019).

Finally, 15 sequences were included for which enhancer chromosomal contact sequencing (4C-seq) data for sorted mesodermal cells were available. Three positive control sequences were included that were previously shown to have mesodermal silencing activity and two types of putative negative controls were included: broadly active mesodermal enhancers and length-matched regions of the E. coli genomic sequence (Gisselbrecht, 2019).

The library of genomic elements was screened for silencer activity in the embryonic mesoderm in 2 rounds. Testing of this library yielded a readily detectable population of mesodermal cells in which GFP expression was reduced; the elements enriched in these cells are referred to as sFS+ elements. Of the 591 sequence elements chosen for inclusion in this library, 501 were genomically integrated into transgenic flies after injection of the pooled library (Gisselbrecht, 2019).

Overlap with transcription start sites (TSSs) were identified as a highly enriched feature of sFS+ elements, which likely reveals the presence of promoter competition. Competition among promoters for association with active enhancers is one mechanism that has been proposed to account for the specificity with which enhancers target genes for activation and has been shown to restrict enhancer-driven activation of gene expression in reporter assays. Overall, the initial set of 41 'hits' that overlapped promoter regions was significantly enriched for mapped instances of the TATA box. While these are technical positives in the silencer screen, since the goal of this study was to analyze CRMs that silence gene expression by other means, any sequences that overlapped promoter regions were omitted from subsequent analyses. Moreover, many of the library elements tested overlapped other library elements; these were mergend for downstream analysis. After filtering to remove elements that overlapped promoters and collapsing overlapping genomic regions, 29 of a total of 352 genomic regions tested for mesodermal silencer activity were positive in the sFS screen (Gisselbrecht, 2019).

To validate the results from the sFS screen, pure transgenic lines were generated from a subset of library elements, and then their silencer activity was assayed by FACS analysis of embryos resulting from these individual reporter strains. Next, whether the silencers detected by the sFS screen could also silence the activity of enhancers other than the strong, ubiquitous enhancer used in the sFS screen was investigated. Thus, silencing by several sFS+ elements was assessed visually by placing these elements upstream of the following mesoderm-specific enhancers and imaging the resulting GFP expression. ChIPCRM2613 is an intronic enhancer of the pan-mesodermal gene heartless, and it drives reporter gene expression throughout the presumptive mesoderm from the beginning of gastrulation (Gisselbrecht, 2013). The Mef2 I-ED5 enhancer drives expression in the fusion-competent myoblasts beginning in late stage 11. In every case examined (15 of 15), at least one of these additional enhancers showed reduced activity in the mesoderm in the context of the tested element. These results not only verify the silencing activity of these sFS+ elements but they also demonstrate that silencers are not specific for a particular enhancer (Gisselbrecht, 2019).

The resulting set of mesodermal silencers was analyzed to determine which genomic features that were explicitly sampled in the design of the element library were predictive of silencer activity. Despite the inclusion in the sFS library of ChIP peaks for transcriptional co-repressors and for a repressive chromatin mark, the only screened element types that were significantly enriched among the active mesodermal silencers were positive controls and non-mesodermal enhancers. In fact, 22 of 29 regions containing mesodermal silencers had been previously reported to have enhancer activity. Testing of the remaining 7 silencers for enhancer activity revealed that 6 of 7 also function as enhancers in the embryo: 5 of 6 were entirely non-mesodermal, while 1 showed restricted mesodermal expression. In total, 28 of 29 of the elements were found to act as mesodermal silencers also exhibited enhancer activity in a different cellular context. Overall, >10% (26 of 200) of the previously known enhancers tested in the assay exhibited mesodermal silencer activity. This constitutes more bifunctional transcriptional regulatory elements than were previously known across all biological systems (Gisselbrecht, 2019).

A different class of bifunctional CRMs was reported recently (Erceg, 2017), in which developmental enhancers have an additional function as Polycomb response elements (PREs). PREs provide genomic binding sites for sequence-specific DNA binding proteins that recruit protein subunits of Polycomb repressive complexes and could, in principle, play a role in silencing target genes. Therefore, this study tested the hypothesis that the silencer activity of enhancers discovered in the current assay resulted from PRE activity. Only 4 of 29 regions displaying mesodermal silencer activity overlapped PREs as defined by Erceg (2017) on the basis of ChIP for the PRE-binding factors Pho and dSfmbt, versus 24 of 323 mesodermal non-silencers, indicating that PREs are not a major source of mesodermal silencers (Gisselbrecht, 2019).

The results suggest a view of enhancers as CRMs with distinct spatiotemporal patterns of both activation and repression. To further assess the generality of this phenomenon, the effects of a subset of the newly discovered mesodermal silencers was visualized on enhancers that are active broadly in the mesoderm at different developmental stages. This enabled simultaneous evaluation of a variety of spatiotemporal domains of silencer and enhancer activity (Gisselbrecht, 2019).

Several elements exhibited apparently uniform silencing activity across the mesoderm and at different stages. The most commonly observed temporal pattern was a lack of silencing activity at gastrulation and strong silencing during the later stages at which sFS was performed. An element, hkb_0.6kbRIRV, was also observed that silenced much more strongly earlier than later in embryonic development; this element simultaneously acted as an enhancer in its previously characterized pattern in the midgut primordia. One element, the oc otd186 enhancer, which was observed to drive expression in the head, exhibited spatially patterned silencing within the mesoderm during gastrulation but not later in development; silencing was moderate in the anterior portion of the germband, but much stronger in the posterior portion, as seen in the context of 2 different early pan-mesodermal enhancers. Two different later-acting mesodermal enhancers showed moderate, uniform silencing across the anteroposterior extent of the germband. Finally, a tested element exhibited enhancer-specific silencing activity. The lz crystal cell enhancer was a moderately weak silencer when tested on different late-acting mesodermal enhancers and a strong silencer at gastrulation in the context of ChIPCRM2613, yet it completely failed to silence activity driven during gastrulation by ChIPCRM7759. These results highlight that silencers exhibit a similarly diverse range of spatiotemporal regulatory patterns as those of enhancers (Gisselbrecht, 2019).

To investigate whether the silencer activity observed in reporter assays reflects the activity of the putative silencer in its native chromosomal context, the average mesodermal transcription was profiled in the genomic neighborhood of silencers or other functional elements. Using published RNA-seq data from sorted mesodermal cells, reads were aggregated within 500-bp windows over a 25-kb region centered on each element, representing the typical size of chromatin state domains observed in a high-resolution Drosophila Hi-C experiment. Then the results were averaged over all of the elements in a class to create a meta-profile of transcript levels surrounding each class of cis-regulatory elements. As expected, transcription near a previously published set of mesodermal enhancers (Gisselbrecht, 2013) is elevated. In contrast, transcription near silencers is below the baseline level of transcription observed near a negative control set of genomic regions. Both effects decay to background levels in the meta-profiles over a scale of ~5 kb, suggesting that silencers act within approximately the same distance range as transcriptional enhancers. The bifunctional PREs reported by Erceg (2017) are also associated with strongly elevated transcription, but this effect appears to spread more broadly on the chromatin domain scale, suggesting that bifunctional PREs may act by a mechanism that is distinct from that of the silencers identified in the sFS screen (Gisselbrecht, 2019).

Next, to further demonstrate the functional importance of the silencers that were identified in the sFS screen, CRISPR-Cas9 genome editing was used to generate a Drosophila strain containing a deletion of the hkb_0.6RIRV element. This element was originally reported as an enhancer driving the expression of the gap gene huckebein (hkb) at the termini of the blastoderm embryo, and this study identified it as a mesodermal silencer in the screen. Mesodermal cells from embryos homozygous for this deletion and from wild-type control embryos were screened and hkb RNA was found to be significantly upregulated in the homozygous mutant mesoderm, which supports a role for this element in silencing its endogenous target gene during normal embryonic development (Gisselbrecht, 2019).

Various types of epigenomic features, including chromatin accessibility, post-translational modifications of histones, and occupancy by TFs and chromatin-modifying enzymes, have been associated with different categories of functional elements in the genome, such as active promoters and enhancers. However, relatively little is known about the chromatin features of active silencers. Therefore the epigenomic environment at the set of 29 mesodermal silencers was explored by assessing the enrichment or depletion of signal from various published epigenomic datasets, as compared to elements that did not display silencer activity in the sFS screen (Gisselbrecht, 2019).

It was hypothesized that since bifunctional elements are more functionally complex than CRMs that act only as enhancers, they may exhibit a more complex suite of TF interactions across various tissues. This study observed that validated silencers are strongly enriched for overlap with highly occupied target (HOT) regions as exceeding a TF complexity score threshold of ~10 overlapping bound factors. Since silencing activity is likely mediated through the effects of bound sequence-specific transcriptional repressors, the set of 29 mesodermal silencers was searched for enriched combinations of evolutionarily conserved DNA binding site motif occurrences for TFs annotated as repressors. The only motif combination that was significantly enriched among silencers was a 3-way combination of the motifs for the TFs Snail, Dorsal, and Tramtrack-PF, which were found together in 12 of 29 mesodermal silencers (Gisselbrecht, 2019).

Snail is a well-known repressor of non-mesodermal genes in the developing mesoderm. Dorsal and Tramtrack have also been shown to play roles in mesodermal gene repression. Analysis of ChIP data for Snail revealed significant enrichment for Snail occupancy at silencers. To validate that this enrichment reflects Snail activity at silencers, predicted Snail binding sites were mutated in 4 silencer elements with high levels of Snail ChIP signal, and the silencer activity of the mutant was compared to wild-type sequences within whole embryos in the FACS-based reporter assay. All 4 elements showed significantly reduced silencer activity. Mutating sites for an unrelated TF, as a negative control, caused no significant reduction in silencer activity. While Snail has been well characterized as a short-range repressor acting within 150 bp, all of the Snail binding sites that were found to be required for full silencer activity are >400 bp away from the silenced enhancer in the reporter construct, indicating that Snail can act as a repressor at distances longer than those described for short-range repression (Gisselbrecht, 2019).

Finally, the evidence was examined for the direct action of Snail binding to silencers on the expression of the silencers' endogenous target genes. Since the majority of elements exhibiting silencer activity in this study were originally reported as enhancers, it was possible to identify published target genes of these bifunctional elements and examine the effect of loss of snail function on their expression. Target genes of 12 elements bound by Snail in ChIP-seq data showed significant derepression in sna mutant embryos. In contrast, no target of any of the 14 Snail-unbound elements was significantly derepressed. Therefore, it is concluded that the known role of Snail in mesodermal repression explains the activity of a large minority (41%) of the observed silencers, while the transcriptional regulators mediating silencing activity through the majority of the silencers remain to be determined (Gisselbrecht, 2019).

In an attempt to identify a 'silencer signature' that is analogous to the previously described chromatin signatures of enhancers and promoters, published ChIP data was assembled from whole embryos or from sorted mesoderm, where available, for several chromatin marks previously associated with active or repressed chromatin states, and hierarchical clustering was performed of all 352 tested genomic regions according to these histone modification chromatin profiles. As expected, clusters of elements with greater signal for the repressive marks H3K27me3 and H3K9me3 are enriched for silencers, while other clusters are depleted of silencers. It was also observed, however, that many non-silencers belonged to these clusters, and that some silencers belonged to other clusters that were instead enriched for non-silencers, suggesting that these commonly profiled chromatin marks do not constitute a general chromatin signature of silencers. Similarly, neither the Groucho nor the CtBP co-repressors were significantly enriched at silencers (Gisselbrecht, 2019).

For a subset of silencers (18 of 29), the individual FACS validation data provided a measure of the strength of silencer activity, in terms of the percentage of cells in the GFPreduced population. Using these quantitative estimates of silencer activity, it was found that H3K27me3 and H3K9ac, a mark that previously had been associated with bivalent promoters and active enhancers, are significantly correlated with silencer strength across these 18 elements, possibly reflecting the fact that mesodermal silencers are active enhancers in other cellular contexts. No single mark or combination of marks that was tested from among the publicly available histone modification profiles accurately discriminates active silencers as a whole from other types of cis-elements (Gisselbrecht, 2019).

It has been well established that enhancers can act directly on their target promoters by looping to create direct, 3-dimensional (3D) physical contacts between genomic elements widely separated in sequence space. Such contacts have also been shown to play a role in repression by heterochromatin and at PREs. Silencers could, in principle, act directly to recruit repressive activities to regulated promoters, or alternatively by sequestering enhancers that would otherwise interact with promoters, or by other mechanisms that do not involve focal contacts to regulated elements, such as nucleating a repressive chromatin state that spreads along the chromosome (Gisselbrecht, 2019).

Therefore data was examined from assays of genomic contacts based on proximity ligation to attempt to distinguish among these hypotheses. Mesodermal silencer activity was observed in a CRM previously characterized by circular chromosome conformation capture (4C). This element makes mesodermally enriched contacts with 2 regions that overlap the promoters of genes that are not expressed in the mesoderm, suggesting the possibility that silencing may be mediated by direct silencer-promoter looping (Gisselbrecht, 2019).

To test the generality of this potential mechanism, Hi-C data was generated from sorted mesodermal and non-mesodermal cells at embryonic stages 11-12, the same developmental stages that was assayed by sFS. Mesodermally enriched contacts were observed at 1-kb resolution in each of 2 paired replicates using the chromoR package and compared the results to the mesodermally enriched 4C contacts previously reported. The frequency of random contacts observed in Hi-C assays is greater with closer genomic distance. Therefore, to control for such nonspecific interactions in this analysis, negative control sets of 'contact regions' were generated by reflecting each observed contact around the viewpoint region. Each Hi-C replicate showed significantly greater overlap with 4C contacts than with negative control regions, indicating that the Hi-C contacts agree with published genomic contacts (Gisselbrecht, 2019).

The features of these mesodermally enriched silencer contacts were examined, as these are potential targets of silencing activity. A list was created of these potential targets by filtering for contacts that were observed in both of the Hi-C replicates and that overlap sFS-tested elements, and then the (epi)genomic features of these regions were examined. Since the TF Snail (Sna) has previously been associated with short-range repression and 'antilooping', the features of regions contacted by Sna-bound mesodermal silencers, Sna-unbound mesodermal silencers, and elements that did not act as silencers in mesoderm were compared. Regions that made mesoderm-specific contacts to Sna-unbound mesodermal silencers are significantly enriched for overlapping TSSs, as compared to those contacting either Sna-bound silencers or non-silencers, indicating that the Sna-unbound mesodermal silencers contact promoters. This suggests that Snail-unbound silencers are targeted primarily by long-range repressors, whereas Snail-bound silencers show almost no promoter contacts, which is consistent with previous reports of short-range repression and antilooping associated with this TF (Gisselbrecht, 2019).

Next, whether the expression levels of silencer-contacted genes were consistent with silencer activity was inspected. For genes whose promoters made contact with any of the 352 tested library elements, RNA-seq data from sorted mesodermal cells was compared with the elements' histone marks that showed significant correlation with silencer activity. The level of H3K27me3 found at a library element was significantly anticorrelated with the mesodermal expression of genes contacted by that element, supporting the model of contact-based repression by silencers (Gisselbrecht, 2019).

Attempts were made to test the alternate model that silencers directly contact enhancers that would otherwise be active. Thus, the contacted regions were examined for overlap with CRMs that have been reported to act as enhancers, according to the REDfly database. Each of the 3 sets of library elements (Sna-bound silencers, Sna-unbound silencers, and non-silencers) were separately tested, and no significant enrichment or depletion of CRM contact was observed in any of the sets. It was further reasoned that direct action by silencers on enhancers would result in enrichment of the enhancer mark H3K4me1 in regions that contact silencers. In this scenario, this enrichment should be apparent in histone mark data from whole embryos and across a broad range of time points, reflecting their enhancer activity in non-mesodermal tissues and/or other developmental stages. Instead a significant depletion of H3K4me1 was observed at Sna-unbound silencer contacts versus non-silencer contacts, which does not support the model of silencers interacting directly with enhancers. The results support models in which distinct classes of transcriptional silencers act by antilooping or by acting directly on the promoters of repressed genes. While the existence of silencers that may sequester enhancers from contacting promoters cannot be ruled out, the results do not support this alternate mechanism (Gisselbrecht, 2019).

This study has developed a highly parallel reporter assay carried out in whole, developing animals to identify a set of transcriptional silencers on the basis of their tissue-specific function. Analysis of RNA-seq data indicated that genes located near these silencers in their endogenous context are expressed at lower levels. Deletion of 1 of these elements at its native genomic locus by CRISPR-Cas9 genome editing demonstrated the importance of that element for the proper expression level of its target gene. This study also integrated a wide variety of data types from previously published datasets, including ChIP of histone modifications and specific factors, with newly generated tissue-specific 3D chromosomal physical interaction data to assess enriched features of the set of tissue-specific silencers and has explored potential mechanisms (Gisselbrecht, 2019).

Many enhancers were found in fact to be bifunctional elements, capable of up- and downregulating gene expression in different cellular contexts. While this phenomenon has been observed previously in studies of individual regulatory elements, the extent of CRM bifunctionality had not been appreciated. It is important to note that many CRMs that failed to show silencing activity in the screen are known enhancers that are not active in the tissue tested. Silencers are therefore an identifiable set of active elements, distinct from 'quenched' or inactive enhancers that neither activate nor repress gene expression (Gisselbrecht, 2019).

While prior studies have found histone modifications associated with enhancer activity, this study suggests that despite the extensive genome-scale ChIP profiling studies by numerous investigators and consortia, the available chromatin profiling data are not sufficient to identify silencers. This is possibly explained by the existence of various silencer classes. Alternatively, there are dozens of chromatin marks that have not been characterized extensively that may mark silencers. Expanded efforts in profiling larger sets of tissue-specific chromatin marks may reveal a signature of active silencers. Similarly, it was surprising that co-repressor occupancy was not a good predictor of silencers. One potential explanation is that many of these elements may be silencers in other cell types or at other developmental stages than were assayed in this study, since co-repressor ChIP data were generated in whole embryos across a broad range of ages. Another possibility is that different subclasses of CRMs with silencer activity may be endowed with subclass-specific chromatin and/or TF signatures. The list of 29 silencers discovered by the sFS assay provides a training set that can be used for the further study of regulatory features that govern silencing (Gisselbrecht, 2019).

Enriched Snail binding was observed at a subset of mesodermal silencers. Snail is a well-characterized short-range repressor protein acting in the mesoderm, and it has been proposed to act by preventing regulated elements from looping to promoters. The current results are consistent with this general model; however, the effects of Snail repression spread over hundreds of base pairs and into neighboring regulatory elements in the reporter assay, in contrast to previously reported limits of short-range repression. Thus, the results indicate that Snail can act by different modes of repression, which had not been observed previously (Gisselbrecht, 2019).

This study provides evidence supporting a model of silencer activity in which a subset of silencers makes direct 3D contacts with the promoters of regulated genes. These physical interactions are important to consider when interpreting genome-wide maps of chromosome conformation. Not all promoter-interacting regions will act as enhancers, and it will be necessary to develop approaches that integrate a wide range of genomic data types to identify and functionally characterize cis-regulatory elements, including distinguishing those acting as enhancers versus silencers (Gisselbrecht, 2019).

It has recently been shown that many developmental enhancers also act as PREs. Despite some common features, including evidence for looping to target promoters, this set of bifunctional enhancer elements is nearly distinct from the elements this study has characterized that act as both enhancers and silencers, and appears to act by distinct mechanisms. It was previously reported that a Drosophila insulator element has a second role in mediating long-range enhancer-promoter interactions. It is suggested that a taxonomy of regulatory elements as enhancers, silencers, insulators, and so forth is likely an oversimplification, and that it is more useful to think generally of CRMs, which can activate and repress, recruit chromatin modifiers and remodelers, and/or structure the 3D genome in a context-sensitive fashion (Gisselbrecht, 2019).

It has been estimated that there may be >50,000 enhancers in the D. melanogaster genome. This study has detected mesodermal silencer activity in >10% of tested non-mesodermal enhancers. If these elements are representative of the broader enhancer population, then this result suggests that there may be thousands of such bifunctional elements across a range of tissues in Drosophila; since many of the elements were tested could be silencers in a cell type that was not examined or at a later developmental stage, these numbers are likely even higher. The sFS approach could be adapted in future studies to screen for bifunctional elements in mammals (Gisselbrecht, 2019).

These results suggest that most, if not all, silencers are also enhancers in a different cell type. CRM bifunctionality complicates the understanding of how gene regulation is specified in the genome and how it is read out in different cell types. The observation that the vast majority of complex trait- and disease-associated variants identified from genome-wide association studies (GWASs) map to noncoding sequences, most of which occur within DNase I hypersensitive sites, emphasizes the importance of understanding these elements. The characterization of bifunctional elements will help in elucidating how precise gene expression patterns are encoded in the genome and aid in the interpretation of cis-regulatory variation (Gisselbrecht, 2019).

Binding partners of the kinase domains in Drosophila obscurin and their effect on the structure of the flight muscle

Drosophila obscurin (Unc-89) is a titin-like protein in the M-line of the muscle sarcomere. Obscurin has two kinase domains near the C-terminus, both of which are predicted to be inactive. This study has identified proteins binding to the kinase domains. Kinase domain 1 bound Ballchen (Ball, an active kinase), and both kinase domains 1 and 2 bound MASK (a 400-kDa protein with ankyrin repeats). Ball was present in the Z-disc and M-line of the indirect flight muscle (IFM) and was diffusely distributed in the sarcomere. MASK was present in both the M-line and the Z-disc. Reducing expression of Ball or MASK by siRNA resulted in abnormalities in the IFM, including missing M-lines and multiple Z-discs. Obscurin was still present, suggesting that the kinase domains act as a scaffold binding Ball and MASK. Unlike obscurin in vertebrate skeletal muscle, Drosophila obscurin is necessary for the correct assembly of the IFM sarcomere. Ball and MASK act downstream of obscurin, and both are needed for development of a well defined M-line and Z-disc. The proteins have not previously been identified in Drosophila muscle (Katzemich, 2015).

A stable lattice of thick and thin filaments in striated muscle is needed to maintain the optimum register of the filaments as the fibres contract. Thin filaments from neighbouring sarcomeres are anchored in the Z-disc by α-actinin and other cross-linking proteins, and thick filaments are held in position by cross-links at the M-line in the middle of the sarcomere. The register of thick filaments is also maintained by elastic links between the ends of the filaments and the Z-disc. Large modular proteins of the titin family, associated with thick filaments, contribute to both the stability and the stiffness of the sarcomere. These proteins are made up of tandem immunoglobulin (Ig) and fibronectin-like (Fn3) domains and can have one, or sometimes two, kinase domains near the C-terminus, and there can also be signalling domains (Katzemich, 2015).

The M-line protein, obscurin, has a similar modular structure in invertebrates and vertebrates, although the number of modules in different isoforms and the position of the signalling domains vary. Both Unc-89 (the obscurin homologue in Caenorhabditis elegans; note that this protein is also known as Unc-89 in Drosophila) and obscurin in Drosophila have SH3 and Rho-GEF signalling domains near the N-terminus and two kinase domains near the C-terminus. In vertebrate obscurin, the signalling domains are near the C-terminus; the isoform obscurin A has an ankyrin-binding domain instead of the two C-terminal kinase domains in obscurin B. Both these isoforms are at the periphery of myofibrils in the M-line region of mature skeletal fibres. Binding of obscurin A to ankyrins creates a link between the sarcoplasmic reticulum (SR) and the myofibril. By contrast, Drosophila obscurin is found throughout the M-line and there is no ankyrin-binding domain, so direct binding to the SR is unlikely. However, in the nematode, loss-of-function mutations in unc89 result in displaced ryanodine receptor and SERCA, as well as abnormal Ca2+ signalling. This suggests that there is a function for Unc-89 in Ca2+ regulation involving the SR. So far, five large isoforms of obscurin have been identified in Drosophila muscles: one expressed in the larva, and four expressed in the pupa and adult. All these isoforms have Ig domains in the tandem Ig region, and at least the first of the kinase domains (denoted Kin1). The indirect flight muscle (IFM) has two isoforms: a major isoform of 475 kDa and a minor isoform that is somewhat smaller. The two remaining isoforms are in other thoracic muscles. Drosophila obscurin is essential for the formation of an M-line, and for the correct assembly of thick and thin filaments in the sarcomere: lack of obscurin in the IFM results in asymmetrical thick filaments and thin filaments of abnormal length and polarity. Paradoxically, vertebrate obscurin is not necessary for normal sarcomere structure, given that obscurin knockout in the mouse had no serious effect on sarcomere assembly or maintenance (Katzemich, 2015).

The kinase domains of titin-like proteins often function as scaffolds binding other proteins, and might or might not be active kinases. In C. elegans, the Unc-89 kinase 1 domain (PK1) is predicted to be inactive because the ATP-binding site lacks essential residues. The Unc-89 kinase 2 domain (PK2) might be active, although a motif contributing to ATP-binding is atypical. Both Unc-89 kinase domains interact with the protein small, C-terminal domain, phosphatase-like 1 (SCPL-1), which is thought to be involved in muscle-specific signalling. Unc-89 PK1 also interacts with the LIM-domain protein, LIM-9; the complex of PK1, SCPL-1 and LIM-9 links Unc-89 to integrin adhesion sites at the cell surface. In Drosophila, both Obscurin kinase domains (denoted Kin1 and Kin2) are predicted to be inactive because the catalytic site lacks the catalytic aspartate and other crucial residues. Both kinase domains in vertebrate obscurin B are predicted to be catalytically active, and can apparently be auto-phosphorylated. The kinase domains are reported to interact with membrane associated proteins: kinase domain 1 (SK1) with the extracellular domain of a Na+/K+-ATPase at adherens junctions, which is not a substrate, and kinase domain 2 (SK2) with the cell-adhesion molecule, N-cadherin, which is an in vitro substrate (Katzemich, 2015).

The kinase domains in titin-like proteins have sequences at the C-terminus that sterically block the active site (the C-terminal regulatory domain). This sequence can inhibit an active kinase, or regulate ligand binding; it can also be part of the structure of the kinase domain, and necessary to maintain the stability of the domain. Titin-like kinases are linked to stretch-activated signalling pathways in muscle. Mechano-sensing by the kinase can result in changes in the C-terminal regulatory domain and transient binding of ligands to the kinase scaffold. The precise mechanism of regulation varies in different species (Katzemich, 2015).

The aim of this study was to identify proteins binding to the two kinase domains in Drosophila obscurin, and to determine the effect of the proteins on the assembly of an ordered sarcomere in IFM. Ball (a protein kinase) was shown to bind to Kin1, and MASK (an ankyrin repeat protein) binds to both Kin1 and Kin2. The kinase ligands are essential for the formation of an intact M-line and Z-disc in the IFM sarcomere (Katzemich, 2015).

The kinase domains of Drosophila Obscurin differ from those of the C. elegans homologue, Unc89, and the vertebrate protein. Both domains are predicted to be inactive as kinases. However, some pseudokinases can become catalytically active by replacing missing residues with residues from neighbouring domains, or from associated ligands. Pseudokinases commonly act as scaffolds for binding proteins involved in signal transduction, and are often tethered to other domains, including Ig and Fn3 domains, which contribute to the binding site. Titin kinase in the M-line region of vertebrate skeletal muscle forms part of a binding site for the autophagy and kinase scaffold proteins, Nbr1 and SQSTM1, and the ubiquitin ligase, MuRF1 (also known as TRIM63). In the case of MuRF1, the site includes the preceding Ig and Fn3 domains, which will also bind MuRF1 without the kinase domain (Katzemich, 2015).

This study has found that Kin1 in Drosophila Obscurin binds Ball, which has the hallmarks of an active serine-threonine kinase. Ball differs from other kinase molecules in having a long extension C-terminal to the kinase sequence. This extension binds to Kin1 of Obscurin, with or without the flanking Ig and regulatory domains. Given that Ball also binds to the Ig domain alone, it is likely the molecule spans a region of Obscurin that includes the Ig domain as well as the kinase. Binding to Kin1 with the regulatory domain was unexpected. However, it is not clear how much of the sequence downstream of the kinase is included in a possible regulatory domain. In pseudokinases that are part of a larger molecule, the association of the regulatory domain with a ligand-binding site can be altered by force applied to the whole molecule. The regulatory domain of Kin1, taken out of its usual context, might not associate with the active site in the same way as it would in vivo. Alternatively, the regulatory domain might be required to stabilise the kinase structure when the muscle is stretched, as suggested for twitchin kinase (Katzemich, 2015).

Ball is found in the Z-disc of IFM, as well as being diffusely distributed in the sarcomere and in some samples, Ball is also detected at the M-line. Ball might migrate to bind to Kin1 in the M-line when the kinase activity of Ball is needed. There are other examples of protein migration in the muscle sarcomere. A transient translocation from the M-line to the Z-disc and cytoplasm has been observed for titin kinase ligands in cardiac muscle. In zebrafish, the myosin chaperone, Unc-45, is associated with myosin during myofibrillogenesis; in the adult, Unc-45 is in the Z-disc in normal fibres and it migrates to the A-band under conditions of stress, where it transiently associates with myosin again. Similarly, Ball might migrate from the Z-disc to the M-line under some conditions. Although this study has shown that Ball is capable of binding to Kin1 in vivo, the conditions necessary for the association are not yet known. Ball is still present in the IFM of obscurin-knockdown flies, though with a less-ordered distribution in the sarcomere, which suggests there are likely to be other binding partners for Ball (Katzemich, 2015).

Kin2 binds MASK, which has two regions with ankyrin repeats, and a relatively long sequence C-terminal to a KH domain. A peptide near the end of the molecule binds to Kin2 with or without the Fn3 domain preceding the kinase. MASK does not bind to the Fn3 domain alone, nor does it bind to Kin2 with sequence C-terminal to the kinase. It is not clear at present whether the C-terminal sequence acts as a regulatory domain (Katzemich, 2015).

The dual position of MASK in both the Z-disc and the M-line of IFM is unlikely to be due to migration of a protein of 400 kDa. The RNA coding for ankyrin-repeat proteins undergoes extensive alternative splicing, which can alter the binding sites of the different ankyrin isoforms. The independence of the binding sites for MASK in the M-line and Z-disc is confirmed by the finding that in obscurin-knockdown flies, MASK is almost eliminated from the M-line but still present in the Z-disc. Thus, Obscurin is needed for MASK to bind in the M-line, but not to the Z-disc. The presence of Obscurin in the M-line of MASK-knockdown flies is consistent with an Obscurin scaffold that binds MASK (Katzemich, 2015).

In addition to MASK, Tropomyosin-1 was identified as a ligand associated with Kin2 expressed in vivo. As tropomyosin is a thin filament protein, the significance of an association with Obscurin, which is at the midline of the thick filament, is not clear. Smaller isoforms of Obscurin have been detected in IFM, and it is possible that Tropomyosin-1 could bind, outside the M-line, to a small isoform containing a Kin2 domain (Katzemich, 2015).

The effect of downregulating Ball or MASK on the structure of the M-line and Z-disc in IFM shows the importance of these proteins in the development of a regular filament lattice. In the IFM of Ball-knockdown flies, the shifted position of the H-zone and M-line is associated with fragmented Z-discs; where the Z-disc is normal within a sarcomere, the H-zone and M-line are at the midline. The aggregates of multiple Z-discs in the IFM of MASK-knockdown flies dominate the sarcomere and there is no regular H-zone. This phenotype differs from the effect of reducing the expression of obscurin in IFM, where the H-zone and M-line are often shifted from the midline, without a corresponding anomaly in the Z-disc. The difference might be due to the presence of Ball and MASK in the Z-disc, whereas Obscurin is solely in the M-line. The high mortality rate at the larval and pupal stages of flies when either Ball or MASK is reduced differs from the survival of flies with reduced obscurin, which is unaffected in RNAi lines. Evidently, the crucial function of Ball and MASK is in binding to the Z-disc. The association of the proteins with Obscurin in the M-line is not necessary for the performance of most muscles, although it is essential for the development of the precise filament lattice that is needed for the performance of the IFM. The function of Obscurin in the assembly of the IFM sarcomere is not seen in vertebrates. Knockouts of obscurin in the mouse have no effect on the assembly of myosin filaments, Z-disc or M-line, but they do impair the assembly of the SR. Whether or not invertebrate and vertebrate obscurin-like proteins are true homologues (with the same functions, rather than just having a similar patterns of domains) is at present uncertain (Katzemich, 2015).

Ball and MASK are reported to be involved in cell proliferation, growth and differentiation in Drosophila. Ball regulates the proliferation and differentiation of germline stem cells and neuroblasts (Herzig, 2014; Yakulov, 2014). However, it is not clear whether this has any relevance to the function of Ball in Drosophila muscle. The VRK family of kinases (of which Ball is a member) phosphorylate the barrier-to-autointegration factor (BAF), which is necessary for the correct assembly of chromatin. Comparison of domains in the sequences of Drosophila Ball (also called NHK-1) and VRK in human, mouse, Xenopus and C. elegans, shows that C. elegans and Drosophila are the only homologues with a long C-terminal sequence extension; the other species have a relatively short stretch of sequence following the kinase domain. This suggests that Ball has a different function in the invertebrates; in Drosophila, the C-terminal sequence binds to Kin1 (Katzemich, 2015).

MASK was first identified in the developing eye of Drosophila, where it is required for proliferation and differentiation of photoreceptors. MASK genetically interacts with Corkscrew (CSW), a protein phosphatase that acts downstream of the epidermal growth factor receptor (EGFR) in a signalling pathway involved in myogenesis in Drosophila. Through these interactions, Obscurin can potentially be linked to a receptor tyrosine kinase (RTK) pathway involved in myogenesis. Obscurin and Kettin are present at an early stage in the development of pupal IFM. Both are large titin-like proteins with tandem Ig domains, which have a dual function in myogenesis and in the mature muscle. Obscurin kinase domains appear to be scaffolds for binding MASK. Ankyrin repeats act as adaptor modules, binding cytoskeletal proteins and signalling molecules. The repeats stabilise protein networks, often together with large structural proteins. Therefore, an interaction between obscurin kinases and MASK could provide a platform for the assembly of signalling proteins, and this could be affected by force on the obscurin molecule. Ankyrin-repeat proteins in vertebrate skeletal muscle (MARPs) interact with the N2A elastic region of titin; in the case of Ankrd2 (also known as Arpp), expression is induced by stretch, and MARPs are thought to be involved in stretch-induced signalling pathways. There are two smaller homologues of MASK in human cells: MASK1 (Ankhd1) and MASK2 (Ankrd17), which have the same domain structure as Drosophila MASK. The Drosophila protein (~400 kDa) is larger than the human proteins (~280 kDa), mainly due to a longer stretch of sequence between the ankyrin repeat domains. MASK is a cofactor of Drosophila Yorkie (Yki) and mammalian Yes-associated protein (YAP) in the Hippo signalling pathway, which controls tissue growth. The signalling function in this pathway is similar for Drosophila and human MASK; however, MASK isoforms have not been found it human muscle. There is a sequence in the C. elegans genome that codes for a protein with homology to Drosophila, mouse and human MASK. The protein, ankyrin repeat and KH domain-containing protein is predicted to be 287 kDa, so similar in size to the human protein. The function is unknown (Katzemich, 2015).

The presence of Ball and MASK in mature IFM suggests the proteins have a signalling function in the adult fly. There is turnover of contractile proteins in Drosophila muscles, including the IFM, throughout the life of a fly. The function of obscurin kinase domains as scaffolds for the assembly of signalling proteins is likely to be important in the continual remodelling of the muscle. During contraction, the M-line experiences shearing stress, due to unbalanced forces in the two half sarcomeres, and the M-line is thought to act as a strain sensor. Ball and MASK might be recruited to the M-line in response to mechanical stress sensed by obscurin. Importantly, mutations in human titin kinase lead to a phenotype (Z-disc streaming) similar to that of Ball and MASK knockdowns, possibly by disrupting protein turnover, which supports the finding that Z-disc abnormalities can be an indirect consequence of mutations in proteins associated with the M-line (Katzemich, 2015).

In summary, this study has identified two proteins, Ball and MASK, that are essential for the assembly of an ordered IFM. The pseudokinase domains of obscurin act as scaffolds binding the proteins. This raises the possibility of investigating the regulation of signalling pathways involved in assembly and maintenance of IFM through interaction with obscurin (Katzemich, 2015).

Characterizing the actin-binding ability of Zasp52 and its contribution to myofibril assembly

In sarcomeres, α-actinin crosslinks thin filaments and anchors them at the Z-disc. Drosophila melanogaster Zasp52 also localizes at Z-discs and interacts with α-actinin via its extended PDZ domain, thereby contributing to myofibril assembly and maintenance, yet the detailed mechanism of Zasp52 function is unknown. This study shows a strong genetic interaction between actin and Zasp52 during indirect flight muscle assembly, indicating that this interaction plays a critical role during myofibril assembly. The results suggest that Zasp52 contains an actin-binding site, which includes the extended PDZ domain and the ZM region. Zasp52 binds with micromolar affinity to monomeric actin. A co-sedimentation assay indicates that Zasp52 can also bind to F-actin. Finally, in vivo rescue assays of myofibril assembly were used to show that the α-actinin-binding domain of Zasp52 is not sufficient for a full rescue of Zasp52 mutants suggesting additional contributions of Zasp52 actin-binding to myofibril assembly (Liao, 2020).

A Novel Mechanism for Activation of Myosin Regulatory Light Chain by Protein Kinase C-Delta in Drosophila

Myosin is an essential motor protein, which in muscle is comprised of two molecules each of myosin heavy-chain (MHC), the essential or alkali myosin light-chain 1 (MLC1), and the regulatory myosin light-chain 2 (MLC2). It has been shown previously that MLC2 phosphorylation at two canonical serine residues is essential for proper flight muscle function in Drosophila; however, MLC2 is also phosphorylated at additional residues for which the mechanism and functional significance is not known. This study found that a hypomorphic allele of Pkcδ causes a flightless phenotype; therefore, it was hypothesized that PKCδ phosphorylates MLC2. Flight disability was rescued by duplication of the wild-type Pkcδ gene. Moreover, MLC2 is hypophosphorylated in Pkcδ mutant flies, but it is phosphorylated in rescued animals. Myosin isolated from Pkcδ mutant flies shows a reduced actin-activated ATPase activity, and MLC2 in these myosin preparations can be phosphorylated directly by recombinant human PKCδ. The flightless phenotype is characterized by a shortened and disorganized sarcomere phenotype that becomes apparent following eclosion. It is concluded that MLC2 is a direct target of phosphorylation by PKCδ, and that this modification is necessary for flight muscle maturation and function (Vaziri, 2020).

Drosophila NUAK functions with Starvin/BAG3 in autophagic protein turnover

The inability to remove protein aggregates in post-mitotic cells such as muscles or neurons is a cellular hallmark of aging cells and is a key factor in the initiation and progression of protein misfolding diseases. While protein aggregate disorders share common features, the molecular level events that culminate in abnormal protein accumulation cannot be explained by a single mechanism. This study shows that loss of the serine/threonine kinase NUAK causes cellular degeneration resulting from the incomplete clearance of protein aggregates in Drosophila larval muscles. In NUAK mutant muscles, regions that lack the myofibrillar proteins F-actin and Myosin heavy chain (MHC) instead contain damaged organelles and the accumulation of select proteins, including Filamin (Fil) (Cheerio) and CryAB. NUAK biochemically and genetically interacts with the cochaperone Starvin (Stv), the ortholog of mammalian Bcl-2-associated athanogene 3 (BAG3). Consistent with a known role for the co-chaperone BAG3 and the Heat shock cognate 71 kDa (HSC70)/HSPA8 ATPase in the autophagic clearance of proteins, RNA interference (RNAi) of Drosophila Stv, Hsc70-4, or autophagy-related 8a (Atg8a) all exhibit muscle degeneration and muscle contraction defects that phenocopy NUAK mutants. It was further demonstrated that Fil/Cheerio is a target of NUAK kinase activity and abnormally accumulates upon loss of the BAG3-Hsc70-4 complex. In addition, Ubiquitin (Ub), ref(2)p/p62, and Atg8a are increased in regions of protein aggregation, consistent with a block in autophagy upon loss of NUAK. Collectively, these results establish a novel role for NUAK with the Stv-Hsc70-4 complex in the autophagic clearance of proteins that may eventually lead to treatment options for protein aggregate diseases (Brooks, 2020).

Proteins must fold into an intrinsic three dimensional structure to perform distinct cellular functions. Denatured or misfolded proteins can be refolded by chaperones or are subject to degradation by the ubiquitin-proteasome system (UPS) and/or the autophagosome-lysosome pathway (ALP). The accumulation of misfolded proteins upon genetic mutation or decreased chaperone function causes protein aggregates that are not effectively cleared by the UPS or the ALP. Environmental insults or aging may exacerbate this accumulation of misfolded proteins, resulting in disease and eventual cell death (Brooks, 2020).

A specialized autophagy pathway, termed chaperone-assisted selective autophagy (CASA), has been verified in both Drosophila and mammalian systems. The CASA complex includes BAG3 in concert with the chaperones HSC70/HSPA8 (HSP70 family), HSPB8 (small HSP family), and the ubiquitin (Ub) ligase CHIP/STUB1. CASA regulates the removal and degradation of Fil from the Z-disc in striated muscle or actin stress fibers in non-muscle cells. The N-terminal actin-binding domain (ABD) in Fil is followed by multiple immunoglobulin (Ig)-like repeats which bind numerous proteins to link the internal cytoskeleton to the sarcolemma. Tension exerted by contractile muscle tissue requires continuous folding and refolding of individual Ig-like domains in Fil, eventually damaging the ability of the protein to sense and transmit mechanical strain. The BAG3-HSC70 protein complex binds to the mechanosensor region (MSR) of Fil and upon detection of protein damage, CHIP ensures the addition of polyubiquitin (polyUb) moieties. Rather than promoting delivery to the proteasome, these Ub chains instead recruit the autophagic Ub adapter protein p62/SQSTM1. p62 interacts with Atg8a/LC3 to induce autophagophore formation and the subsequent clearance of Fil through lysosomal degradation. Fil aggregates and a block in autophagosome-lysosome fusion are present in lysosomal associated membrane protein 2 (LAMP2)-deficient muscles, thus linking impaired autophagy to abnormal protein deposits (Brooks, 2020).

Drosophila NUAK encodes for a conserved serine/threonine kinase that is homologous to the mammalian kinases NUAK1/ARK5 and NUAK2/SNARK. These proteins comprise a family of twelve AMP-activated protein kinase (AMPK)-related kinases (NUAK1 and 2, BRSK 1 and 2, QIK, QSK, SIK, MARK 1-4, and MELK) that share a conserved N-terminal kinase domain activated by the upstream liver kinase B1 (LKB1). NUAK1 and NUAK2 proteins are broadly expressed, but enriched in cardiac and skeletal muscle. Muscle contraction and LKB1 phosphorylation can activate both NUAK proteins. NUAK2 activity is additionally stimulated by oxidative stress, AMP, and glucose deprivation in various cell types. Interestingly, NUAK2 expression increases during muscle differentiation and in response to stress or in aging muscle tissue, whereas dominant-negative (DN)-NUAK2 induces atrophy. Homozygous NUAK1 KO mice are embryonic lethal and <10% of NUAK2 homozygotes survive, precluding analysis of post-embryonic contributions. Because of this embryonic lethality, conditional NUAK1 KO mice were generated to examine muscle function . However, no change was observed in muscle mass or fiber size between control or muscle-specific NUAK1 KO mice, likely due to functional redundancy (Brooks, 2020).

The presence of single NUAK orthologs in worms (Unc-82) or flies (NUAK/CG43143) allows for the study of NUAK protein function without compensation from additional family members that may mask cellular roles. unc-82 associates with Paramyosin and likely Myosin B to promote proper myofilament assembly in C. elegans. The kinase domain in Drosophila NUAK shares 61% identity and 80% similarity to human NUAK1 and NUAK2. In flies, RNAi knockdown of NUAK phenocopies weak Lkb1 defects in regulating cell polarity during ommatidial formation and actin cone formation in spermatogenesis. NUAK kinase targets or additional functions in other tissues have not been reported (Brooks, 2020).

This study identified Drosophila NUAK as a key regulator of autophagic protein clearance in muscle tissue. NUAK physically interacts with and phosphorylates Fil [encoded by Drosophila cheerio (cher)]. NUAK also genetically and biochemically interacts with the Stv-Hsc-70-4 complex and Stv overexpression is sufficient to rescue NUAK-mediated muscle deterioration. The identification of Fil as a cargo protein that abnormally accumulates in muscle tissue deficient for NUAK, Stv, Hsc70-4, and Atg8a links protein aggregation to defects in autophagic disposal (Brooks, 2020).

Prior to this study, few substrates of NUAK kinase activity had been uncovered. One of these is Myosin phosphatase targeting-1 (MYPT1), a regulatory subunit of myosin light-chain phosphatase. Two Drosophila regulatory subunits, MYPT75D and Myosin binding subunit (Mbs), were tested in Stv NUAK sensitized genetic assay and no protein aggregation and/or muscle degeneration was observed. While negative, this data nevertheless argues that this family of phosphatases likely does not function with NUAK in muscle tissue. Since the mammalian NUAK1-MYPT1 interaction was identified in vitro and further validated in HEK293 cells, NUAK likely has cell and tissue-specific targets that regulate diverse biological outputs (Brooks, 2020).

Based upon the discovery of Fil as a novel NUAK substrate, two scenarios are envisioned that are not mutually exclusive to explain the molecular function of NUAK in preventing protein aggregation. First, the increase in sarcomere number upon muscle-specific NUAK RNAi suggests that at least one role of NUAK may be to negatively regulate the addition of proteins (such as Fil) into sarcomeres. This data is consistent with studies that show C. elegans unc-82 regulates myofilament assembly. Notably, one key feature of the misincorporated proteins in unc-82 mutants is their inclusion into aggregate-like structures, similar to the accumulation of Fil and CryAB in NUAK-/- muscles. An additional, or alternative possibility, is that NUAK phosphorylates unfolded or 'damaged' Fil for removal from the sarcomere, thereby triggering the Stv-Hsc70-4 complex to promote autophagic turnover. Thus, proteins such as Fil that fail to get incorporated into sarcomeres and/or sustain damage due to repeated rounds of tension-induced muscle contraction, may destabilize myofilament architecture and trigger abnormal protein (Brooks, 2020).

In both contractile muscle tissue and in adherent cells subjected to mechanical force, BAG3 acts as a hub to coordinate Fil-induced tension-sensing and autophagosome formation. The MSR of Fil is comprised of Ig repeats whose conformational transitions between open and closed states dictate differential protein-protein interactions and biological outputs. While the chaperones Hsc70/HSPA8 and HSPB8 weakly bind to the MSR of Fil, this biochemical interaction is greatly enhanced in the presence of BAG3. Interestingly, BAG3 interacts with Ig repeats 19-21 in the MSR, while the selected interaction domain of NUAK with Fil comprises Ig repeats 15-18. These data suggest that NUAK and Stv each bind to a separate region of the MSR in Fil (Brooks, 2020).

It remains to be determined if NUAK-mediated phosphorylation is a prerequisite for the removal of damaged Fil protein by BAG3. The rescue results suggest that this phosphorylation event is not required as Stv overexpression alleviates protein aggregation and muscle degeneration upon a loss of NUAK. An alternative possibility is that this excess Stv protein is present in sufficient amounts to interact with Fil and overcome the necessity for phosphorylation by NUAK. The inability of NUAK overexpression to restore muscle defects due to knockdown of Stv, Hsc70-4, or Atg8a suggests that NUAK functions upstream or parallel to this pathway. It seems likely that NUAK has additional target substrates for kinase activity that may regulate autophagic protein clearance in muscle tissue (Brooks, 2020).

Recent studies demonstrate that increased autophagic degradation of Fil by BAG3 also induces fil transcription as a compensatory mechanism to ensure steady-state Fil levels. Thus, whether loss of NUAK or Stv alters gene expression upon a block in protein clearance was investigated. While the mRNA levels of cher, CryAB, Hsc70-4, or Atg8a were not altered in NUAK or stv mutants, there was a large increase in p62 transcripts. Thus, this increase in p62 mRNA synthesis may contribute to the elevated p62 protein levels observed upon loss of NUAK or Stv as multiple stress conditions increase p62 transcription, including proteasome inhibition, starvation and atrophic muscle conditions. Data that support a role for an autophagic block include the localization of p62 and Atg8a to regions of protein aggregation (Brooks, 2020).

A model for NUAK is proposed that incorporates these new findings with existing roles for BAG3. Fil and CryAB are physically associated at the Z-disc in Drosophila larval muscle. The phosphorylation of Fil by NUAK may control the incorporation of Fil into the Z-disc during myofibril assembly and/or may be required for the disposal of damaged Fil protein. BAG3 and chaperones such as Hsc70/HSPA8 are thought to monitor the MSR of Fil to detect force-induced damage and to promote the addition of K63-linked polyUb chains. Recruitment of the ubiquitin autophagic adapter p62/SQSTM1 induces autophagosome initiation through the accumulation of Atg8a. Eventual fusion of these autophagosomes with lysosomes promotes protein client complex destruction (Brooks, 2020).

Upon loss of NUAK, excess Fil protein that fails to be incorporated into the Z-disc and/or is damaged due to tension-induced muscle contraction begins to accumulate near the Z-disc. The presence of CryAB in Fil-like aggregates may be due to the normal association of CryAB with Fil at the Z-disc, either to monitor Fil protein damage, or to prevent protein aggregation. It is interesting that while both Fil and CryAB contain actin-binding domains, these associations are lost in NUAK-/- muscle tissue as F-actin is displaced from regions of Fil-CryAB accumulation. At this point it cannot be determined if NUAK preferentially binds to the short (~90kD) and/or long (~240 kD) Fil isoforms since the mapped interaction domains (Ig domains 15-18) are present in both isoforms (Brooks, 2020).

In the initial stages of aggregate formation, nearly all Fil puncta are decorated with Ub. It is hypothesized that the observed decrease in Ub-Fil colocalization in large regions of aggregate formation may be due to intrinsic properties of aggregation-prone proteins whereby protein misfolding triggers aggregation of Fil with itself and other proteins. The accumulation of p62 and circular structures that stain positive for Atg8a in regions of Fil accumulation demonstrate that the autophagosome machinery is recruited to BAG3-client complexes. The absence of lysosomes in these aggregate regions suggest that either fusion and/or transport to sites of degradation are compromised (Brooks, 2020).

CASA-mediated autophagy via the BAG3-client complex includes Hsc70-4/HSPA8, HSPB8, and the E3 ligase CHIP/STUB1, the latter of which ubiquitinates Fil for the subsequent recruitment of p62 to initiate autophagosome formation. However, fibroblasts deficient for CHIP are not defective in autophagy and mice or flies lacking CHIP/STUB1 are viable. A failure to enhance protein aggregation defects upon CHIP RNAi knockdown in the sensitized NUAK+/- or stv+/- backgrounds suggests that additional Ub ligases cooperate with the Stv/BAG3 complex to remove damaged proteins. Future studies will also determine which Drosophila protein is the equivalent of HSPB8 since no genetic interactions were observed with putative CG14207 or Hsp67Bc RNAi lines. This negative data does not rule out the possibility that protein levels are not reduced enough to see phenotypes upon RNAi induction or possible functional redundancy exists between CG14207 and Hsp67Bc (Brooks, 2020).

An interesting hallmark of protein aggregate diseases is the accumulation of specific proteins in affected cells or tissues. Thus, proteins susceptible to aggregation in vivo may possess specific structural characteristics or shared biological functions. This latter feature is evident in a group of protein aggregate diseases termed myofibrillar myopathies (MFM). Laser microdissection of aggregates from normal or affected muscles reveal specificity in the types of proteins that accumulate in patients afflicted with MFMs. Common proteins present in these aggregates include Filamin C (FILC), αB-crystallin (CRYAB), BAG3, and Desmin (DES), among others. The inability of MFM patients to clear these aggregates results in myofibrillar degeneration and a decline in muscle function. Interestingly, mutations in Drosophila NUAK phenocopy both structural and functional deficits observed in MFM patients, including Fil and CryAB accumulation, muscle degeneration, and locomotor defects. The discovery of cellular degeneration and protein aggregation in muscle tissue upon loss of the single fly NUAK ortholog highlights the power of Drosophila as a model. Future studies will focus on identifying kinase targets of NUAK and defining additional proteins that function in NUAK and stv-mediated autophagy for the eventual development of therapeutic targets to treat MFMs and other protein aggregate diseases (Brooks, 2020).

Muscle-derived Myoglianin regulates Drosophila imaginal disc growth

Organ growth and size are finely tuned by intrinsic and extrinsic signaling molecules. In Drosophila, the BMP family member Dpp is produced in a limited set of imaginal disc cells and functions as a classic morphogen to regulate pattern and growth by diffusing throughout imaginal discs. However, the role of TGFβ/Activin-like ligands in disc growth control remains ill-defined. This study demonstrates that Myoglianin (Myo), an Activin family member, and a close homolog of mammalian Myostatin (Mstn), is a muscle-derived extrinsic factor that uses canonical dSmad2-mediated signaling to regulate wing size. It is proposed that Myo is a myokine that helps mediate an allometric relationship between muscles and their associated appendages. Although Babo/dSmad2 signaling has been previously implicated in imaginal disc growth control, the ligand(s) responsible and their production sites(s) have not been identified. Previous in situ hybridization and RNAi knockdown experiments suggested that all three Activin-like ligands contribute to control of wing size. However, no expression of these Activin-like ligands was found in imaginal discs, with the exception of Actβ which is expressed in differentiating photoreceptors of the eye imaginal disc. It is concluded that the small wing phenotypes caused by RNAi knockdown of Actβ or daw are likely the result of off-target effects and that Myo is the only Activin-type ligand that regulates imaginal disc growth (Upadhyay, 2020).

Although Babo/dSmad2 signaling has been previously implicated in imaginal disc growth control, the ligand(s) responsible and their production sites(s) have not been identified. Previous in situ hybridization and RNAi knockdown experiments suggested that all three Activin-like ligands (Myoglianin, Activinβ, and Dawdle) contribute to control of wing size. However, this study found no expression of these Activin-like ligands in imaginal discs, with the exception of Actβ which is expressed in differentiating photoreceptors of the eye imaginal disc. More importantly, using genetic null mutants, this study showed that only loss of myo affects imaginal disc size. The discrepancy in phenotypes between tissue-specific knockdown results and the genetic nulls is often noted and not fully understood. In addition to simple off-target effects within the wing disc itself, one possible explanation is that many GAL4 drivers are expressed in tissues other than those reported, potentially resulting in deleterious effects for the animal that indirectly affect imaginal disc size. Another possibility is that in Actβ and daw genetic null backgrounds a non-autonomous compensatory signal is generated by another tissue and this signal is not activated in the case of tissue-specific knockdown. Both of these explanations are thought unlikely in this instance since it was demonstrated that only the Babo-A receptor isoform is expressed and required in discs. Since it was previously shown that Daw only signals through isoform Babo-C, it is unclear why knockdown of daw in the wing disc would result in a small wing phenotype as previously reported. It is concluded that the small wing phenotypes caused by RNAi knockdown of Actβ or daw are likely the result of off-target effects and that Myo is the only Activin-type ligand that regulates imaginal disc growth (Upadhyay, 2020).

The signaling ability of TGFβ ligands is modulated by the specific combinations of receptors and co-receptors to which they bind. In Drosophila, the receptor requirements for effective signaling through dSmad2 likely vary for each ligand and tissue. This study found that Myo signaling in the wing disc requires Punt as the type II receptor and Babo-A as the type I receptor. Furthermore, it was establish that Myo is the exclusive Activin-like ligand signaling to the discs since loss of Myo eliminated detectable phosphorylation of dSmad2 in the wing imaginal disc. Since Babo-A is the only isoform expressed in wing discs, it is also concluded that Myo is able to signal through this isoform in the absence of other isoforms. Whether Myo can also signal through Babo-B or C is not yet clear, but in the context of mushroom body remodeling Babo-A also appears to be the major receptor isoform utilized. The co-receptor Plum (see Plum, an Immunoglobulin Superfamily Protein, Regulates Axon Pruning by Facilitating TGF-β Signaling) is also required for mushroom body remodeling, suggesting that Plum and Babo-A are both necessary for efficient Myo signaling. However, it is noteworthy that Plum null mutants are viable while Myo null mutants are not. This observation suggests that Plum is not required for all Myo signaling during development. Further studies will be required to evaluate whether Plum is essential to mediate Myo signaling in imaginal discs (Upadhyay, 2020).

The requirement of Punt as a type II receptor for production of an efficient signaling complex with Myo may be context dependent. In the mushroom body, indirect genetic evidence suggests that the two Type II receptors function redundantly. Although both punt and wit are expressed in imaginal discs, only loss of punt produces a phenotype in the brk reporter assay. To date, clear signaling has not been seen in S2 cells expressing Punt and Babo-A when Myo is added. It is also notable that a previous attempt to study Myo signaling in a heterotypic cell culture model also failed. In that study, Myo was found to form a complex with Wit and Babo-A in COS-1 cells but no phosphorylation of dSmad2 was reported. One explanation is that effective signaling by Myo requires Punt, and Babo-A, and perhaps another unknown co-receptor that substitutes for Plum. Despite this caveat, the current results provide in vivo functional evidence for a Myo signaling complex that requires Babo-A and Punt to phosphorylate dSmad2 for regulation of imaginal disc growth (Upadhyay, 2020).

Final tissue size is determined by several factors including cell size, proliferation, death rates, and duration of the growth period. While cell size changes were observed upon manipulation of Myo signaling, the direction of change depended on the genotype. In myo mutants, estimation of cell size via apical surface area indicates that the cells are ~20% smaller than wild-type. Although this measurement does not indicate the actual volume of the cells, it gives an indication of cell density in the epithelial sheet of the wing pouch, which is analogous to counting cells in the adult wing. RNAi knock down of babo-a in the entire disc produced smaller adult wings with larger (less dense) cells. This result differs from the myo mutant, but is similar to the reported adult wing phenotypes of babo mutants and larval disc phenotypes of dSmad2 mutants. When babo-a is knocked down in one compartment, that compartment is reduced in size with smaller cells. It is concluded that tissue size reduction is the consistent phenotype upon loss of Myo signaling, but cell size changes depend on the specific type of manipulation (Upadhyay, 2020).

While cell size effects may be context dependent, it is notable that neither reduction in size of imaginal discs nor adult wing surface area can be explained solely by a cell size defect. Since no apoptotic increase was seen in myo mutant discs, and because dSmad2 knockdown also fails to alter apoptotic rate, the mostly likely cause is an altered proliferation rate. Consistent with this view is that the large disc phenotype exhibited by dSmad2 protein null mutants is clearly dependent on Myo and it has been previously shown that this is the result of enhanced proliferation. Similarly earlier studies also showed that expression of activated Babo or activated dSmad2 in wing discs also leads to larger wings with slightly smaller cells which is most easily explained by an enhanced proliferation rate. It is worth noting that this proposed enhanced proliferation rate is difficult to detect since cell division is random with regard to space and time during development. Thus a ~ 20% reduction in adult wing size caused by a proliferation defect translates into about 1/5th of disc cells dividing on average one less time throughout the entire time course of larval development. Therefore, without prolonged live imaging, this small reduction in proliferation rate will not be detectable using assays that provide only static snapshots of cell division. It is worth noting, however, that previous clonal studies also concluded that dSmad2 or Babo loss in wing disc clones resulted in a reduced proliferation rate (Upadhyay, 2020).

One attempt to shed light on the transcriptional output of TGFβ signaling responsible for wing disc size employed microarray mRNA profiling of wild-type versus dSmad2 gain- and loss-of-function wing discs. However, this study did not reveal a clear effect on any class of genes including cell cycle components, and it was concluded that the size defect is the result of small expression changes of many genes. Consistent with this view are dSmad2 Chromatin Immunoprecipitation experiments in Kc cells which revealed that dSmad2 is associated with many genomic sites and thus may regulate a myriad of genes (Upadhyay, 2020).

Insect myoglianin is a clear homolog of vertebrate Myostatin (Mstn/GDF8), a TGFβ family member notable for its role in regulating skeletal muscle mass. Mstn loss-of-function mutants lead to enlarged skeletal muscles. Mstn is thought to affect muscle size through autocrine signaling that limits muscle stem cell proliferation, as well as perturbing protein homeostasis via the Insulin/mTOR signaling pathways. Similarly, Gdf11, a Mstn paralog, also regulates size and proliferation of muscles and adipocytes, and may promote healthy aging. Mstn and Gdf11 differ in where they are expressed and function. Mstn is highly expressed in muscles during development while Gdf11 is weakly expressed in many tissues. Both molecules are found to circulate in the blood as latent complexes in which their N-terminal prodomains remain associated with the ligand domain. Activation requires additional proteolysis of the N-terminal fragment by Tolloid-like metalloproteases to release the mature ligand for binding to its receptors. Interestingly in Drosophila, the Myoglianin N-terminal domain was also found to be processed by Tolloid-like factors, but whether this is a prerequisite for signaling has not yet been established. In terms of functional conservation in muscle size control, the results of both null mutants and RNAi depletion indicates that it has little effect on muscle size. This contradicts a previous study in which muscle-specific RNAi knockdown of myo was reported to produce larger muscles similar to the vertebrate observation. The discrepancy between the tissue-specific RNAi knockdown and previous studies is not clear, but the current null mutant analysis strongly argues that Drosophila Myo does not play a role in muscle size control. Intriguingly however, this study found that loss of Actβ, another ligand that signals through Babo and dSmad2, results in a smaller muscles) contrary to that produced by loss of vertebrate Mstn and various other vertebrate Activin family members. Recent data has shown that Drosophila Actβ is the only Activin-like ligand that affects muscle growth, and it does so, in part, by regulating Insulin/Tor signaling in the opposite direction compared to vertebrates. Thus, in Drosophila the Myo/Activin pathway promotes muscle growth while in vertebrates it inhibits muscle growth (Upadhyay, 2020).

The most intriguing finding of this study is that muscle-derived Myo acts non-autonomously to regulate imaginal disc growth. This is in stark contrast to the two BMP ligands, Dpp and Gbb, which are produced by disc cells and act autonomously within the disc itself to regulate both growth and pattern. The fact that a TGFβ ligand can act in an endocrine-like manner is not particularly novel since many vertebrate members of the TGFβ family, including Myostatin, the closest homolog to Drosophila Myoglianin, are found in the blood. Even the disc intrinsic molecule Dpp has been recently shown to be secreted into the hemolymph where it circulates and signals to the prothoracic gland to regulate a larval nutritional checkpoint. Several additional reports indicate that ligands from the Drosophila Activin-like subfamily also circulate in the hemolymph and function as inter-organ signals. For example, muscle-derived Actβ and Myo signal to the fat body to regulate mitochondrial function and ribosomal biogenesis, respectively. In addition, Daw produced from many tissue sources can signal to the Insulin producing cells and the midgut to stimulate Insulin secretion and repress expression of sugar metabolizing genes, respectively. Thus, many TGFβ type factors act as both paracrine and endocrine signals depending on the tissue and process involved (Upadhyay, 2020).

The phenotype of the myo mutant animal supports the claim that endogenous Myo contributes to imaginal disc growth. The ectopic expression assay produced various wing disc sizes when Myo was expressed in different tissues, indicating that the growth response likely depends on the level of Myo being produced in the distal tissue. Loss of glial derived Myo is not sufficient to suppress overgrowth of dSmad2 mutant discs, but overexpression of Myo in glia did partially rescue size of myo null wing discs, likely because the repo-Gal4 driven overexpression produces more ligand than endogenous glia. Likewise, expression from a large tissue such as muscle or fat body likely produces more Myo than glia leading to normal disc growth or even overgrowth. It is also possible that Myo signaling activity is modified depending on the tissue source. Like other TGFβ family members, Myo requires cleavage by a furin protease at its maturation site to separate the C-terminal ligand from the prodomain. Myo may also require a second cleavage by a Tolloid protease family member to achieve full dissociation of the prodomain from the ligand to ensure complete activation. Either of these cleavage reactions, or any other step impacting the bioavailability of active Myo ligand, may vary with tissue or may be modulated by environmental conditions (Upadhyay, 2020).

What is the rationale for larval muscle regulating imaginal discs size? A possible reason is that for proper appendage function, the muscle and the structure (leg, wing, and haltere) that it controls should be appropriately matched to ensure optimal organismal fitness for the environmental niche the adult occupies. For example, a large muscle powering a small wing might result in diminished fine motor control. Conversely, a small muscle may not be able to power a large wing to support flight. However, the multi-staged nature of muscle and appendage development complicates this picture. Larval muscles are histolysed during metamorphosis and do not contribute to the adult muscle. However, remnants of larval muscles in the thoracic segment are preserved as fibers that act as scaffolds upon which the larval myoblasts infiltrate and fuse to become the adult indirect flight muscles. Thus, at least for the indirect flight muscles, the size of the larval muscle scaffold might contribute to the building of a bigger adult muscle. Another possibility invokes a signal relay system. Wg and Serrate/Notch signaling from the wing disc epithelial cells control myoblast proliferation during larval development. Thus it may be that Myo signaling from the larval muscles stimulates proliferation of the disc epithelial layer which in turn enhances Wnt and Serrate/Notch signaling to myoblasts to increase their number thereby coordinating the adult appendage size with muscle size. A final scenario is that, since muscle is a major metabolic and endocrine organ, Myo production may be regulated by the general metabolic state of the larva. If healthy, high levels of Myo, in concert with other growth signals such as insulin, leads to a bigger fly with large wings, and if the metabolic state is poor then lower Myo levels leads to diminished proliferation and a smaller cell size resulting in a smaller fly with small wings (Upadhyay, 2020).

Regardless of the precise mechanism, the ability of the muscle to control appendage size has interesting implications in terms of evolutionary plasticity. The proportionality of insect wing size to body size can vary over a large range, but the mechanism responsible for determining this particular allometric relationship for a given species is not understood. It was recently demonstrated that in Drosophila, motor neuron derived Actβ, another TGFβ superfamily member, can dramatically affect muscle/body size (Moss-Taylor, 2019). Therefore, it is tempting to speculate that evolutionary forces might modulate the activity of these two genes to produce an appropriate body-wing allometry that is optimal for that species' ecological niche (Upadhyay, 2020).

Deconstruction of the beaten Path-Sidestep interaction network provides insights into neuromuscular system development

An 'interactome' screen of all Drosophila cell-surface and secreted proteins containing immunoglobulin superfamily (IgSF) domains discovered a network formed by paralogs of Beaten Path (Beat) and Sidestep (Side), a ligand-receptor pair that is central to motor axon guidance. This study describes a new method for interactome screening, the Bio-Plex Interactome Assay (BPIA), which allows identification of many interactions in a single sample. Using the BPIA, four more members of the Beat-Side network were 'deorphanized'. Interactions were confirmed using surface plasmon resonance. The expression patterns of beat and side genes suggest that Beats are neuronal receptors for Sides expressed on peripheral tissues. side-VI is expressed in muscle fibers targeted by the ISNb nerve, as well as at growth cone choice points and synaptic targets for the ISN and TN nerves. beat-V genes, encoding Side-VI receptors, are expressed in ISNb and ISN motor neurons (Li, 2017).

Protein-protein interactions (PPIs) control a vast array of processes in metazoans, ranging from signal transduction and gene regulation within cells to signaling between cells via cell surface and secreted proteins (CSSPs). The strength of PPIs varies widely, from high-affinity interactions that create stable protein complexes to weak transient interactions. Defining global PPI patterns ('interactomes') has been the focus of much recent research. Progress has been made in generating high-throughput protein interaction data for a variety of organisms, including S. cerevisiae, C. elegans and D. melanogaster. Methods used to create interactomes include affinity purification/mass spectrometry (AP-MS) and the yeast two-hybrid assay (Y2H) (Li, 2017).

It is estimated that up to one sixth of human genes encode CSSPs. CSSPs control signaling from the extracellular milieu to cells and the flow of information between cells. Due to their importance and accessibility, CSSPs are often the targets for therapeutic agents, including humanized monoclonal antibody drugs such as checkpoint inhibitors, the non-Hodgkin's lymphoma drug Rituxan, and the breast cancer drug Herceptin. However, the biochemical properties of many CSSP interactions prevent them from being detected by commonly used techniques employed in high-throughput PPI screens, and CSSPs are underrepresented in global interactomes. There are several reasons for this. First, these proteins are usually glycosylated and have disulfide bonds, so they need to be expressed in the extracellular compartment. CSSP interactions between monomers are also often weak, with KDs in the μM range, making them difficult to capture due to their short half-lives. Lastly, insoluble transmembrane domains on cell surface proteins preclude their purification with standard biochemical techniques, which makes them difficult to study using methods such as AP-MS (Li, 2017).

Despite these difficulties, recent advances have been made in the study of global CSSP interaction patterns. Interactions among cell-surface proteins (CSPs) often occur between clusters of proteins on cell surfaces, and avidity effects (stronger binding due to clustering) can make these cell-cell interactions stable even when monomers bind only weakly. To facilitate detection of interactions among CSSP extracellular domains (ECDs) in vitro, several groups have taken advantage of avidity effects by attaching ECDs to protein multimerization domains and expressing ECD fusions as soluble secreted proteins. These methods have been shown to be effective, allowing detection of interactions that otherwise would not have been observable (Li, 2017).

Özkan (2013) scaled up the avidity-based approach, developing a high-throughput ELISA-like screening method, the Extracellular Interactome Assay (ECIA). The ECIA was used to define interactions among 202 Drosophila CSSPs, comprising all CSSPs within three ECD families. These were the immunoglobulin superfamily (IgSF), fibronectin type III (FNIII) and leucine-rich repeat (LRR) families. The ECIA utilized dimers as 'bait' and pentamers as 'prey'. It detected 106 interactions, 83 of which were previously unknown (Li, 2017).

The most striking finding from the ECIA interactome was that a subfamily of 21 2-IgSF domain CSPs, the Dprs, selectively interacts with a subfamily of 9 3-IgSF domain CSPs, the DIPs, forming a network called the 'Dpr-ome' (Özkan, 2013). Each Dpr and DIP that has been examined is expressed by a small and unique subset of neurons at each stage of development. One Dpr-DIP pair is required for normal synaptogenesis and influences neuronal cell fate. In the pupal optic lobe, neurons expressing a Dpr are often presynaptic to neurons expressing a DIP to which that Dpr binds in vitro. The Dpr-ome initially defined by the global interactome contained several 'orphans', proteins with no binding partner (Özkan, 2013). By expressing new versions of Dprs and DIPs, including chimeras, and using these to conduct a 'mini-interactome' analysis of the Dpr-ome, it was possible to find partners for all but one orphan. That protein, Dpr18, has changes to conserved binding interface residues and may lack the capacity to bind to any DIPs (Li, 2017).

The ECIA also identified a second IgSF network, formed among members of the Beaten Path (Beat) and Sidestep (Side) protein subfamilies. Beat-Ia and Side were identified by genetic screens for motor axon defects, and were later shown to have a ligand-receptor relationship. They control the projection of motor axons to muscle targets. Beat-Ia is expressed on motor axons, where it binds to Side, which is expressed on muscles. This binding causes motor axons to decrease their adhesion to each other, allowing them to leave their bundles and turn onto the muscle fibers. beat-Ia and side have strong motor axon phenotypes. In the absence of either protein, motor axons often remain in their fascicles and never leave to arborize on their target muscles (Li, 2017).

The ECIA detected the known Beat-Ia::Side interaction, and also uncovered other interactions between members of the Beat and Side subfamilies. Seven of the 14 Beats were found to bind to four of the eight Sides. The remaining Beats and Sides were still orphans with no binding partners in the other subfamily. The functions of the newly defined interactions between Beats and Sides were unknown. Most beat genes are expressed in embryonic neurons. Some Beats were genetically characterized using deletion mutations and RNAi, but loss of these Beats did not cause strong motor axon phenotypes. None of the other Side subfamily members had been examined (Li, 2017).

This paper describes the development of a new method for interactome screening, which is called the BPIA (Bio-Plex Interactome Assay). This method uses the 'Bio-Plex' system, which employs Luminex xMAP technology. This method detects binding of a prey protein to many bait proteins, each conjugated to a bead of a different color, in each assay well. For the ECIA, the number of assays required for the interactome screen was the square of the number of proteins examined, while with the Bio-Plex the number of assays could be equal to the number of proteins. In principle, then, the Bio-Plex might greatly speed up interactome screening, and might also be more sensitive, since the available signal-to-background ratio is much greater for the Bio-Plex than for the ECIA. As a test of the method, a Bio-Plex 200 was used to do a mini-interactome screen of the Beat-Side network. Based on the the fact that the Dprs and DIPs that were initially orphans were later shown to have binding partners, it was hypothesized that some of the orphan Beats should have Side partners, and vice versa. Consistent with this hypothesis, it was possible to deorphanize two more Beats and two Sides using the BPIA (Li, 2017).

To further understanding of Beat and Side function during embryonic development, this study characterized expression of several Beats and Sides, focusing primarily on Side-VI and the three Beat-Vs, which were the strongest interactors in both the ECIA and BPIA screens. The three beat-Vs exhibit differential expression in identified motor neurons, while side-VI is expressed at motor axon choice points and in a subset of target muscle fibers (Li, 2017).

In principle, the Bio-Plex system can allow 500 unique protein-protein (bait-prey) interaction pairs to be analyzed in a single well. In this method, capture of proteins from media with streptavidin-coupled beads bypasses the purification step for bait proteins. The assay is also compatible with the use of unpurified prey proteins, thereby reducing the workload for multiplexed screenings. The small size of the beads, the ability to probe multiple interactions simultaneously, and the small volume of the binding reactions all help reduce the amount of protein and reagents needed for the assay. It was possible to produce enough bait and prey proteins for the mini-interactome described in this study (a 23 x 23 matrix) with a single 10 cm dish transfection per protein (Li, 2017).

As a test of the system, the BPIA was used to examine interactions between the Drosophila Beat and Side IgSF protein subfamilies. Beat-Ia is a receptor on motor growth cones that recognizes Side expressed on muscles, and in the absence of Beat-Ia or Side motor axons fail to leave their bundles and arborize on their muscle targets. There are 14 Beat subfamily members and 8 Side subfamily members, but all of these proteins except Beat-Ia and Side itself were orphans until the global IgSF interactome revealed interactions between six other Beats and three Sides (Li, 2017).

In the Dpr-ome, the other IgSF network uncovered by the interactome, every Dpr protein likely to be capable of binding has an interaction partner in the DIP subfamily. Based on this, it is predicted that there should be additional interactions to be discovered within the Beat-Side subfamily network. Using the BPIA, three new interactions were found: Beat-VI::Side-II, Beat-Ic::Side-III, and Beat-Ic::Side. These results suggest that the BPIA is more sensitive than the ECIA. Like the ECIA, the BPIA should be able to find new receptor-ligand interactions even if proteins not previously known to have any interactions were tested. Of course, for both assays any candidate receptor-ligand pairs need to be confirmed as genuine using other methods. For the Beat-Side network, all three new interactions found by the BPIA, as well as the interactions between the three Beat-Vs and Side-VI found by the ECIA, were verified by SPR. This study also demonstrated that Beat-Vs and Side-VI interact using cell-based binding assays and binding to live-dissected embryos (Li, 2017).

There are still five Beats and two Sides that remain orphans. Since the structure of Beat-Side complexes is unknown, it cannot be determined whether these Beats and Sides are likely to be able to bind, but it is speculated that at least the three Beat-IIIs are likely to have Side partners. The Beat-II and Beat-V clusters each interact with a single Side partner, and perhaps the Beat-IIIs interact with one of the two orphan Sides. It is possible that changes in methodology, such as using more highly multimerized preys and/or baits, could increase sensitivity and allow detection of additional interactions (Li, 2017).

The expression patterns of side and beat genes were examined in order to obtain insights into their possible functions. Most sides are expressed in cells in the periphery as well as in the CNS, while most beats are expressed only by CNS neurons, including motor neurons. Beat-Ia::Side interactions are required for normal motor axon guidance, and highly penetrant motor axon defects in which muscles remain uninnervated are observed in mutants lacking either protein. By contrast, partial loss of function of beat-Ib, beat-Ic, both beat-IIs, or beat-VI causes motor axon defects with less than 20% penetrance. Genetic redundancy is a common theme in motor axon guidance , so it is not surprising that only low-penetrance defects are observed when Beat paralogs are not expressed. Given that Beat-Ia and Side both interact with other partners, it is perhaps remarkable that beat-1a and side have such strong phenotypes as single mutants (Li, 2017).

Beat-V and Side-VI also have redundant functions in motor axon guidance. side-VI is expressed in motor axon targets, including muscles 12 and 13 and interacts with the three Beat-Vs, at least two of which are expressed in subsets of motor neurons. Beat-V::Side-VI interactions produced the strongest signals in both the ECIA and BPIA. Sow-penetrance (~1/5 of stage 17 embryonic hemisegments affected) muscle 12 innervation defects were observed in side-VI insertion mutants or in deletion mutants lacking all three beat-V genes. There were also low-penetrance ISN guidance defects in both mutants. The fact that most muscle 12 s are innervated normally in beat-V or side-VI mutants indicates that, while Beat-V::Side-VI interactions may contribute to correct targeting of the RP5 axon to muscle 12, other cues must also be involved. Muscles 12 and/or 13 also express Wnt-4 (a repulsive ligand) and the LRR protein Capricious (Caps; probably an adhesion molecule), and low-penetrance RP5 targeting defects are observed in Wnt-4 and caps mutants. Perhaps muscle 12 is distinguished from other nearby muscles by a set of partially redundant cues, so that strong targeting phenotypes are not observed in any single mutant (Li, 2017).

Although Beat and Side paralogs may not be central to motor axon guidance, their expression patterns suggest that they could be important for determining synaptic connections within the CNS. side-VIII, encoding an orphan Side, is expressed in a small subset of embryonic CNS neurons. In the optic lobe of the pupal brain, an RNAseq analysis of two photoreceptors (R7 and R8) and five types of lamina neurons (L1-L5) revealed that beats and sides have highly specific expression patterns. For example, beat-VII is specific to L2, beat-VI to L5, beat-IIa to L3 (with lower levels in L4), and beat-IIIc to R8, being expressed at much higher levels in those cells relative to all other cells. side and side-III are specific to L3, side-II is specific to L1, side-IV is specific to L2, and side-V is specific to L5. Each of the L neuron types as well as R7 and R8 synapse with different sets of neurons in the medulla, a ten-layered structure that processes visual information from the retina and lamina. It has been observed that R and L neurons expressing specific Dprs often form synapses on medulla neurons expressing DIPs to which those Dprs bind in vitro. In a similar manner, perhaps some of the medulla neurons that are postsynaptic to L or R neurons expressing specific Sides or Beats express their in vitro binding partners, and these Beat-Side interactions might be important for synapse formation or maintenance (Li, 2017).

Drosophila adult muscle precursor cells contribute to motor axon pathfinding and proper innervation of embryonic muscles

Despite several decades of studies on the neuromuscular system, the relationship between muscle stem cells and motor neurons remains elusive. Using the Drosophila model, evidence is provided that adult muscle precursors (AMPs), the Drosophila muscle stem cells, interact with the motor axons during embryogenesis. AMPs not only hold the capacity to attract the navigating intersegmental (ISN) and segmental a (SNa) nerve branches, but are also mandatory to the innervation of muscles in the lateral field. This so-far-ignored AMP role involves their filopodia-based interactions with nerve growth cones. In parallel, the expression of the guidance molecule-encoding genes sidestep and side IV in AMPs is reported. Altogether, these data support the view that Drosophila muscle stem cells represent spatial landmarks for navigating motor neurons and reveal that their positioning is crucial for the muscles innervation in the lateral region. Furthermore, AMPs and motor axons are interdependent, as the genetic ablation of SNa leads to a specific loss of SNa-associated lateral AMPs (Lavergne, 2020).

During Drosophila embryogenesis, a stereotypical pattern of AMPs per abdominal hemisegment in ventral (V-AMP), lateral (L-AMPs), dorsolateral (DL-AMPs) and dorsal (D-AMPs) positions can be distinguished. This study has investigated the relationship between AMPs and motor axons, and their dynamics, during development using embryos carrying the M6-gapGFP transgene, which allows visualization of the membrane of AMP cells. The intersegmental nerve (ISN) established contacts with the DL-AMPs during the embryonic stage 13 and then navigated toward the D-AMP to contact it at stage 15. Within the lateral field, the segmental nerve a (SNa) is sub-divided into two branches, dorsal (D-SNa), which innervates the lateral transverse muscles (LTs 1-4), and lateral (L-SNa), which targets the segmental border muscle (SBM). The SNa sub-division takes place during stage 15 and it was observed that the L-SNa branch migrated towards the L-AMPs before innervating the SBM. In parallel, the anterior L-AMP underwent shape changes and directional migration towards the L-SNa. In a similar way, one of the DL-AMPs moves dorsally following ISN migration and the D-AMPs appear to extend toward the ISN. However, AMPs survival and behavior are not affected in the absence of motor axons, as shown in the prospero mutant, where motor axons fail to exit the CNS. To better characterize dynamics of AMP-motor axons interactions, the M6-GAL4; UAS-Life-actin GFP reporter line was used that allows in vivo visualization of both the motor axons and the AMPs. The M6-Gal4 and M6-gapGFP lines are both driven by the same regulatory elements; however, the expression in motor axons, which is low and difficult to distinguish in M6-gapGFP embryos, is enhanced by the GAL4/UAS system and is clearly present in the M6>lifeActGFP context. Live imaging revealed that, among the numerous oriented cytoplasmic projections sent out by the AMPs, those contacting the growth cones of motor axons became stabilized. In particular, stabilization of filopodial connections between L-AMPs and SNa coincided with the setting of the SNa branching point and specification of its lateral branch. Oriented filopodial dynamics were found in the dorsal region with the contact between D-AMP projections and ISN growth cone prior to ISN migration toward the D-AMP. As previously demonstrated, muscle founders are needed for terminal defasciculation of the main motor axon branches. In this context, AMP positioning and the fact that they actively engage with the navigating motor axons might also participate in this process by acting as spatial check-points that either induce and or attract targeted defasciculation of ISN and SNa (Lavergne, 2020).

To investigate the impact of L-AMPs on the SNa pathway and branching, the effect of a genetic ablation of the AMP cells was assessed using the M6-GAL4-driven expression of the pro-apoptotic gene reaper. This enabled targeted induction of apoptosis in AMPs, leading to AMP cell loss without strong defects in the ISN and SNa trajectory, despite the expression of M6-GAL4 in the motor neurons. This differential effect could be due to a lower expression level of M6-GAL4 in motor neurons than in AMPs, and/or a stronger resistance of neural cells to the Reaper-induced apoptosis. Importantly, in 86% of hemisegments, complete loss of L-AMPs was associated with absence of the lateral branch of SNa (L-SNa), strongly suggesting that L-AMPs play an instructive role in L-SNa formation and/or stabilization. In contrast, loss of L-SNa in hemisegments where the L-AMPs were still present occurred in 5.6% of analyzed hemisegments. The L-SNa loss in this context was thus higher than the one observed in the M6-GAL4 line with only 1.8% of hemisegments without L-SNa. To explain this difference, an effect of Reaper expression in the motor system cannot be excluded, but this could also be a consequence of early stages of apoptosis in L-AMPs. Thus, M6-GAL4-induced apoptosis created a context in which loss of the L-SNa branch was observed in L-AMP-devoid segments where the L-SNa target muscle (SBM) was still present. This suggests that L-SNa branch formation might not be dependent on its muscle target, and so prompted a test of whether the L-SNa would form or persist in an SBM-devoid context. reaper expression was targeted to the developing SBM using the SBM(lbl)-GAL4 driver. The SBM(lbl)-GAL4-driven apoptosis resulted in a systematic loss of the SBM and only sporadic loss of the L-AMPs (12% hemisegments). In the SBM-devoid context but with L-AMP cells correctly located, the L-SNa branch was still present (73% of hemisegments). Additionally, in a subset of SBM-deficient embryos, L-AMPs shifted toward the navigating SNa, leading to a shortened L-SNa (13% of the hemisegments). These observations thus suggest an instructive role for AMPs in L-SNa establishment, and reveal that SBM might not be needed for this process and is at least dispensable for its stabilization. To further test the role of L-AMPs in lateral defasciculation of the SNa, different genetic contexts were analyzed in which AMP specification is affected. First a perturbation of asymmetric cell divisions was induced. To adversely affect divisions of progenitor cells that give rise to AMPs, the asymmetry determinant Numb was ectopically expressed using the pan-mesodermal driver Twist-GAL4. In the lateral region, this led predominantly to the loss of one of the L-AMPs and a duplication of the SBM with no major impact on L-SNa formation compared with the control Twist-GAL4 line. However, in a small subset of hemisegments, loss of both L-AMPs but not SBM (often duplicated) was observed. In this rare context, the L-SNa was absent in 88% of hemisegments, supporting the view that L-AMPs are required for L-SNa branching. These findings are also consistent with the effects of generalized mesodermal expression of the identity gene Pox meso (Poxm), which can lead to a loss of L-AMPs without affecting SBM. In such a context, the L-SNa formation is impaired in 89% of L-AMP-devoid segments against 37% in random Twi>Poxm hemisegments. Interestingly, pan-mesodermal expression of Pox meso can also induce misplacement of L-AMPs along the SBM, leading to aberrant L-SNa trajectory. Hence, L-AMPs and their spatial positioning appear crucial to achieve the formation and correct pathfinding of the L-SNa (Lavergne, 2020).

The findings described above suggest that L-AMPs are a source of attractive signals that promote lateral sub-branching of the SNa, making it competent to innervate the SBM. Interestingly, the SBM is the only lateral muscle innervated by the Connectin-positive SNa, which does not express this homophilic target recognition molecule. In such a context, L-AMP-mediated lateral sub-branching of SNa offers a way to drive L-SNa to its specific muscle target. As L-AMPs seem not to express Connectin either, in contrast to previous suggestions, their role in attracting SNa and inducing the L-SNa sub-branching might rely on other guidance molecules (Lavergne, 2020).

It has been previously shown that the mutants of sidestep and beat-1a, which encode interacting membrane proteins of the immunoglobulin superfamily, displayed loss of L-SNa, a phenotype similar to the one observed when L-AMPs are missing. However, the mechanisms leading to the loss of the L-SNa in sidestep and beat-1a mutants have not been elucidated. In addition, the embryonic expression pattern of sidestep has been only partially characterized. By using in situ hybridization, this study found that sidestep mRNA is strongly enriched in all the AMPs, suggesting its potential involvement in the dialogue between AMPs and motor axons. It was therefore decided to test Sidestep protein distribution at the time when L-SNa sub-branching is taking place. By examining stage 14 to 15 embryos, a previously unreported faint and transient expression of Sidestep was found specifically in L-AMPs. To confirm this observation, the expression of Sidestep was analyzed in a mutant for beat-1a. It has been reportedthat the contact of Beat-1a-expressing motor axons with Sidestep-expressing cells leads to a negative regulation of the expression of sidestep. If this contact is missing, cells normally expressing sidestep transiently and at a low level will continue to do so, leading to continuous and higher Sidestep level in these cells. Analyses of beat-1a mutants confirmed that the L-AMPs are Sidestep-expressing cells and that Sidestep expression onset coincides with L-SNa sub-branching. The high Sidestep expression resulting from the lack of beat-1a was still detected in late-stage embryos in which it became gradually restricted to the most anterior L-AMPs. This late differential Sidestep expression may point to a leading role for the anterior L-AMPs in the process of interaction with SNa and in its lateral sub-branching. Additionally, an increased Sidestep expression in L-AMPs was also observed in SNa-devoid pros mutants and in the Duf-GAL4; UAS-NetrinB (NetB) context. Importantly, the SBM does not express Sidestep, making the L-AMPs the only Sidestep-expressing cells in the L-SNa pathway. Thus, this newly reported expression pattern suggests that L-AMPs could attract the L-SNa through the temporally and spatially restricted expression of sidestep (Lavergne, 2020).

Interestingly, the sidestep mutants also display a stall phenotype of the ISN suggestive of a potential role of the D-AMPs. Indeed, this study observed that the aberrantly located D-AMPs, after the mesodermal overexpression of the activated form of the Notch receptor (NICD), are able to attract the ISN, suggesting that they are a source of guiding signals. However, because only faint sidestep transcript expression was observed in D-AMPs and Sidestep protein was not detected, it is expected that other guiding cues may be in play. It is important to notice that to visualize the attractive potential of mis-positioned D-AMPs induced pan-mesodermal expression of NICD was observed via a GAL4/UAS system known to be thermo-sensitive. High mesodermal expression of NICD induced at 29°C leads to the loss of majority of muscles but, as is shown in this study, several muscles persist in Twi-GAL4;UAS-NICD embryos incubated at 25°C, thus allowing uncouple effects of delocalized D-AMPs from potential influence of muscles loss on ISN trajectories. However, loss of D-AMPs, observed in this study in a Poxm gain-of-function context, appears to have only a minor effect on the capacity of ISN motor axons to target dorsal muscles, which are correctly innervated by the ISN in 65% of hemisegments without D-AMP. These results highlight differential requirements of AMPs for motor axons defasciculation and navigation with L-AMPs being mandatory for L-SNa branching and D-AMPs acting as guiding cells for the ISN. However, the functional significance underlying the guidance of motor nerves by muscle stem cells remains to be determined (Lavergne, 2020).

It is also important to state that the loss of L-SNa in the sidestep mutants is not fully penetrant (observed in less than 10% of hemisegments), suggesting that sidestep is not the only player in L-SNa sub-branching. This could be due to functional redundancy between several members of Side and Beat families comprising 8 and 14 members, respectively. Expression and function of Side and Beat family members remain largely unexplored, but the fact that Sidestep labels L-AMPs and its paralog, side VI, is expressed in the DL-AMPs suggests there might be a 'Side expression code' that operates in AMPs and makes them competent to interact with navigating motor axons. In support of this hypothesis, this study found that side IV, another member of the Side family, is also expressed in AMPs with a higher transcript levels detected in L-AMPs, suggesting it could contribute to the interactions between L-AMPs and the SNa. To gain insight into AMP functions of sidestep and side IV in setting interactions with motor neurons, the selective AMP-targeting tools need to be developed to generate AMP-specific mutant rescue (Lavergne, 2020).

It has previously been shown that the nervous system is required for the establishment of the adult muscle pattern and that motor axons serve as a support for migration of AMPs during larval and pupal development. More recently, it has also been suggested that the nervous system could be involved in the selection of founder cells from the pool of AMPs. This study took advantage of a previously described genetic context (pan-muscular expression of NetB) to affect the SNa and tested impact of SNa loss on L-AMPs. In stage 16 DUF>NetB embryos, loss of the SNa observed in 84% of the hemisegments does not affect the number of L-AMPs. However, in surviving 3rd instar larvae in hemisegments lacking the SNa, number of L-AMPs is dramatically reduced. Interestingly, a specific depletion was observed of normally associated with SNa anterior L-AMPs (complete loss in 10 out of 26 hemisegments analyzed), while the posterior L-AMPs associated with the transverse nerve (TN) remained unaffected. Thus, this data provides evidence for a cross-talk between AMPs and motor axons, with the AMPs attracting navigating motor axons, which in turn are required for AMP maintenance during larval stages. The loss of anterior L-AMPs in SNa-devoid larva suggests that SNa-derived signals promote survival of associated L-AMPs, but precise underlying mechanisms remain to be elucidated (Lavergne, 2020).

Thus, in Drosophila, the dynamic interactions and close association between AMPs and the motor axon network contribute to setting ISN trajectory and are required for SNa sub-branching and proper innervation of lateral muscles, which is itself needed in larvae for the maintenance of anterior L-AMPs. In vertebrates, it has previously been described that muscle pioneers can impact motor axon pathfinding in zebrafish and more recently in mice that muscle stem cells activate and contribute to neuromuscular junction regeneration in response to denervation, and that depletion of muscle stem cells induced neuromuscular junction degeneration. This study, conducted in Drosophila, represents the first demonstration that, during development of neuromuscular system, muscle stem cells interact with motor neurons and contribute to proper muscle innervation (Lavergne, 2020).

Genetic screen in Drosophila muscle identifies autophagy-mediated T-tubule remodeling and a Rab2 role in autophagy

Transverse (T)-tubules make-up a specialized network of tubulated muscle cell membranes involved in excitation-contraction coupling for power of contraction. Little is known about how T-tubules maintain highly organized structures and contacts throughout the contractile system despite the ongoing muscle remodeling that occurs with muscle atrophy, damage and aging. This study uncovered an essential role for autophagy in T-tubule remodeling with genetic screens of a developmentally regulated remodeling program in Drosophila abdominal muscles. It was shown that autophagy is both upregulated with and required for progression through T-tubule disassembly stages. Along with known mediators of autophagosome-lysosome fusion, the screens uncover an unexpected shared role for Rab2 with a broadly conserved function in autophagic clearance. Rab2 localizes to autophagosomes and binds to HOPS complex members, (Jiang, 2014; Takáts, 2014) suggesting a direct role in autophagosome tethering/fusion. Together, the high membrane flux with muscle remodeling permits unprecedented analysis both of T-tubule dynamics and fundamental trafficking mechanisms (Fujita, 2017).

Differentiated muscle cells, or myofibers, are highly organized in order to coordinate the roles of specialized subcellular structures involved in contraction. Myofibril bundles of sarcomeres provide the contractile force. The power of contraction, however, requires synchronous sarcomere function under control of the 'excitation-contraction coupling' system that includes two membranous organelles, the sarcoplasmic reticulum (SR) and Transverse (T)-tubules (Al-Qusairi, 2011). The T-tubule membrane network is continuous with the muscle cell plasma membrane, with tubulated membranes that invaginate radially inward in a repeated pattern at each sarcomere. With excitation-contraction coupling, neuromuscular action potentials are transmitted along the muscle T-tubule membrane to the SR junction, or dyad/triad, triggering coordinated SR Ca2+ release and synchronous sarcomere contractions (Al-Qusairi, 2011). Formation of organized T-tubule membranes is thus critical for muscle function (Takeshima, 2015). Mechanisms must also remodel the T-tubule membrane network with ongoing myofiber reorganization in response to muscle use, damage, atrophy and aging. However, the extent and mechanisms of T-tubule remodeling remain largely unknown, in part due to challenges with observing T-tubule membrane network dynamics within intact mammalian myofibers (Fujita, 2017).

The T-tubule network includes both transversal and longitudinal tubular membrane elements that form and mature with myofiber differentiation and growth. In mouse skeletal muscle, mostly longitudinal tubular membranes initially present in embryonic muscle are remodeled postnatally with expansion to predominantly transversal tubular elements. In contrast, both longitudinal and transversal T-tubule elements are maintained in adult mammalian cardiac muscle and in insect muscles. Relatively few molecular factors are known to shape the T-tubule network, and perhaps not surprisingly, all of which so far encode for membrane-associated functions (CAV3, DYSF, BIN1/Amph2, MTM1, DNM2). Mutations in each also are associated with human myopathy and/or cardiomyopathy with T-tubule disorganization, pointing to the critical importance of membrane-mediated mechanisms to maintain the T-tubule membrane network (Fujita, 2017).

Drosophila is a powerful system for insights into the functional requirements for T-tubule formation and remodeling. The BIN1 BAR-domain protein has a conserved function involved in membrane tubulation required for T-tubule formation that was first described for the single Drosophila homolog, Amphiphysin. The amph null mutant flies lack transversal T-tubule element membranes in myofibers at all developmental stages, corresponding with both larval and adult mobility defects. In contrast, the myotubularin (mtm) fly homolog of mammalian MTM1/MTMR2/MTMR1 subfamily of phosphatidylinositol 3-phosphate phosphatases is required only at later stages in development for T-tubule remodeling. While mtm loss of function has no obvious effects on larval muscle T-tubule organization or function, mtm-depleted post-larval stage muscles lack transversal T-tubule membranes with adult mobility defects in eclosion and flight. Together, the amph and mtm mutant conditions that both lack transversal T-tubule elements in post-larval stage muscle yet different early development requirements underscores that distinct mechanisms are involved in T-tubule formation (amph-dependent) versus maintenance/remodeling (amph- and mtm-dependent) (Fujita, 2017).

In Drosophila, a set of larval body wall muscles that persist as viable pupal abdominal muscles, called dorsal internal oblique muscles (IOMs), are essential for adult eclosion. During metamorphosis, changes in IOM cell size and myofibril content have been noted. Previous studies have shown that wildtype IOMs undergo dramatic cortical and membrane remodeling with costamere integrin adhesion complex disassembly and reassembly at discrete pupal stages (Ribeiro, 2011). In contrast, the mtm-depleted IOMs exhibited persistent disassembly or a block in reassembly of integrin costameres along with the loss of transversal T-tubule membranes at late pupal stages, but without any precocious cell death (Ribeiro, 2011). A striking feature in the mtm-depleted IOMs was the accumulation of endosomal-like membranes decorated with integrin and T-tubule markers, Amph and Discs large (Dlg1, a PDZ protein). Altogether, these results suggest that T-tubule membranes may undergo disassembly-reassembly with normal myofiber remodeling, including the delivery of disassembled T-tubule membrane into an endomembrane trafficking pathway. The role for a molecular-cellular program in control of T-tubule remodeling that is at least partially distinct from that involved in initial T-tubule formation raises many questions about possible mechanisms, including the regulation of T-tubule organization and dynamics, the membrane fate(s) and source(s) with disassembly-reassembly, respectively, and the specific membrane trafficking routes and effectors involved. Possible hints may come from studies of other specialized dynamic cell membrane invaginations shown to involve endosomal and Golgi membrane trafficking pathways, such as cellularization of Drosophila syncytial embryos and the tubulated demarcation membrane system in megakaryocyte platelet formation (Fujita, 2017).

Membrane trafficking relies on the large family of Rab GTPases, with over sixty Rabs in humans and thirty in flies. The different Rabs are under distinct spatiotemporal regulation for recruitment, activation and functions at specific membrane compartments or domains. Guanine nucleotide exchange factors (GEFs) convert specific inactive GDP-bound Rabs to an active GTP-bound form. Active Rab-GTP then recruits a range of specific effector proteins to the membrane that mediate key trafficking functions, including cargo selection, vesicle budding, transport, tethering and fusion. Subsequently, GTPase-activating proteins (GAPs) deactivate Rabs by promoting GTP hydrolysis. Many membrane compartments have been defined by well-established localized functions of specific Rabs, for example: ER (Rab1), Golgi (Rab1, Rab6), secretory vesicles (Rab8), early endosomes (Rab5, Rab21), recycling endosomes (Rab11, Rab35), late endosomes (Rab7, Rab9), lysosomes (Rab7) and others. Thus, identifying the specific Rabs required for a cellular process can provide clues to potential underlying membrane trafficking mechanisms involved. However, examples exist of Rabs with multiple known sites of function or yet unknown functions, and conversely, certain cellular processes - like T-tubule remodeling - lack defined roles yet for any Rabs (Fujita, 2017).

This study utilized the advantages of Drosophila IOMs to screen for Rab GTPases and related membrane trafficking functions required for T-tubule remodeling in intact muscle. The results show that the entire contractile and excitation-contraction coupling system, including T-tubules, are disassembled and reassembled in IOMs during Drosophila metamorphosis. Autophagy, the membrane trafficking process for degradation of cytoplasmic contents by delivery to lysosomes, is upregulated with IOM remodeling where it plays an indispensable role for progression through T-tubule disassembly to reassembly. Genetic analysis of IOM remodeling also reveals an unexpected and broad role for Rab2 in autophagy in flies and mammals. From these data, it is proposed that Rab2 localizes to autophagosomes where it interacts with the HOPS complex, which in turn, mediates tethering and trans-SNARE complex formation with Rab7-marked lysosomes to promote autophagosome-lysosome fusion. Together, these results show that Drosophila IOM remodeling provides an unprecedented in vivo context for discovery and analysis of T-tubule dynamics with relevance to human myopathy, as well as an ideal system due to high membrane flux to study fundamental trafficking pathways (Fujita, 2017).

This study has characterized a wildtype myofiber remodeling program by confocal and electron microscopy in intact muscles in vivo. In Drosophila IOMs during metamorphosis, the entire contractile and excitation-contraction coupling system, including T-tubules, are disassembled and then reassembled. This process highlights that myofibers harbor distinct programs for initial T-tubule formation versus regulated T-tubule remodeling. This likely includes additional mechanisms for T-tubule membrane disassembly and renovation, features that reflect those seen with mammalian myofiber atrophy and recovery. The Drosophila body wall muscles provide an unprecedented system permitting a combination of powerful visualization and systematic perturbation analysis, including the first genetic screens, of T-tubule dynamics and organization (Fujita, 2017).

Autophagy is upregulated with the onset of IOM remodeling during metamorphosis. Further, disruption of autophagy initiation, autophagosome formation or clearance all induced loss of T-tubules with a block in IOM remodeling at/after T-tubule disassembly. This is the first report of a non-cell death role of autophagy in Drosophila metamorphosis. The role of autophagy in IOMs that persist and redifferentiate during metamorphosis is clearly different from its roles in pupal midgut and salivary gland cells that undergo autophagic forms of cell death. There are multiple speculative direct or indirect role(s) for autophagy specifically in T-tubule membrane remodeling: (1) a direct role in T-tubule membrane recycling, as a means to deliver disassembled T-tubule membrane via autophagosomes to lysosomes or related organelles for intracellular storage, then later redeployed to contribute to T-tubule reassembly; (2) an indirect role in cell renovation, including T-tubule membrane clearance, to permit cell space for redifferentiation; or (3) an indirect role in cell metabolism, to support cell survival and/or the energy cost of redifferentiation with starvation during metamorphosis. Most likely, autophagy serves some combination of these roles in IOM remodeling (Fujita, 2017).

How could autophagy play a direct role in T-tubule remodeling? It was surprising that mCD8:GFP-positive small vesicles accumulated to a similar degree as autophagosome numbers in IOMs when autophagosome-lysosome fusion was blocked. This suggests that mCD8:GFP localizes to autophagosomes during IOM remodeling. It is possible that T-tubule membranes are a source of autophagosomal membrane, at least in part: mCD8:GFP labels the muscle plasma membrane and T-tubules in larval muscle precursor cells of IOMs, and T-tubule disassembly coincides with the upregulation in autophagy early in metamorphosis. Also, disruption of autophagy induction blocked normal progression in disassembly and remodeling of T-tubule-derived mCD8:GFP-marked membranes. In the absence of autophagy initiation, mCD8:GFP-positive stacked membranes were observed, likely retained or partially disassembled T-tubules. It is proposed that T-tubules are remodeled through autophagosomes. It is important to note that T-tubules are not an apparent autophagic cargo, but instead, a possible source of autophagosome membrane. In this scenario, T-tubules are disassembled into autophagosomes and then reassembled from subsequent autolysosome-related structures, both of which successively increased in numbers during wildtype IOM remodeling (Fujita, 2017).

Alternatively or additionally, other roles for autophagy could indirectly impact T-tubule remodeling. Extensive IOM atrophy with nearly complete disassembly of the contractile and excitation-contraction systems by 1d APF is followed by a rapid re-differentiation within hours after 3.5d APF. Autophagy could be required to simply clear away and degrade the old contraction systems in order to make space to rebuild and realign new systems, as well as permit the normal central repositioning of nuclei away from the myofiber cortex. However, the persistent block in early IOM remodeling with autophagy disruption suggests that the remodeling normally proceeds through a progression of interrelated steps rather than independent programs for disassembly and reassembly. Autophagy also has a well-established role in metabolic homeostasis through the recycling of amino acids and turnover of damaged mitochondria in the lysosome. The current data suggest that mitochondria are a major autophagic cargo with IOM remodeling. In conditions that disrupted autophagy initiation (Atg1, Atg18 RNAi), the cytoplasm was abnormally filled with mitochondria in IOMs at 4d APF. Consistent with that, a significant portion of autophagosomes harbored intact mitochondria when autophagosome-lysosome fusion was blocked (Rab2, Rab7 or Stx17 RNAi). This is different from observations in larval muscle, in which mitochondria were notably absent in autophagosomes that accumulated with a block in autophagy. It is possible that mitophagy, a selective form of autophagy for mitochondrial turnover, is upregulated and could play both metabolic and cell renovation roles in IOM remodeling. Interestingly, the autophagy-blocked IOMs remained viable throughout metamorphosis, suggesting that autophagy is not absolutely required for cell survival through the starvation with metamorphosis (Fujita, 2017).

Through a systemic screen of all Drosophila Rab GTPases, an unexpected role was uncovered for Rab2 in autophagy. The striking Rab2 RNAi IOM phenotype was shared with RNAi of other functions known to be specifically required for autophagosome-lysosome fusion. Genetic blockade of autophagosome-lysosome fusion resulted in a dramatic phenotype, with massive accumulations of autophagosomes within IOMs. Previously, autophagosome-lysosome fusion was shown to involve the cooperative functions of Rab7, the HOPS tethering complex, and a trans-SNARE complex between Stx17, SNAP29 and VAMP7/8. Among these tethering and fusion functions, it has been shown that Stx17 (a hairpin SNARE) is recruited to autophagosomal membranes, while Rab7 and VAMP7/8 localize to endolysosomal membranes. Stx17 localizes to autophagosomes as well as to the ER and mitochondria, but the HOPS complex directly associates and colocalizes with Stx17 only at autophagosomes (Jiang et al., 2014; Takáts et al., 2014). This suggests that Stx17 is not a sole determinant for HOPS complex recruitment (Fujita, 2017).

It is proposed that Rab2 is required for the autophagosomal recruitment of the HOPS complex. Rab2 specifically localized to completed autophagosomes, and Rab2 had an affinity with the HOPS complex, as does Stx17. It is envisioned that upon completion of autophagosome biogenesis/maturation, Rab2 and Stx17 are recruited to the outer autophagosomal membrane. Then, the HOPS complex is subsequently recruited to autophagosomes in a Rab2-depedent manner through coincident interactions with both Stx17 and Rab2 (see Hierarchal analysis of Rab2 and factors involved in autophagosome-lysosome fusion). At the same time, the HOPS complex binds Rab7 on lysosomes. In turn, the HOPS complex tethers autophagosomes and lysosomes to promote trans-SNARE complex formation between Stx17, SNAP29 and Vamp7/8 and ultimately autophagosome-lysosome fusion (Fujita, 2017).

Rab2 role in autophagy discovered in fly muscle relates to a broader autophagy requirement in other cell types and across species. The localization of Rab2 on autophagosomes in Drosophila IOMs was conserved for both Rab2A and Rab2B in mouse embryonic fibroblasts (MEFs). As in flies, the Rab2A/2B double knockout led to a delay or block in autophagy clearance as indicated by accumulation of LC3/Atg8. However, the specific Rab2 loss-of-function phenotypes were not identical. While Rab2 was required for autophagosome-lysosome fusion in fly IOMs, the Rab2A/2B double knockouts in MEFs indicated a requirement at a later step in autophagic clearance. Interestingly, this disparity in autophagy phenotypes across species is also seen with Rab7. In flies and yeast, Rab7/Ypt7 is essential for autophagosome-lyososome/vacuole fusion, while mammalian Rab7 knockdowns more clearly indicate a required role in autolysosome maturation. Other examples indicate that the autophagosome-lysosome fusion machinery is not highly evolutionarily conserved. The Stx17-SNAP29-VAMP7/8 trans-SNARE complex is conserved in Drosophila and mammals, but not in yeast, where no autophagosomal SNARE has been reported so far. Moreover, budding yeast do not encode for Rab2 (Fujita, 2017).

Altogether, it is plausible that Rab2 is required for autophagosome-lysosome fusion efficiency, and Rab2-dependency is variable across different tissues or species. Two possible models could explain the different Rab2 autophagy requirements in flies and mouse cells. First, it is suggested that autophagosomes sequentially fuse with endosomes then lysosomes to become amphisomes and autolysosomes, respectively. If either of the steps requires Rab2A/2B, then intermediates with partially degraded contents could accumulate in double knockout MEFs. Alternatively, an autophagosome may normally fuse with multiple lysosomes to ensure full degradation of its contents. In the absence of Rab2A/2B in MEFs, autophagosomes could still fuse but not with a sufficient number of lysosomes, resulting in an accumulation of partially digested autolysosomes (Fujita, 2017).

Rab2 has been previously associated with transport events at the Golgi apparatus, ER-to-Golgi traffic and secretory granule formation, as well as in a C. elegans endocytic/phagocytic pathway. Gillingham et al. systematically explored Rab effectors in Drosophila cultured cells, and found that Rab2 interacts with the HOPS complex besides known Golgi-resident effectors (Gillingham, 2014). The interaction between Rab2 and HOPS complex is also conserved in mammals, and the unexpected Rab2 localization to autophagosomes was found. Thus, it is likely that Rab2 exerts multiple functions through interaction with different effectors at different places. A possible Rab2 function in the endosome-lysosome system that affects autophagic flux cannot be excluded, although no clear lysosomal defects were detected in Rab2A/B knockout MEFs. Several other factors that localize to autophagosomes or late endosomes-lysosomes, including Atg14, PLEKHM1 and EPG5, have been shown to control autophagosome maturation. It is plausible that Rab2 contributes to autophagosome maturation through both a direct role in the fusion mechanism and an indirect role in endo-lysosome maturation, the same as Rab7 and the HOPS complex (Fujita, 2017).

How Rab2 localizes to autophagosomes remains unclear. Localization of Rab2 on autophagosomes in IOMs did not depend on HOPS complex subunits, Vps39 and Vps41, or on Stx17. Further studies will be needed to determine the identities of the Rab2 guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP) that regulate Rab2 GTPase activity in autophagosome-lysosome fusion. A conserved TBC domain protein, OATL1/TBC1D25, is a strong candidate for a Rab2 GAP, given OATL1 localization to autophagosomes and involvement in autophagosome-lysosome fusion. Further, it was reported that OATL1 directly bound to and showed GAP activity for Rab2A (Fujita, 2017).

Autophagy is critical for the maintenance of myofiber homeostasis in mammalian skeletal muscle. It is known that several myopathies are associated with excess accumulation of autophagic structures in muscle. Further, loss of autophagy in mouse skeletal muscle shows anomalies, including abnormal mitochondria, disassembled sarcomeres and disorganized triads, as also seen in aged muscle. It is established that autophagy is down-regulated during the course of aging. This evidence points to a possible significance of autophagy in myofiber remodeling and in T-tubule maintenance. Jumpy/MTMR14 PI3-phosphatase and Dynamin-2 (DNM2) GTPase, two causative genes of human centronuclear myopathy, are required for not only T-tubule maintenance but also proper progression of autophagy. Based on these reports and the current findings, it is speculated that their roles in T-tubule maintenance are mediated, at least in part, through autophagy (Fujita, 2017).

Signaling pathways that regulate atrophy and hypertrophy in Drosophila have been identified, however, the mechanisms and direct mediators of muscle remodeling remain largely unknown. IOM remodeling is a good model to study the mechanisms of muscle remodeling, given that the signaling pathways that control muscle remodeling are conserved between Drosophila and mammals. Advantages of the IOM system are not only its genetic tractability, but also its reproducibility and structure. As a relatively giant single cell along the body wall, IOMs enable tracking of a single cell and its subcellular organization during metamorphosis. The results show that studies in IOMs can provide new insights into the mechanisms of muscle remodeling as well as regulation of fundamental membrane trafficking pathways, such as autophagy and endocytosis (Fujita, 2017).

The Drosophila formin Fhos is a primary mediator of sarcomeric thin-filament array assembly

Actin-based thin filament arrays constitute a fundamental core component of muscle sarcomeres. This study used formation of the Drosophila indirect flight musculature for studying the assembly and maturation of thin-filament arrays in a skeletal muscle model system. Employing GFP-tagged actin monomer incorporation, several distinct phases in the dynamic construction of thin-filament arrays were identified. This sequence includes assembly of nascent arrays after an initial period of intensive microfilament synthesis, followed by array elongation, primarily from filament pointed-ends, radial growth of the arrays via recruitment of peripheral filaments and continuous barbed-end turnover. Using genetic approaches, the single Drosophila homolog of the FHOD sub-family of formins, Fhos, was identified as a primary and versatile mediator of IFM thin-filament organization. Localization of Fhos to the barbed-ends of the arrays, achieved via a novel N-terminal domain, appears to be a critical aspect of its sarcomeric roles (Shwartz, 2016).

Spatial pattern analysis of nuclear migration in remodelled muscles during Drosophila metamorphosis

During metamorphosis in Drosophila, multi-nucleated larval dorsal abdominal muscles either undergo cell death or are remodeled to temporary adult muscles. Muscle remodeling is associated with anti-polar nuclear migration and atrophy during early pupation followed by polar migration and muscle growth during late pupation. Loss of Cathepsin-L has been shown to inhibit anti-polar movements, while knockdown of autophagy-related genes affected nuclear positioning along the medial axis in late metamorphosis. This study has developed new descriptors of myonuclear distribution. Nuclear tracks were used to distinguish between fast moving nuclei associated with fragments of dead muscles (sarcolytes) and slow-moving nuclei inside remodelled muscles. Anti-polar migration leads to a longitudinal nuclear spread decrease. Unexpectedly, the decrease in longitudinal nuclear spread was significantly enhanced by Atg9, Atg5 and Atg18 silencing, indicating that the loss of autophagy promotes the migration and clustering of nuclei. In vivo imaging and quantitative image analysis of Drosophila metamorphosis promise to provide novel insights into the relationship between muscle wasting and myonuclear positioning (Kuleesha, 2017).

Identification of the essential protein domains for Mib2 function during the development of the Drosophila larval musculature and adult flight muscles

The proper differentiation and maintenance of myofibers is fundamental to a functional musculature. Disruption of numerous mostly structural factors leads to perturbations of these processes. Among the limited number of known regulatory factors for these processes is Mind bomb2 (Mib2), a muscle-associated E3 ubiquitin ligase, which was previously established to be required for maintaining the integrity of larval muscles. This study has examined the mechanistic aspects of Mib2 function by performing a detailed functional dissection of the Mib2 protein. The ankyrin repeats, in its entirety, and the hitherto uncharacterized Mib-specific domains (MIB), are shown to be important for the major function of Mib2 in skeletal and visceral muscles in the Drosophila embryo. Furthermore, novel mib2 alleles were identified that have arisen from a forward genetic screen aimed at identifying regulators of myogenesis. Two of these alleles are viable, but flightless hypomorphic mib2 mutants, and harbor missense mutations in the MIB domain and RING finger, respectively. Functional analysis of these new alleles, including in vivo imaging, demonstrates that Mib2 plays an additional important role in the development of adult thorax muscles, particularly in maintaining the larval templates for the dorsal longitudinal indirect flight muscles during metamorphosis (Domsch, 2017).

Cell death regulates muscle fiber number

Cell death can have both cell autonomous and non-autonomous roles in normal development. Previous studies have shown that the central cell death regulators grim and reaper are required for the developmentally important elimination of stem cells and neurons in the developing central nervous system (CNS). This study shows that cell death in the nervous system is also required for normal muscle development. In the absence of grim and reaper, there is an increase in the number of fibers in the ventral abdominal muscles in the Drosophila adult. This phenotype can be partially recapitulated by inhibition of cell death specifically in the CNS, indicating a non-autonomous role for neuronal death in limiting muscle fiber number. It was also shown that FGFs produced in the cell death defective nervous system are required for the increase in muscle fiber number. Cell death in the muscle lineage during pupal stages also plays a role in specifying fiber number. Altogetger, data suggests that FGFs from the CNS act as a survival signal for muscle FCs. Thus, proper muscle fiber specification requires cell death in both the nervous system and in the developing muscle itself (Sarkissian, 2016).

A comprehensive anatomical map of the peripheral octopaminergic/tyraminergic system of Drosophila melanogaster

The modulation of an animal's behavior through external sensory stimuli, previous experience and its internal state is crucial to survive in a constantly changing environment. In most insects, octopamine (OA) and its precursor tyramine (TA) modulate a variety of physiological processes and behaviors by shifting the organism from a relaxed or dormant condition to a responsive, excited and alerted state. Even though OA/TA neurons of the central brain are described on single cell level in Drosophila melanogaster, the periphery was largely omitted from anatomical studies. Given that OA/TA is involved in behaviors like feeding, flying and locomotion, which highly depend on a variety of peripheral organs, it is necessary to study the peripheral connections of these neurons to get a complete picture of the OA/TA circuitry. This study describes the anatomy of this aminergic system in relation to peripheral tissues of the entire fly. OA/TA neurons arborize onto skeletal muscles all over the body and innervate reproductive organs, the heart, the corpora allata, and sensory organs in the antennae, legs, wings and halteres underlining their relevance in modulating complex behaviors (Pauls, 2018).

The adrenergic system of mammals influences various aspects of the animal's life. Its transmitters/hormones, adrenaline and noradrenaline, modulate a variety of physiological processes and behaviors. They are secreted into the bloodstream by the adrenal glands in response to stress. In addition, they are synthesized and released by axonal terminals in the central nervous system (CNS) as well as sympathetic fibers of the autonomic nervous system. Adrenaline and noradrenaline have been described as modulators to shift the organism from a relaxed or dormant state to a responsive, excited and alerted state. Stressful stimuli induce a metabolic and behavioral adaptation, leading to enhanced energy supply, increased muscle performance, increased sensory perception and a matched behavior. This so-called 'fight or flight' response can be seen in vertebrates and invertebrates. In insects, the stress response is mediated -- among others -- by octopamine (OA) and its precursor tyramine (TA). TA is synthesized from tyrosine by the action of a tyrosine decarboxylase enzyme (Tdc) and functions as an independent neurotransmitter/-modulator as well as the intermediate step in OA synthesis. For this, TA is catalyzed by the tyramine-β-hydroxylase (TΒH). Similar to the vertebrate adrenergic system, OA and TA act through specific G-protein coupled receptors. Besides structural similarities between OA/TA and adrenaline/noradrenaline and the corresponding receptors, functional similarities are illustrated by the action of these transmitters/hormones in the regulation of physiological processes and behaviors. OA and TA are known to modulate muscle performance, glycogenolysis, fat metabolism, heart rate, and respiration in insects (Pauls, 2018).

While the role of TA as an independent signaling molecule was underestimated for a long time, OA has been extensively studied and was shown to have effects on almost every organ, sensory modality and behavior in a great variety of insects. The most intensively studied peripheral organs regarding the modulatory role of OA are muscles. Here, OA is thought to not exclusively modulate muscle performance or motor activity. OA rather modulates muscle action according to metabolic and physiological processes, for example by promoting energy mobilization directly from the fat body, or indirectly by promoting the release of adipokinetic homones (AKH) from neuroendocrine cells in the corpora cardiaca (CC, a homolog of the vertebrate anterior pituitary gland and an analog of mammalian pancreatic alpha cells). In addition to the impact of OA/TA on muscles, fat body and AKH cells, OA is shown to modulate the heart, trachea and air sacs, gut, hemocytes, salivary glands, Malpighian tubules and ovaries in insects, mainly to induce a general stress or arousal state. However, in total OA seems to modulate a vast number of behaviors, which are not necessarily coupled to stress responses. The OA/TA system is shown to also act on inter alia (i.a.) learning and memory, sleep, feeding, flight, locomotion, and aggression (Pauls, 2018).

As mentioned above, OA and TA act as neurotransmitters and neuromodulators, allowing them to act in a paracrine, endocrine or autocrine fashion. In the fruit fly Drosophila, huge efforts were made to describe OA/TA neurons (OANs/TANs) in the brain and ventral nervous system (VNS) down to the single cell level. Nevertheless, although knowledge about physiological processes and behaviors modulated by the OA/TA system in the brain is rich, less is known about how OA and TA reach all its target organs and tissues in the periphery (exceptions: reproductive organs and muscles) (Pauls, 2018).

This study used the genetically tractable fruit fly Drosophila melanogaster to describe the arborizations of Tdc2-Gal4-positive, and therefore OANs and TANs in the periphery, as the Drosophila Tdc2 gene is expressed neurally. OANs/TANs were found to be widespread distributed throughout the fly's body with innervations in the skeletal muscles, reproductive organs, corpora allata, antennae, legs, wings, halteres and the heart. This diverse innervation pattern reflects the modulatory role of OA/TA in many different behaviors and physiological processes. These results provide, for the very first time, a complete and comprehensive map of the OA/TA circuitry in the entire insect body. This map allows assumptions about the type of OA/TA signaling (paracrine or endocrine) to a specific organ and, at the same time, it provides a deeper understanding to what extend the OA/TA-dependent activity of peripheral organs is altered, for example by genetically manipulating Tdc2-Gal4-positive neurons in the brain and VNS (Pauls, 2018).

This study used the Tdc2-Gal4 line, allowing Gal4 expression under the control of a regulatory sequence of the tyrosine decarboxylase enzyme. As this enzyme is essential for the synthesis of TA from tyrosine, the Tdc2-Gal4-line labels both TANs and OANs. Within the Drosophila brain, Tdc2-Gal4 labels in total about 137 cells, while additional 39 cells are located in the VNS. The small number of Tdc2Ns lead to arborizations in large parts of the central brain, optic lobes and the thoracic and abdominal ganglion. Based on the profound innervation of Tdc2Ns in the brain and VNS, the variety of behaviors modulated by the OA/TA system including learning and memory, feeding, vision, and sleep, are not surprising. Beyond the brain and VNS, OANs and TANs massively innervate regions within the periphery of the fly. This study described arborizations on most skeletal muscles, the antennae, wings, halteres and reproductive system and parts of the circulatory system and stomodaeal ganglion (Pauls, 2018).

The findings are in line with previous reports focusing on the expression of different OA and TA receptors in the fly. Accordingly, the OA receptor OAMB is expressed in reproductive organs (in both male and female flies) and muscles, which are directly innervated by Tdc2Ns. Additionally, the midgut and trachea contain OA and TA receptors, but do not seem to be innervated by Tdc2Ns, even though axons run in close vicinity to these organs. Likewise, the OA receptor Octβ2R is expressed in the fat body, salivary glands and Malpighian tubules, tissues that seem not to be innervated by Tdc2Ns, while the expression of Octβ1R and Octβ3R is more specific56,57. The three tyramine receptors TyrR, TyrRII and TyrRIII show a broad expression in the periphery, also in tissues not innervated by Tdc2Ns56. The lack of direct innervation of these peripheral tissues might argue for volume transmission or hemolymph released OA/TA from Tdc2Ns. Alternatively, Drosophila TDC1, the product of the non-neurally expressed Tdc gene, is expressed in the gut musculature, rectal papillae, Malpighian tubules and two small clusters in the thoracic nervous system and might be the source for peripheral TA. Interestingly, TyrR seems to be the only receptor expressed in the heart, suggesting that only TA modulates heart function. Contrary, OA has a modulatory effect on the heart of other insect species including honeybees, olive fruit flies and cockroaches. This is also in line with a previous report providing evidence that OA modulates the heart rate of the Drosophila fly and pupa, but not the larva (Pauls, 2018).

OA-dependent modulation of organs and tissues is mainly elicited through muscle action, especially in terms of its impact on the 'fight or flight' response. In line with this, it was observed Tdc2-Gal4-positive arborizations on nearly all skeletal muscles and many visceral muscles. In both Drosophila and desert locusts OA and TA is expressed in type II terminals of skeletal muscles50. OA has an excitatory effect on Drosophila flight muscles, while TA was shown to inhibit excitatory junction potentials, and thereby reduce muscle contractions and locomotion at least in the larva. In addition, flies lacking OA show severe deficits in flight initiation and maintenance. Interestingly, in an antagonistic effect to serotonin, OA reduces crop muscle activity presumably via Octβ1R, suggesting that OA has different effects on muscle activity dependent on the type of muscle. However, the data do not provide any convincing evidence of a direct innervation of Tdc2Ns of the crop, even though many fibers run in close vicinity, suggesting that OA might target the crop by volume transmission (Pauls, 2018).

Furthermore, OA modulates ovulation and fertilization in insects. Flies lacking OA display a severe egg-laying phenotype. Remarkably, within the female reproductive organ two different OA receptors, OAMB and Octβ2R, are necessary. Again, OA has a strong impact on muscle activity within the reproductive system. Octβ2R is expressed in the visceral oviduct muscle and elicits muscle relaxation through an increase of intracellular cAMP levels. Such an OA-dependent modulation appears to be conserved as OA is found in dorsal unpaired median neurons of locusts innervating oviduct muscles through the oviducal nerve. However, the data suggest that OA-positive fibers not only innervate oviduct muscles, but also enter the organs themselves. The OAMB receptor is expressed in epithelial cells inducing fluid secretion through increasing intracellular Ca2+ levels. Thus, OA affects different processes within the female reproductive organ due to the expression of different receptors and their coupled signaling pathways, which may be a general mechanism of the OA/TA system to fulfill an extensive modulatory function (Pauls, 2018).

OA does not exclusively modulate muscle activity, but also sensory neurons of external tissues like the antennae, halteres and wings. OA has also been shown to increase the spontaneous activity of olfactory receptor neurons (ORN). The modulation of ORNs allows OA to modulate the innate response to attractive stimuli like fruit odors or pheromones. Further, this modulation helps nestmate recognition in ants. In addition to Tdc2-Gal4-positive arborizations in the funiculus, Tdc2-Gal4-positive sensory neurons were found in the JO, a chordotonal organ sensitive to mechanosensory stimuli and thus important for hearing in insects. In mosquitos, OA modulates auditory frequency tuning and thereby affects mating behavior. In locusts, OA similarly modulates the response of chordotonal neurons in the legs to encode proprioceptive information. These data suggest that chordotonal neurons in the legs, wings, halteres and thorax are included in the Tdc2-Gal4 line suggesting a conserved modulatory role of OA/TA for insect proprioception (Pauls, 2018).

Taken together, this study suggests that the OA/TA system massively modulates various organs and tissues in the periphery of Drosophila. Through distinct receptors and coupled signaling pathways OANs/TANs mainly induce 'fight or flight' responses by modulating muscle activity, proprioception, and heart rate. As a result, the innervation pattern in the periphery supports the idea that the OA/TA system is crucial for insects to switch from a dormant to an excited state, by a positive modulation of muscle activity, heart rate and energy supply, and a simultaneous negative modulation of physiological processes like sleep (Pauls, 2018).

Myofibril diameter is set by a finely tuned mechanism of protein oligomerization in Drosophila

Myofibrils are huge cytoskeletal assemblies embedded in the cytosol of muscle cells. They consist of arrays of sarcomeres, the smallest contractile unit of muscles. Within a muscle type, myofibril diameter is highly invariant and contributes to its physiological properties, yet little is known about the underlying mechanisms setting myofibril diameter. This study shows that the PDZ and LIM domain protein Zasp, a structural component of Z-discs, mediates Z-disc and thereby myofibril growth through protein oligomerization. Oligomerization is induced by an interaction of its ZM domain with LIM domains. Oligomerization is terminated upon upregulation of shorter Zasp isoforms which lack LIM domains at later developmental stages. The balance between these two isoforms, which are call growing and blocking isoforms sets the stereotyped diameter of myofibrils. If blocking isoforms dominate, myofibrils become smaller. If growing isoforms dominate, myofibrils and Z-discs enlarge, eventually resulting in large pathological aggregates that disrupt muscle function (Gonzalez-Morales, 2019).

Inside cells, proteins are assembled into complex functional units. The correct assembly of these units is crucial for their function. Myofibrils are highly organized assemblies of cytoskeletal proteins forming an array of sarcomeres that are embedded in the cytosol of myotubes and mediate contractility. Sarcomeres are composed of actin-containing thin filaments and myosin-containing thick filaments arranged into antiparallel cables. Thin filaments are anchored to a large multiprotein complex called the Z-disc, and thick filaments are anchored to another large multiprotein complex called the M-line. Anchoring of myofibrils to the exoskeleton provides the mechanical tension that aligns sarcomeres into myofibrils and coordinates their development. Once aligned, sarcomeres grow to their final size. Electron and confocal microscopy studies showed that sarcomeres form initially from small structures called Z-bodies that grow eventually into mature Z-discs to which thin filaments are anchored. The size of the Z-disc therefore sets the diameter of the myofibril. While mechanisms have been proposed that set the length of sarcomeres, Z-disc growth and growth termination is poorly understood (Gonzalez-Morales, 2019b).

A hallmark of genetically caused myopathies is the appearance of large aggregates composed mainly of Z-disc proteins. Interestingly, many myopathy-associated mutations encode Z-disc proteins. Mutations in any of the four α-actinin genes or in Zasp and other Alp/Enigma family genes in humans cause myopathies characterized by the presence of large aggregates. The aggregation phenotype is conserved among animals: fruit flies with mutations in myopathy-related genes also develop Z-disc aggregates (Gonzalez-Morales, 2019b).

α-Actinin and Z-disc Alternatively Spliced Protein (Zasp) are conserved proteins that coordinate Z-disc formation. α-Actinin forms a rod-shaped antiparallel homodimer at the Z-disc, where it crosslinks and serves as an attachment point for actin filaments. Zasp and other members of the Alp/Enigma family of proteins are scaffolding proteins with an α-actinin-binding PDZ domain, an uncharacterized Zasp Motif (ZM; InterPro: IPR031847 and IPR006643) domain, and one to four protein-protein interaction LIM domains (InterPro: IPR001781). Zasp and α-actinin proteins are present at the earliest stages of Z-disc formation, and are required for Z-disc assembly (Gonzalez-Morales, 2019b).

Vertebrates have seven Alp/Enigma genes, each encoding several isoforms. The Drosophila genome has three Zasp genes, Zasp52, Zasp66, and Zasp67, which encode 21, 12, and 4 isoforms, respectively. Zasp66 and Zasp67 are duplications of Zasp52 and resemble the smallest isoforms of Zasp52 (González-Morales, 2019a). The number of isoform variants and genes adds an additional layer of complexity and regulation to sarcomere formation (Gonzalez-Morales, 2019b).

Drosophila indirect flight muscles wee used, and it was asked if the mechanism that controls Z-disc size relates to the pathological aggregation behavior known for Z-disc-related myopathies. This study shows that accumulation of multivalent Zasp growing isoforms (with multiple LIM domains) causes Z-disc growth, whereas upregulation of monovalent blocking isoforms at later developmental time points terminates Z-disc growth. An imbalance of growing and blocking Zasp isoforms results either in aggregate formation, enlarged Z-disc size or reduced Z-disc size. It is proposed that this mechanism has wide implications for diseases caused by aggregate formation (Gonzalez-Morales, 2019b).

These findings indicate that Z-disc formation and growth is driven by multivalent oligomerization of Zasp proteins with multiple LIM domains, and eventually terminated at the proper Z-disc size by the upregulation of blocking isoforms without LIM domains. The process of Z body and Z-disc formation is reminiscent of membraneless organelles with compositions distinct from the surrounding cytosol, which form through a mechanism of phase separation (e.g. Cajal bodies or P bodies) (Boeynaems, 2018; Weber, 2017). Both have sharp boundaries between themselves and the cytoplasm, they form and organize as discrete puncta in the cytosol, and multivalent protein domains are often involved in their formation (Gonzalez-Morales, 2019b).

Sarcomere size is stereotyped in a given muscle type but distinct among different muscles. How can the current model explain differences in sarcomere sizes? The sarcomere grows, while the Zasp growing isoforms dominate the Zasp isoform pool. Different sarcomere sizes can be achieved in two ways. First, the sarcomere growth period (i.e., the window of time in which Zasp growing isoforms dominate) might vary among muscle types. Second, the speed at which new Zasp molecules are recruited to the Z-disc, might be different among muscle types, while the growth period remains constant. Given the diversity of muscle types and therefore sarcomere sizes that exist, it is likely that a combination of these two strategies occur simultaneously (Gonzalez-Morales, 2019b).

Finally, apart from the ZM-LIM mechanism described in this study, additional redundant mechanisms to control Z-disc growth might exist, as evidenced by the observation that Zasp52-PR overexpression makes big Z-discs and aggregates, while the mutant removing Zasp52 LIM domains reduces Z-disc size to a comparatively small degree. Redundant mechanisms might operate through other LIM domain proteins, or the coordination of Z-disc and M-line growth, all of which may provide important buffering functions to ensure proper myofibril size, which is crucial for fully functional muscles (Gonzalez-Morales, 2019b).

The ZM domain is a conserved domain without a clearly defined function. On its own, the ZM domain from two mouse Zasp proteins localizes to the Z-disc (Klaavuniemi, 2004; Klaavuniemi, 2006). The data suggest that Z-disc localization is a conserved feature of ZM domains from vertebrates to insects. ZM-containing proteins are tethered to the Z-disc by the physical interaction with the LIM domains of other Zasp proteins. In sum, the LIM domain serves as a recruitment signal for Zasp proteins and potentially other unidentified ZM-containing proteins to join the Z-disc. In addition, given the appearance of the ZM domain in bilateral animals with canonical Z-discs, it is postulated that a conserved mechanism involving LIM-ZM binding underlies Z-disc growth and growth termination (Gonzalez-Morales, 2019b).

In vertebrates, the Zasp proteins are very diverse and are better known as Alp/Enigma family: ZASP/Cypher/Oracle/LDB3/PDLIM6, ENH/PDLIM5, PDLIM7/ENIGMA/LMP-1, CLP36/PDLIM1/Elfin/hCLIM1, PDLIM2/Mystique/SLIM, ALP/PDLIM3, and RIL/PDLIM4. The ZM/DUF4749 motif occurs in ZASP, CLP36, PDLIM2, ALP and RIL. The LIM domain occurs in all Zasp proteins, either as one domain in Alp family members or as three domains in Enigma family members. Two Zasp genes were identified that encode only blocking isoforms in fruit flies: Zasp66 and Zasp67, and one gene, Zasp52, that encodes blocking and growing isoforms. Although Zasp66 and Zasp67 genes are insect-specific (Gonzalez-Morales, 2019a), vertebrate Alp/Enigma genes also express isoforms without LIM domains that could fulfill a blocking isoform function. In addition, because Zasp52-PK, which only contains one LIM domain, behaves as a blocking isoform, the Alp members with only one LIM domain might also behave as blocking isoforms (Gonzalez-Morales, 2019b).

The function of the growing isoforms of Zasp requires multiple functional LIM domains. As the Enigma family members contain three C-terminal LIM domains, they are the ideal candidates to fulfill the growing role in vertebrates. Three Enigma proteins exist in vertebrates: PDLIM7/Enigma/LMP-1, ENH/PDLIM5 and ZASP/Cypher/Oracle/LDB3/PDLIM6. Functional redundancy between them at the Z-disc is likely common and demonstrated in one case. In Cypher knockout mice sarcomere assembly occurs normally during development, followed by immediate sarcomere failure after postnatal onset of contractility. ENH mutants exhibit cardiac dilation and abnormal Z-disc structure in the heart. Intriguingly, in both Cypher and ENH single mutants, as well as Cypher ENH double mutants, sarcomeres look considerably smaller in diameter in electron microscopy images. Thus, a similar role for Enigma proteins in setting sarcomere diameter in vertebrates appears likely (Gonzalez-Morales, 2019b).

Is the mechanism that controls Z-disc size related to the protein aggregation defects in human myopathies? The Z-disc oligomerization hypothesis agrees well with the observation that many myopathies present aggregates, and several human ZASP mutations have been linked to aggregate-forming myopathies. Many ZASP mutations linked to disease lie within the ZM domain or one of the LIM domains. Protein aggregation in myopathy patients might be a consequence of an imbalance in the mechanism that controls sarcomere size, favoring the growing over the blocking isoforms. If this were the case, the data points to a potential therapeutic avenue: blocking the growing isoforms with short peptides containing a ZM domain (Gonzalez-Morales, 2019b).

In conclusion, it is proposed that a conserved mechanism involving LIM-ZM binding underlies Z-disc growth and therefore myofibril diameter (Gonzalez-Morales, 2019b).

Reverse engineering forces responsible for dynamic clustering and spreading of multiple nuclei in developing muscle cells

How cells position organelles is a fundamental biological question. During Drosophila embryonic muscle development, multiple nuclei transition from being clustered together, to splitting into two smaller clusters, to spreading along the myotube's length. Perturbations of microtubules and motor proteins disrupt this sequence of events. These perturbations do not allow intuiting which molecular forces govern the nuclear positioning; therefore computational screening was used to reverse engineer and identify these forces. The screen reveals three models: two suggest that the initial clustering is due to the nuclear repulsion from the cell poles, while the third, most robust, model poses that this clustering is due to a short-ranged internuclear attraction. All three models suggest that the nuclear spreading is due to the long-ranged internuclear repulsion. The robust model was quantitatively tested by comparing it to data from perturbed muscle cells. The model was also tested by using agent-based simulations with elastic dynamic microtubules and molecular motors. The model predicts that, in longer mammalian myotubes with a great number of nuclei, the spreading stage would be preceded with segregation of the nuclei into a large number of clusters, proportional to the myotube length, with a small average number of nuclei per cluster (Manhart, 2020).

Microtubules provide guidance cues for myofibril and sarcomere assembly and growth

Muscle myofibrils and sarcomeres present exceptional examples of highly ordered cytoskeletal filament arrays, whose distinct spatial organization is an essential aspect of muscle cell functionality. This study utilized ultra-structural analysis to investigate the assembly of myofibrils and sarcomeres within developing myotubes of the indirect flight musculature of Drosophila. A temporal sequence composed of three major processes was identified: subdivision of the unorganized cytoplasm of nascent, multi-nucleated myotubes into distinct organelle-rich and filament-rich domains; initial organization of the filament-rich domains into myofibrils harboring nascent sarcomeric units; and finally, maturation of the highly-ordered pattern of sarcomeric thick (myosin-based) and thin (microfilament-based) filament arrays in parallel to myofibril radial growth. Significantly, organized microtubule arrays were present throughout these stages and exhibited dynamic changes in their spatial patterns consistent with instructive roles. Genetic manipulations confirm these notions, and imply specific and critical guidance activities of the microtubule-based cytoskeleton, as well as structural interdependence between the myosin- and actin-based filament arrays. These observations highlight a surprisingly significant, behind-the-scenes role for microtubules in establishment of myofibril and sarcomere spatial patterns and size, and provide a detailed account of the interplay between major cytoskeletal elements in generating these essential contractile myogenic units (Dhanyasi, 2020).

Diet-MEF2 interactions shape lipid droplet diversification in muscle to influence Drosophila lifespan

Using Drosophila, a role for was uncovered for myocyte enhancer factor 2 (MEF2) in modulating diet-dependent lipid droplet diversification within adult striated muscle, impacting mortality rates. Muscle-specific attenuation of MEF2, whose chronic activation maintains glucose and mitochondrial homeostasis, leads to the accumulation of large, cholesterol ester-enriched intramuscular lipid droplets in response to high calorie, carbohydrate-sufficient diets. The diet-dependent accumulation of these lipid droplets also correlates with both enhanced stress protection in muscle and increases in organismal lifespan. Furthermore, MEF2 attenuation releases an antagonistic regulation of cell cycle gene expression programs, and up-regulation of Cyclin E is required for diet- and MEF2-dependent diversification of intramuscular lipid droplets. The integration of MEF2-regulated gene expression networks with dietary responses thus plays a critical role in shaping muscle metabolism and function, further influencing organismal lifespan. Together, these results highlight a potential protective role for intramuscular lipid droplets during dietary adaptation (Zhao, 2020).

Systemic muscle wasting and coordinated tumour response drive tumourigenesis

Cancer cells demand excess nutrients to support their proliferation, but how tumours exploit extracellular amino acids during systemic metabolic perturbations remain incompletely understood. This study used a Drosophila model of high-sugar diet (HSD)-enhanced tumourigenesis to uncover a systemic host-tumour metabolic circuit that supports tumour growth. Coordinate induction is demonstrated of systemic muscle wasting with tumour-autonomous Yorkie-mediated SLC36-family amino acid transporter expression as a proline-scavenging programme to drive tumourigenesis. Indole-3-propionic acid was identified as an optimal amino acid derivative to rationally target the proline-dependency of tumour growth. Insights from this whole-animal Drosophila model provide a powerful approach towards the identification and therapeutic exploitation of the amino acid vulnerabilities of tumourigenesis in the context of a perturbed systemic metabolic network (Newton, 2020).

Cancer cells require a constant supply of metabolic intermediates to support their proliferation. To meet the biosynthetic demands associated with tumourigenesis, cancer cells actively acquire nutrients from the extracellular space. Cancer is a systemic disease that associates with a range of host metabolic abnormalities such as obesity, insulin resistance and cancer-associated cachexia; each of which alters the host systemic nutritional environment. These changes in both nutrient composition and availability may have profound effects on cancer development and progression. However, how cancer cells sense and respond to nutritional changes in the context of organismal metabolic alterations remains an underexplored area in cancer biology (Newton, 2020).

Cancer-associated cachexia is a systemic metabolic syndrome of weight loss associated with progressive skeletal muscle wasting. The multifactorial and heterogeneous condition of cachexia involves a complex multi-organ interplay, which has impeded its comprehensive understanding at the molecular level. A series of recent studies using Drosophila melanogaster have shown that tumour-derived factors modulate host metabolism. In addition, tumour-derived factors promote the release of nutrients from the tumour microenvironment to promote tumour growth. This study has leveraged a Drosophila model of high-sugar diet (HSD)-enhanced tumourigenesis and demonstrates that HSD-enhanced tumours induce SLC36-family transporter expression as a coordinated mechanism to exploit exogenous proline for tumourigenesis during systemic muscle wasting. Furthermore, these mechanistic insights were used to rationally target the proline dependency of tumours as an approach to inhibit tumour growth (Newton, 2020).

Muscle wasting is observed in chronic muscle wasting diseases due to cancer (cachexia), ageing/senescence (sarcopenia), myopathies and other metabolic diseases, as well as in acute conditions due to burns and sepsis. This study demonstrates that the combination of diet-induced obesity and tumour growth induces systemic muscle wasting, associated with functional locomotion defect. This muscle phenotype was not due to the inability to properly form muscle during development; the muscles are wholly formed at the early stage of larval development, and waste progressively at the later larval stage, as the tumours develop. In addition, it was demonstrated that the muscle phenotype observed in this model is unrelated to a developmentally related degeneration process (Newton, 2020).

This study reveals a systemic amino acid-utilising circuit whereby HSD-enhanced tumours induce muscle wasting as a systemic metabolic network to drive tumourigenesis. A consequence of muscle wasting in ras1G12V;csk-/- animals raised on HSD was increased release of proline into the circulation. Plasma amino acid profiling of patients with sarcopenia - a condition of muscle wasting associated with aging—showed elevated plasma proline levels, indicating that elevated free circulating proline is a common feature of muscle wasting. This study identified a proline vulnerability of HSD-enhanced tumours; SLC36-family amino acid transporter Path is required for tumour growth and exogenous proline promotes tumourigenesis through Path. This study highlights two layers of coordination in tumour metabolic response: (1) at the whole organism level, by promoting muscle wasting and systemic amino acid availability, and (2) at the tumour-autonomous level, by altering amino acid transporter repertoire (Newton, 2020).

Path and CG1139 have different transport characteristics for proline, with Path having a lower transport capacity compared to CG1139. Despite this, labelled proline uptake experiments indicated uptake of extracellular proline in Ras/Src/Path-tumours but not in Ras/Src/CG1139-tumours, suggesting that CG1139 and Path may exhibit different activities in the context of an oncogenic background. Consistent with a previous report (Goberdhan, 2005), the current data support the signalling role of Path through activation of the Tor-S6K pathway. Proline metabolism has been demonstrated to support cancer clonogenicity and metastasis. Furthermore, proline promotes cancer cell survival under nutrient-limited or hypoxic microenvironments. The current data extend these studies to reveal a tumour-promoting role of proline in response to systemic host metabolic changes (Newton, 2020).

Indole-3-propionic acid (IPA) specifically targets the proline dependency of tumour growth in Drosophila. Furthermore, this study demonstrates a functional effect of IPA on human cells. A recent study demonstrated that proline uptake in Ras-driven tumour cells is much higher in spheroids in three-dimensional cultures—which better mimic conditions in vivo—compared to monolayers in two-dimensional cultures, suggestive of a potentially stronger effect in tumours that are dependent on proline for growth in vivo. Proline is a limiting amino acid for protein synthesis in kidney cancers. Therefore, reducing proline uptake with SLC36-inhibitors -- as exemplified here by use of IPA -- may warrant consideration as a therapeutic strategy to break the nutritional circuit between systemic muscle wasting and tumour growth in proline vulnerable cancers (Newton, 2020).

Mechanical tension and spontaneous muscle twitching precede the formation of cross-striated muscle in vivo

Muscle forces are produced by repetitive stereotyped acto-myosin units called sarcomeres. Sarcomeres are chained into linear myofibrils spanning the entire muscle fiber. In mammalian body muscles, myofibrils are aligned laterally resulting in their typical cross-striated morphology. Despite this detailed textbook knowledge about the adult muscle structure, it is still unclear how cross-striated myofibrils are built in vivo. This study investigated the morphogenesis of Drosophila abdominal muscles and establishrf them as in vivo model for cross-striated muscle development. Using live imaging, ong immature myofibrils lacking a periodic acto-myosin pattern were found to be built simultaneously in the entire muscle fiber and then align laterally to mature cross-striated myofibrils. Interestingly, laser micro-lesion experiments demonstrate that mechanical tension precedes the formation of the immature myofibrils. Moreover, these immature myofibrils do generate spontaneous Ca2+ dependent contractions in vivo, which when chemically blocked result in cross-striation defects. Together, these results suggest a myofibrillogenesis model, in which mechanical tension and spontaneous muscle twitchings synchronise the simultaneous self-organisation of different sarcomeric protein complexes to build highly regular cross-striated myofibrils spanning throughout large muscle fibers (Weitkunat, 2017).

Dynamics of transcriptional (re)-programming of syncytial nuclei in developing muscles

A stereotyped array of body wall muscles enables precision and stereotypy of animal movements. In Drosophila, each syncytial muscle forms via fusion of one founder cell (FC) with multiple fusion competent myoblasts (FCMs). The specific morphology of each muscle, i.e. distinctive shape, orientation, size and skeletal attachment sites, reflects the specific combination of identity transcription factors (iTFs), such as Apterous, Even-Skipped and Slouch/S59, expressed by its FC. This study addressed three questions: Are FCM nuclei naive? What is the selectivity and temporal sequence of transcriptional reprogramming of FCMs recruited into growing syncytium? Is transcription of generic myogenic and identity realisation genes coordinated during muscle differentiation? The tracking of nuclei in developing muscles shows that FCM nuclei are competent to be transcriptionally reprogrammed to a given muscle identity, post fusion. In situ hybridisation to nascent transcripts for FCM, FC-generic and iTF genes shows that this reprogramming is progressive, beginning by repression of FCM-specific genes in fused nuclei, with some evidence that FC nuclei retain specific characteristics. Transcription of identity realisation genes is linked to iTF activation and regulated at levels of both transcription initiation rate and period of transcription. The generic muscle differentiation programme is activated independently. It is concluded that transcription reprogramming of fused myoblast nuclei is progressive, such that nuclei within a syncytial fibre at a given time point during muscle development are heterogeneous with regards to specific gene transcription. This comprehensive view of the dynamics of transcriptional (re)programming of post-mitotic nuclei within syncytial cells provides a new framework for understanding the transcriptional control of the lineage diversity of multinucleated cells (Bataille, 2017).

Five Alternative Myosin converter domains influence muscle power, stretch activation, and kinetics

Muscles have evolved to power a wide variety of movements. A protein component critical to varying power generation is the myosin isoform present in the muscle. However, how functional variation in muscle arises from myosin structure is not well understood. The influence of the converter, a myosin structural region at the junction of the lever arm and catalytic domain, was studied using Drosophila because its single myosin heavy chain gene expresses five alternative converter versions (11a-e). Five transgenic fly lines were generated, each forced to express one of the converter versions in their indirect flight muscle (IFM) fibers. Electron microscopy showed that the converter exchanges did not alter muscle ultrastructure. The four lines expressing converter versions (11b-e) other than the native IFM 11a converter displayed decreased flight ability. IFM fibers expressing converters normally found in the adult stage muscles generated up to 2.8-fold more power and displayed up to 2.2-fold faster muscle kinetics than fibers with converters found in the embryonic and larval stage muscles. Small changes to stretch-activated force generation only played a minor role in altering power output of IFM. Muscle apparent rate constants, derived from sinusoidal analysis of the chimeric converter fibers, showed a strong positive correlation between optimal muscle oscillation frequency and myosin attachment kinetics to actin, and an inverse correlation with detachment related cross-bridge kinetics. This suggests the myosin converter alters at least two rate constants of the cross-bridge cycle with changes to attachment and power stroke related kinetics having the most influence on setting muscle oscillatory power kinetics (Glasheen, 2018).


Al-Qusairi, L. and Laporte, J. (2011). T-tubule biogenesis and triad formation in skeletal muscle and implication in human diseases. Skelet Muscle 1(1): 26. PubMed ID: 21797990

Artero, R., et al. (2003). Notch and Ras signaling pathway effector genes expressed in fusion competent and founder cells during Drosophila myogenesis. Development 130: 6257-6272. PubMed ID: 14602676

Atreya, K. B. and Fernandes, J. J. (2008). Founder cells regulate fiber number but not fiber formation during adult myogenesis in Drosophila. Dev. Biol. 321(1): 123-40. PubMed ID: 18616937

Bataille, L., Boukhatmi, H., Frendo, J. L. and Vincent, A. (2017). Dynamics of transcriptional (re)-programming of syncytial nuclei in developing muscles. BMC Biol 15(1): 48. PubMed ID: 28599653

Belu, M. and Mizutani, C. M. (2011). Variation in mesoderm specification across drosophilids is compensated by different rates of myoblast fusion during body wall musculature development. PLoS One 6(12): e28970. PubMed ID: 22194964

Boeynaems, S., Alberti, S., Fawzi, N. L., Mittag, T., Polymenidou, M., Rousseau, F., Schymkowitz, J., Shorter, J., Wolozin, B., Van Den Bosch, L., Tompa, P. and Fuxreiter, M. (2018). Protein phase separation: a new phase in cell biology. Trends Cell Biol 28(6): 420-435. PubMed ID: 29602697

Brooks, D., Naeem, F., Stetsiv, M., Goetting, S. C., Bawa, S., Green, N., Clark, C., Bashirullah, A. and Geisbrecht, E. R. (2020). Drosophila NUAK functions with Starvin/BAG3 in autophagic protein turnover. PLoS Genet 16(4): e1008700. PubMed ID: 32320396

Busser, B. W., Taher, L., Kim, Y., Tansey, T., Bloom, M. J., Ovcharenko, I. and Michelson, A. M. (2012). A machine learning approach for identifying novel cell type-specific transcriptional regulators of myogenesis. PLoS Genet 8: e1002531. PubMed ID: 22412381

Cannavò, E., Khoueiry, P., Garfield, D.A., Geeleher, P., Zichner, T., Gustafson, E.H., Ciglar, L., Korbel, J.O. and Furlong, E.E. (2015). Shadow enhancers are pervasive features of developmental regulatory networks. Curr Biol [Epub ahead of print]. PubMed ID: 26687625

Cao, T., Sujkowski, A., Cobb, T., Wessells, R. J. and Jin, J. P. (2020). The glutamic acid-rich long C-terminal extension of troponin T has a critical role in insect muscle functions. J Biol Chem. PubMed ID: 32024695

Chandel, I., Baker, R., Nakamura, N. and Panin, V. (2019). Live imaging and analysis of muscle contractions in Drosophila embryo. J Vis Exp(149). PubMed ID: 31355800

Chaturvedi, D., Reichert, H., Gunage, R. D. and VijayRaghavan, K. (2017). Identification and functional characterization of muscle satellite cells in Drosophila. Elife 6. PubMed ID: 29072161

Colombani, J., et al. (2003). A nutrient sensor mechanism controls Drosophila growth. Cell 114: 739-749. PubMed ID: 14505573

Cusanovich, D. A., Reddington, J. P., Garfield, D. A., Daza, R. M., Aghamirzaie, D., Marco-Ferreres, R., Pliner, H. A., Christiansen, L., Qiu, X., Steemers, F. J., Trapnell, C., Shendure, J. and Furlong, E. E. M. (2018). The cis-regulatory dynamics of embryonic development at single-cell resolution. Nature. PubMed ID: 29539636

Deng, S., Bothe, I. and Baylies, M. K. (2015). The formin Diaphanous regulates myoblast fusion through actin polymerization and Arp2/3 regulation. PLoS Genet 11: e1005381. PubMed ID: 26295716

de Velasco, B., Mandal, L., Mkrtchyan, M. and Hartenstein, V. (2006). Subdivision and developmental fate of the head mesoderm in Drosophila melanogaster. Dev. Genes Evol. 216(1): 39-51. PubMed ID: 16249873

Dhanyasi, N., Segal, D., Shimoni, E., Shinder, V., Shilo, B.Z., VijayRaghavan, K. and Schejter, E.D. (2015). Surface apposition and multiple cell contacts promote myoblast fusion in Drosophila flight muscles. J Cell Biol 211: 191-203. PubMed ID: 26459604

Dhanyasi, N., VijayRaghavan, K., Shilo, B. Z. and Schejter, E. D. (2020). Microtubules provide guidance cues for myofibril and sarcomere assembly and growth. Dev Dyn. PubMed ID: 32725855

Domsch, K., Acs, A., Obermeier, C., Nguyen, H. T. and Reim, I. (2017). Identification of the essential protein domains for Mib2 function during the development of the Drosophila larval musculature and adult flight muscles. PLoS One 12(3): e0173733. PubMed ID: 28282454

Erceg, J., Pakozdi, T., Marco-Ferreres, R., Ghavi-Helm, Y., Girardot, C., Bracken, A. P. and Furlong, E. E. (2017). Dual functionality of cis-regulatory elements as developmental enhancers and Polycomb response elements. Genes Dev 31(6): 590-602. PubMed ID: 28381411

Fernandes, J. J. and Keshishian, H. (1998). Nerve-muscle interactions during flight muscle development in Drosophila. Development 125: 1769-1779. PubMed ID: 9521914

Fernandes, J. J. and Keshishian, H. (1999). Development of the adult neuromuscular system, Int. Rev. Neurobiol. 43: 221-239. PubMed ID: 10218161

Fernandes, J. J. and Keshishian, H. (2005). Motoneurons regulate myoblast proliferation and patterning in Drosophila. Dev. Biol. 277(2): 493-505. PubMed ID: 15617689

Fujita, N., Huang, W., Lin, T.H., Groulx, J.F., Jean, S., Kuchitsu, Y., Koyama-Honda, I., Mizushima, N., Fukuda, M. and Kiger, A.A. (2017). Genetic screen in Drosophila muscle identifies autophagy-mediated T-tubule remodeling and a Rab2 role in autophagy. Elife pii: e23367. PubMed ID: 28063257

Furlong, E. E. M., Andersen, E. C., Null, B., White, K. P. and Scott, M. P. (2001). Patterns of gene expression during Drosophila mesoderm development. Science 293: 1629-1633. PubMed ID: 11486054

Gillingham, A. K., Sinka, R., Torres, I. L., Lilley, K. S. and Munro, S. (2014). Toward a comprehensive map of the effectors of rab GTPases. Dev Cell 31(3): 358-373. PubMed ID: 25453831

Gisselbrecht, S. S., Barrera, L. A., Porsch, M., Aboukhalil, A., Estep, P. W., Vedenko, A., Palagi, A., Kim, Y., Zhu, X., Busser, B. W., Gamble, C. E., Iagovitina, A., Singhania, A., Michelson, A. M. and Bulyk, M. L. (2013). Highly parallel assays of tissue-specific enhancers in whole Drosophila embryos. Nat Methods 10(8): 774-780. PubMed ID: 23852450

Gisselbrecht, S. S., Palagi, A., Kurland, J. V., Rogers, J. M., Ozadam, H., Zhan, Y., Dekker, J. and Bulyk, M. L. (2019). Transcriptional silencers in Drosophila serve a dual role as transcriptional enhancers in alternate cellular contexts. Mol Cell. 77(2):324-337 PubMed ID: 31704182

Glasheen, B. M., Ramanath, S., Patel, M., Sheppard, D., Puthawala, J. T., Riley, L. A. and Swank, D. M. (2018). Five Alternative Myosin converter domains influence muscle power, stretch activation, and kinetics. Biophys J 114(5): 1142-1152. PubMed ID: 29539400

Gonzalez-Morales, N., Marsh, T. W., Katzemich, A., Marescal, O., Xiao, Y. S. and Schock, F. (2019a). Different evolutionary trajectories of two insect-specific paralogous proteins involved in stabilizing muscle myofibrils. Genetics 212(3): 743-755. PubMed ID: 31123042

Gonzalez-Morales, N., Xiao, Y. S., Schilling, M. A., Marescal, O., Liao, K. A. and Schock, F. (2019b). Myofibril diameter is set by a finely tuned mechanism of protein oligomerization in Drosophila. Elife 8. PubMed ID: 31746737

Hara, Y., Koganezawa, M. and Yamamoto, D. (2015). The Dmca1D channel mediates Ca inward currents in Drosophila embryonic muscles. J Neurogenet: 1-28. PubMed ID: 26004544

Holz, A., et al. (2003). The two origins of hemocytes in Drosophila. Development 130: 4955-4962. PubMed ID: 12930778

Hudson, A. M., Petrella, L. N., Tanaka, A. J. and Cooley, L. (2008). Mononuclear muscle cells in Drosophila ovaries revealed by GFP protein traps. Dev. Biol. 314(2): 329-40. PubMed ID: 18199432

Jablonska, J., Dubinska-Magiera, M., Jagla, T., Jagla, K. and Daczewska, M. (2018). Drosophila Hsp67Bc hot-spot variants alter muscle structure and function. Cell Mol Life Sci. PubMed ID: 30032358

Jiang, P., Nishimura, T., Sakamaki, Y., Itakura, E., Hatta, T., Natsume, T. and Mizushima, N. (2014). The HOPS complex mediates autophagosome-lysosome fusion through interaction with syntaxin 17. Mol Biol Cell 25(8): 1327-1337. PubMed ID: 24554770

Johnston, J. S., Zapalac, M. E. and Hjelmen, C. E. (2020). Flying high-muscle-specific underreplication in Drosophila. Genes (Basel) 11(3). PubMed ID: 32111003

Jung, S. H., Evans, C. J., Uemura, C. and Banerjee, U. (2005). The Drosophila lymph gland as a developmental model of hematopoiesis. Development 132(11): 2521-33. PubMed ID: 15857916

Kamada, Y., Funakoshi, T., Shintani, T., Nagano, K., Ohsumi, M. and Ohsumi, Y. (2000). Tor-mediated induction of autophagy via an Apg1 protein kinase complex. J. Cell Biol. 150: 1507-1513. PubMed ID: 10995454

Kantorovitz, M. R., Kazemian, M., Kinston, S., Miranda-Saavedra, D., Zhu, Q., Robinson, G. E., Gottgens, B., Halfon, M. S. and Sinha, S. (2009). Motif-blind, genome-wide discovery of cis-regulatory modules in Drosophila and mouse. Dev Cell 17: 568-579. PubMed ID: 19853570

Katzemich, A., West, R. J., Fukuzawa, A., Sweeney, S. T., Gautel, M., Sparrow, J. and Bullard, B. (2015). Binding partners of the kinase domains in Drosophila obscurin and their effect on the structure of the flight muscle. J Cell Sci 128(18): 3386-3397. PubMed ID: 26251439

Kirisako, T., Baba, M., Ishihara, N., Miyazawa, K., Ohsumi, M., Yoshimori, T., Noda, T. and Ohsumi, Y. (1999). Formation process of autophagosome is traced with Apg8/Aut7p in yeast. J. Cell Biol. 147: 435-446. PubMed ID: 10525546

Klaavuniemi, T., Kelloniemi, A. and Ylanne, J. (2004). The ZASP-like motif in actinin-associated LIM protein is required for interaction with the alpha-actinin rod and for targeting to the muscle Z-line. J Biol Chem 279(25): 26402-26410. PubMed ID: 15084604

Klaavuniemi, T. and Ylanne, J. (2006). Zasp/Cypher internal ZM-motif containing fragments are sufficient to co-localize with alpha-actinin--analysis of patient mutations. Exp Cell Res 312(8): 1299-1311. PubMed ID: 16476425

Kuleesha, Y., Puah, W. C. and Wasser, M. (2016). A model of muscle atrophy based on live microscopy of muscle remodelling in Drosophila metamorphosis. R Soc Open Sci 3: 150517. PubMed ID: 26998322

Kuleesha, Feng, L. and Wasser, M. (2017). Spatial pattern analysis of nuclear migration in remodelled muscles during Drosophila metamorphosis. BMC Bioinformatics 18(1): 329. PubMed ID: 28693471

Lavergne, G., Zmojdzian, M., Da Ponte, J. P., Junion, G. and Jagla, K. (2020). Drosophila adult muscle precursor cells contribute to motor axon pathfinding and proper innervation of embryonic muscles. Development 147(4). PubMed ID: 32001438

Li, H., Watson, A., Olechwier, A., Anaya, M., Sorooshyari, S. K., Harnett, D. P., Lee, H. P., Vielmetter, J., Fares, M. A., Garcia, K. C., Ozkan, E., Labrador, J. P. and Zinn, K. (2017). Deconstruction of the beaten Path-Sidestep interaction network provides insights into neuromuscular system development. Elife 6. PubMed ID: 28829740

Liao, K. A., Gonzalez-Morales, N. and Schock, F. (2020). Characterizing the actin-binding ability of Zasp52 and its contribution to myofibril assembly. PLoS One 15(7): e0232137. PubMed ID: 32614896

Madan, A., Thimmaiya, D., Franco-Cea, A., Aiyaz, M., Kumar, P., Sparrow, J. C. and Nongthomba, U. (2017). Transcriptome analysis of IFM-specific actin and myosin nulls in Drosophila melanogaster unravels lesion-specific expression blueprints across muscle mutations. Gene 631: 16-28. PubMed ID: 28739398

Manhart, A., Azevedo, M., Baylies, M. and Mogilner, A. (2020). Reverse engineering forces responsible for dynamic clustering and spreading of multiple nuclei in developing muscle cells. Mol Biol Cell: mbcE19120711. PubMed ID: 32129712

McMahon, A., Supatto, W., Fraser S. E. and Stathopoulos, A. (2008). Dynamic analyses of Drosophila gastrulation provide insights into collective cell migration. Science 322: 1546-1550. PubMed ID: 19056986

McMahon, A., Reeves, G. T., Supatto, W. and Stathopoulos, A. (2010). Mesoderm migration in Drosophila is a multi-step process requiring FGF signaling and integrin activity. Development 137(13): 2167-75. PubMed ID: 20530544

Moss-Taylor, L., Upadhyay, A., Pan, X., Kim, M. J. and O'Connor, M. B. (2019). Body size and tissue-scaling is regulated by motoneuron-derived activinbeta in Drosophila melanogaster. Genetics. PubMed ID: 31585954

Newton, H., Wang, Y. F., Camplese, L., Mokochinski, J. B., Kramer, H. B., Brown, A. E. X., Fets, L. and Hirabayashi, S. (2020). Systemic muscle wasting and coordinated tumour response drive tumourigenesis. Nat Commun 11(1): 4653. PubMed ID: 32938923

Ordan, E. and Volk, T. (2015). A non-signaling role of Robo2 in tendons is essential for Slit processing and muscle patterning. Development 142: 3512-3518. PubMed ID: 26400093

Ozkan, A. and Berberoglu, H. (2013). Cell to substratum and cell to cell interactions of microalgae. Colloids Surf B Biointerfaces 112: 302-309. PubMed ID: 24004676

Pauls, D., Blechschmidt, C., Frantzmann, F., El Jundi, B. and Selcho, M. (2018). A comprehensive anatomical map of the peripheral octopaminergic/tyraminergic system of Drosophila melanogaster. Sci Rep 8(1): 15314. PubMed ID: 30333565

Riechmann, V., et al. (1998). The genetic control of the distinction between fat body and gonadal mesoderm in Drosophila. Development 125(4): 713-723. PubMed ID: 9435291

Rouault, H., Mazouni, K., Couturier, L., Hakim, V. and Schweisguth, F. (2010). Genome-wide identification of cis-regulatory motifs and modules underlying gene coregulation using statistics and phylogeny. Proc Natl Acad Sci U S A 107: 14615-14620. PubMed ID: 20671200

Ribeiro, I., Yuan, L., Tanentzapf, G., Dowling, J. J. and Kiger, A. (2011). Phosphoinositide regulation of integrin trafficking required for muscle attachment and maintenance. PLoS Genet 7(2): e1001295. PubMed ID: 21347281

Safi, F., Shteiman-Kotler, A., Zhong, Y., Iliadi, K. G., Boulianne, G. L. and Rotin, D. (2016). Drosophila Nedd4-long reduces Amphiphysin levels in muscles and leads to impaired T-tubule formation. Mol Biol Cell 27(6):907-18. PubMed ID: 26823013

Sandmann, T., et al. (2007). A core transcriptional network for early mesoderm development in Drosophila melanogaster. Genes Dev. 21: 436-449. PubMed ID: 17322403

Sarkissian, T., Arya, R., Gyonjyan, S., Taylor, B. and White, K. (2016). Cell death regulates muscle fiber number. Dev Biol [Epub ahead of print]. PubMed ID: 27131625

Scott, R. C., Schuldiner, O. and Neufeld, T. P. (2004). Role and regulation of starvation-induced autophagy in the Drosophila fat body. Dev Cell. 7: 167-178. PubMed ID: 15296714

Shwartz, A., Dhanyasi, N., Schejter, E.D. and Shilo, B.Z. (2016). The Drosophila formin Fhos is a primary mediator of sarcomeric thin-filament array assembly. Elife 5. PubMed ID: 27731794

Szikora, S., Gajdos, T., Novak, T., Farkas, D., Foldi, I., Lenart, P., Erdelyi, M. and Mihaly, J. (2020). Nanoscopy reveals the layered organization of the sarcomeric H-zone and I-band complexes. J Cell Biol 219(1). PubMed ID: 31816054

Takats, S., Pircs, K., Nagy, P., Varga, A., Karpati, M., Hegedus, K., Kramer, H., Kovacs, A. L., Sass, M. and Juhasz, G. (2014). Interaction of the HOPS complex with Syntaxin 17 mediates autophagosome clearance in Drosophila. Mol Biol Cell 25(8): 1338-1354. PubMed ID: 24554766

Takeshima, H., Hoshijima, M. and Song, L. S. (2015). Ca(2)+ microdomains organized by junctophilins. Cell Calcium 58(4): 349-356. PubMed ID: 25659516

Tixier, V., Bataille, L., Etard, C., Jagla, T., Weger, M., Daponte, J. P., Strahle, U., Dickmeis, T. and Jagla, K. (2013). Glycolysis supports embryonic muscle growth by promoting myoblast fusion. Proc Natl Acad Sci U S A 110: 18982-18987. PubMed ID: 24191061

Togel, M., Meyer, H., Lehmacher, C., Heinisch, J. J., Pass, G., Paululat, A. (2013). The bHLH transcription factor Hand is required for proper wing heart formation in Drosophila. Dev Biol 381: 446-459. PubMed ID: 23747982

Upadhyay, A., Peterson, A. J., Kim, M. J. and O'Connor, M. B. (2020). Muscle-derived Myoglianin regulates Drosophila imaginal disc growth. Elife 9:e51710. PubMed ID: 32633716

Vaziri, P., Ryan, D., Johnston, C. A. and Cripps, R. M. (2020). A Novel Mechanism for Activation of Myosin Regulatory Light Chain by Protein Kinase C-Delta in Drosophila. Genetics 216(1): 177-190. PubMed ID: 32753389

Viswanathan, M. C., Schmidt, W., Rynkiewicz, M. J., Agarwal, K., Gao, J., Katz, J., Lehman, W. and Cammarato, A. (2017). Distortion of the Actin A-triad results in contractile disinhibition and cardiomyopathy. Cell Rep 20(11): 2612-2625. PubMed ID: 28903042

Wang, F., Minakhina, S., Tran, H., Changela, N., Kramer, J. and Steward, R. (2018). Tet protein function during Drosophila development. PLoS One 13(1): e0190367. PubMed ID: 29324752

Weber, S. C. (2017). Sequence-encoded material properties dictate the structure and function of nuclear bodies. Curr Opin Cell Biol 46: 62-71. PubMed ID: 28343140

Weitkunat, M., Lindauer, M., Bausch, A. and Schnorrer, F. (2017). Mechanical tension and spontaneous muscle twitching precede the formation of cross-striated muscle in vivo. Development [Epub ahead of print]. PubMed ID: 28174246

Wolfstetter, G., Pfeifer, K., van Dijk, J. R., Hugosson, F., Lu, X. and Palmer, R. H. (2017). The scaffolding protein Cnk binds to the receptor tyrosine kinase Alk to promote visceral founder cell specification in Drosophila. Sci Signal 10(502). PubMed ID: 29066538

Williams, J., Boin, N. G., Valera, J. M. and Johnson, A. N. (2015). Noncanonical roles for Tropomyosin during myogenesis. Development [Epub ahead of print]. PubMed ID: 26293307

Zhao, X., Li, X., Shi, X. and Karpac, J. (2020). Diet-MEF2 interactions shape lipid droplet diversification in muscle to influence Drosophila lifespan. Aging Cell 19(7). PubMed ID: 32537848

Zhong, Y., Shtineman-Kotler, A., Nguyen, L., Iliadi, K. G., Boulianne, G. L. and Rotin, D. (2011). A splice isoform of DNedd4, DNedd4-long, negatively regulates neuromuscular synaptogenesis and viability in Drosophila. PLoS One 6(11): e27007. PubMed ID: 22110599

Return: Genes expressed in mesoderm

Genes involved in tissue development

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.