InteractiveFly: GeneBrief

myoglianin: Biological Overview | References


Gene name - myoglianin

Synonyms -

Cytological map position - 102D4-102D4

Function - ligand

Keywords - glia, mesoderm, larval brain, neuronal remodeling, regulation of Ecdysone receptor

Symbol - myo

FlyBase ID: FBgn0026199

Genetic map position - chr4:711,890-716,675

Classification - Transforming growth factor-beta family

Cellular location - secreted



NCBI links: EntrezGene

Myoglianin orthologs: Biolitmine

Recent literature
Ishimaru, Y., Tomonari, S., Matsuoka, Y., Watanabe, T., Miyawaki, K., Bando, T., Tomioka, K., Ohuchi, H., Noji, S. and Mito, T. (2016). TGF-beta signaling in insects regulates metamorphosis via juvenile hormone biosynthesis. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 27140602
Summary:
Although butterflies undergo a dramatic morphological transformation from larva to adult via a pupal stage (holometamorphosis), crickets undergo a metamorphosis from nymph to adult without formation of a pupa (hemimetamorphosis). Despite these differences, both processes are regulated by common mechanisms that involve 20-hydroxyecdysone (20E) and juvenile hormone (JH). JH regulates many aspects of insect physiology, such as development, reproduction, diapause, and metamorphosis. Consequently, strict regulation of JH levels is crucial throughout an insect's life cycle. However, it remains unclear how JH synthesis is regulated. This study reports that in the corpora allata of the cricket, Gryllus bimaculatus, Myoglianin (Gb'Myo), a homolog of Drosophila Myoglianin/vertebrate GDF8/11, is involved in the down-regulation of JH production by suppressing the expression of a gene encoding JH acid O-methyltransferase, Gb'jhamt. In contrast, JH production is up-regulated by Decapentaplegic (Gb'Dpp) and Glass-bottom boat/60A (Gb'Gbb) signaling that occurs as part of the transcriptional activation of Gb'jhamt. Gb'Myo defines the nature of each developmental transition by regulating JH titer and the interactions between JH and 20E. When Gb'myo expression is suppressed, the activation of Gb'jhamt expression and secretion of 20E induce molting, thereby leading to the next instar before the last nymphal instar. Conversely, high Gb'myo expression induces metamorphosis during the last nymphal instar through the cessation of JH synthesis. Gb'myo also regulates final insect size. Because Myo/GDF8/11 and Dpp/bone morphogenetic protein (BMP)2/4-Gbb/BMP5–8 are conserved in both invertebrates and vertebrates, the present findings provide common regulatory mechanisms for endocrine control of animal development.
Langerak, S., Kim, M. J., Lamberg, H., Godinez, M., Main, M., Winslow, L., O'Connor, M. B. and Zhu, C. C. (2018). The Drosophila TGF-beta/Activin-like ligands Dawdle and Myoglianin appear to modulate adult lifespan through regulation of 26S proteasome function in adult muscle. Biol Open 7(4). PubMed ID: 29615416
Summary:
The Drosophila Activin signaling pathway employs at least three separate ligands - Activin-β (Actβ), Dawdle (Daw), and Myoglianin (Myo) - to regulate several general aspects of fruit fly larval development, including cell proliferation, neuronal remodeling, and metabolism. This study provides experimental evidence indicating that both Daw and Myo are anti-ageing factors in adult fruit flies. Knockdown of Myo or Daw reduced mean lifespan, while overexpression of either ligand in adult muscle enhanced mean lifespan. An examination of ubiquitinated protein aggregates in adult muscles revealed a strong inverse correlation between Myo- or Daw-initiated Activin signaling and the amount of ubiquitinated protein aggregates, demonstrating that the lifespan extension effect caused by overexpression of wild-type Daw or Myo in adult muscle tissues can be completely abrogated by knockdown of a 26S proteasome regulatory subunit Rpn1 in adult fly muscle, and that the prolonged lifespan caused by overexpression of Daw or Myo in adult muscle could be due to enhanced protein levels of the key subunits of 26S proteasome.
Marmor-Kollet, N., Gutman, I., Issman-Zecharya, N. and Schuldiner, O. (2019). Glial derived TGF-beta instructs axon midline stopping. Front Mol Neurosci 12: 232. PubMed ID: 31611773
Summary:
A fundamental question that underlies the proper wiring and function of the nervous system is how axon extension stops during development. However, mechanistic understanding of axon stopping is currently poor. The stereotypic development of the Drosophila mushroom body (MB) provides a unique system in which three types of anatomically distinct neurons (gamma, alpha'/beta', and alpha/beta) develop and interact to form a complex neuronal structure. All three neuronal types innervate the ipsi-lateral side and do not cross the midline. Plum, an immunoglobulin (Ig) superfamily protein that was previously shown to function as a TGF-beta accessory receptor, is required within MB alpha/beta neurons for their midline stopping. Overexpression of Plum within MB neurons is sufficient to induce retraction of alpha/beta axons. As expected, rescue experiments revealed that Plum likely functions in alpha/beta neurons and mediates midline stopping via the downstream effector RhoGEF2. Finally, glial-derived Myoglianin (Myo) was identified as the major TGF-beta ligand that instructs midline stopping of MB neurons. Taken together, this study strongly suggests that TGF-beta signals originating from the midline facilitate midline stopping of alpha/beta neuron in a Plum dependent manner.
Upadhyay, A., Peterson, A. J., Kim, M. J. and O'Connor, M. B. (2020). Muscle-derived Myoglianin regulates Drosophila imaginal disc growth. Muscle-derived Myoglianin regulates Drosophila imaginal disc growth. Elife 9:e51710. PubMed ID: 32633716
Summary:
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. Ig 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.

BIOLOGICAL OVERVIEW

Glia secrete myoglianin, a TGF-β ligand, to instruct developmental neural remodeling in Drosophila. Glial myoglianin upregulates neuronal expression of an ecdysone nuclear receptor that triggers neurite remodeling following the late-larval ecdysone peak. Thus glia orchestrate developmental neural remodeling not only by engulfment of unwanted neurites but also by enabling neuron remodeling (Awasaki, 2011).

To establish and refine functional neural circuits, neurons alter connections as the organism matures. In Drosophila, larval brain neural circuits are remodeled into adult ones during metamorphosis. Neurons forming functional larval neural circuits prune their neural projections by local degeneration in early metamorphosis and re-extend their neurites to form the adult-specific neural circuits. This phenomenon requires activation of TGF-β signaling in the remodeling neurons. TGF-β signaling upregulates expression of the B1 isoform of the ecdysone receptor (EcR-B1) at the late larval stage. The pruning of larval projections is then triggered by the steroid molting hormone ecdysone (Awasaki, 2011).

The activin-β gene (Actβ), which encodes a Drosophila activin/TGF-β family molecule, is expressed in the developing larval brain. Temporal inhibition of Actβ with its dominant-negative form or double-stranded RNA (dsRNA) partially suppresses the expression of EcR-B1 in the wandering larvae. In a previous study, it was proposed that Actβ is a candidate ligand for TGF-β signaling in neuronal remodeling (Zheng, 2003). However, the recently isolated Actβ null mutant, Actβed80 (Zhu, 2008), had no developmental defects in the remodeling of the mushroom body γ neurons. This observation excludes Actβ as a principal ligand for TGF-β–dependent remodeling of mushroom body neurons. In addition, the Actβ null mutant grows normally until the pharate adult stage (Zhu, 2008)., contrasting the embryonic lethality that results from the ubiquitous induction of the dominant-negative form or dsRNA of Actβ. These contradictory phenomena suggest that off-target effects occur when Actβ is suppressed with dominant-negative proteins or RNA interference (Awasaki, 2011).

Notably, myoglianin (myo), which encodes another Drosophila TGF-β ligand (Lo, 1999), is temporally expressed in the brain of third instar larvae. Although no myo transcripts could be detected in the brain of early larvae, intense signals for myo transcripts were seen in subsets of glial cells in the cortex and inner regions of the central brain after the mid third instar larval stage. myo is selectively expressed in two subtypes of larval glial cells: the larval cortex and astrocyte-like glial cells. The cortex glia surround the cell body of each mature neuron and the astrocyte-like glia infiltrate into brain neuropile. The glial processes of both types are in the vicinity of, if not directly contacting, the larval mushroom body γ neurons (Awasaki, 2011).

To determine whether myo governs mushroom body remodeling, the glial expression of myo was silenced by targeted RNAi. dsRNA or microRNA (miRNA) against myo was selectively expressed in glia using the pan-glial GAL4 driver repo-GAL4. myo transcripts were no longer detectable after induction of myo dsRNA in pan-glial cells. The pruning and re-extension of mushroom body γ axons were examined by immunostaining with antibody to Fasciclin 2 (Fas2). In wild-type animals, the perpendicular γ axonal branches in the larval mushroom body lobes are completely pruned by 18 h after puparium formation (APF). γ neurons subsequently re-extended axons horizontally to form the midline-projecting γ lobe in adult brains. This remodeling was blocked by pan-glial induction of myo RNAi. The perpendicular axonal branches of γ neurons persisted through early metamorphosis, and the abnormally retained larval neurites coexisted with the α/β lobes in the adult mushroom bodies that failed to remodel. Direct visualization of mushroom body γ neurons validated the above observations with antibody to Fas2. The myo-silenced brains, including their glial network, were otherwise grossly normal. These observations indicate that a loss of myo expression in glia has no detectable effect on glial cells but adversely affects mushroom body remodeling (Awasaki, 2011).

myo was knocked down using glial subtype–specific drivers. Notably, only cortex glia–specific silencing could marginally block mushroom body remodeling and elicit mild mushroom body lobe defects in about 60% of adult mushroom bodies. However, silencing myo in both larval cortex and astrocyte-like glia fully recapitulated the mushroom body remodeling defects caused by the pan-glial induction of myo RNAi. These findings indicate that myo from two glial sources acts redundantly to govern mushroom body remodeling (Awasaki, 2011).

Next, to determine whether glial-derived myo is required for upregulation of EcR-B1 in remodeling mushroom body γ neurons, EcR-B1 expression in late-larval mushroom bodies in wild-type larvae was compared with expression in myo RNAi larvae. Although the characteristic pattern of EcR-B1 enrichment was detected in wild-type larvae, no such enrichment was detected in myo RNAi-expressing larvae. For example, the strong nuclear signal of EcR-B1 in the mushroom body γ neurons was no longer discernible. When EcR-B1 expression was selectively restored in the mushroom body γ neurons of animals expressing myo RNAi in glia, no defect in mushroom body remodeling could be detected. These findings suggest that the neuronal phenotypes resulting from glial myo RNAi can be effectively rescued by neuronal induction of EcR-B1. These results indicate that the glia-derived Myo instructs mushroom body remodeling via upregulation of neuronal EcR-B1 (Awasaki, 2011).

Remodeling of larval olfactory projection neurons is under the control of the same TGF-β and ecdysone signaling as the mushroom body γ neurons (Marin, 2005). The loss of glial myo blocked EcR-B1 expression and neurite remodeling of projection neurons, and the remodeling defect was substantially rescued by projection neuron–specific induction of transgenic EcR-B1. These results suggest that glia-derived Myo acts broadly to pattern neuronal remodeling via upregulation of EcR-B1 (Awasaki, 2011).

Using an FRT-mediated inter-chromosomal recombination technique, a deletion mutant, myoΔ1 was generated. Organisms homozygous for myoΔ1 showed no developmental delay through the third instar larval stage. However, mutant larvae prepupate on the surface of or in the food and are developmentally arrested before head inversion. In the myo mutant pupae (0 h APF) or prepupae (2–6 h APF), no enhancement of EcR-B1 expression was detected in the clustered cell bodies of mushroom body γ neurons. When myo expression was restored using myo-GAL4 to drive UAS-myo, the myo mutants grew into pharate or eclosed adults. These animals showed enriched EcR-B1 in the larval brain and they underwent normal mushroom body remodeling. In contrast, when myo expression was restored with myo-GAL4, but excluded in glia by using repo-GAL80 to selectively block GAL4 function in all glial cells, no enrichment of EcR-B1 was detected in the late larval or prepupal brains, and no evidence was found of neuronal remodeling in the pharate adults. These results with myo null mutants substantiate the notion that myo expression in glia governs neuronal remodeling via upregulation of neuronal EcR-B1 expression (Awasaki, 2011).

Does Myo activate TGF-β signaling through the Baboon (Babo) receptor that, contrasting with Myo, acts cell-autonomously to enable neuronal remodeling? There are three Babo isoforms with different ligand-binding domains (Jensen, 2009). Babo-A has been implicated in governing neuron remodeling. To determine whether Myo activity requires Babo-A, their relationship was examined by epistasis. Ubiquitous expression of Myo induced larval lethality. If Myo signals through Babo-A, silencing babo-A should suppress the Myo-induced larval lethality. Attempts were made to deplete specific Babo isoforms by miRNAs to isoform-specific exons. Targeted induction of babo-A miRNA, but not babo-B or babo-C miRNA, effectively blocked mushroom body remodeling. When such isoform-specific miRNAs were co-induced with the myo transgene, only miRNA against babo-A potently suppressed the larval lethality that resulted from ectopic Myo expression. These epistasis results provide in vivo evidence that Myo and Babo-A act in a linear pathway to upregulate EcR-B1 and enable neuronal remodeling (Awasaki, 2011).

Remodeling of larval neurons occurs promptly as the larvae cease activity and become pupae. The tight temporal control of this developmental neuronal remodeling requires a timely induction of the EcR-B1 in these neurons. Three pathways, including TGF-β signaling, the cohesin protein complex and the FTZ-F1 nuclear receptor, are essential for the late-larval upregulation of EcR-B1. The nature and source of the TGF-β signaling become increasingly clear with the finding that Myo, in addition to Babo and dSmad2, is indispensable for the upregulation of EcR-B1. Myo can bind with the Babo/Wit receptor complex in culture (Lee-Hoeflich, 2005). Notably, Myo is produced by glia and is required in glia for neuronal expression of EcR-B1. Namely, glial cells directly instructed the neural remodeling through secretion of Myo. Glial cells further participate in the execution of neuronal remodeling through facilitation of neurite pruning by engulfment of the unwanted neuronal processes. Thus, glial cells orchestrate developmental neural remodeling and may have active roles in dynamically modulating mature neuronal connections (Awasaki, 2011).

Modulators of hormonal response regulate temporal fate specification in the Drosophila brain

How a progenitor sequentially produces neurons of different fates and the impact of extrinsic signals conveying information about developmental progress or environmental conditions on this process represent key, but elusive questions. Each of the four progenitors of the Drosophila mushroom body (MB) sequentially gives rise to the MB neuron subtypes. The temporal fate determination pattern of MB neurons can be influenced by extrinsic cues, conveyed by the steroid hormone ecdysone. This study shows that the activation of Transforming Growth Factor-beta (TGF-beta) signalling via glial-derived Myoglianin regulates the fate transition between the early-born alpha'beta' and the pioneer alphabeta MB neurons by promoting the expression of the ecdysone receptor β1 isoform (EcR-β1). While TGF-beta signalling is required in MB neuronal progenitors to promote the expression of EcR-β1, ecdysone signalling acts postmitotically to consolidate the alpha'beta' MB fate. Indeed, it is proposed that if these signalling cascades are impaired alpha'beta' neurons lose their fate and convert to pioneer alphabeta. Conversely, an intrinsic signal conducted by the zinc finger transcription factor Kruppel-homolog 1 (Kr-h1) antagonises TGF-beta signalling and acts as negative regulator of the response mediated by ecdysone in promoting alpha'beta' MB neuron fate consolidation. Taken together, the consolidation of alpha'beta' MB neuron fate requires the response of progenitors to local signalling to enable postmitotic neurons to sense a systemic signal (Marchetti, 2019).

This study reveals a fundamental role for Myo-mediated TGF-β signalling in regulating fate specification of MB neurons. This signalling is initiated in the neuronal progenitors and it is proposed that it is necessary to consolidate the identity of newly born neurons by enabling them to sense and integrate the ecdysone hormonal signal. As modulator of this consolidation fate program, the factor Kr-h1 negatively regulates ecdysone signalling response and antagonises the TGF-β pathway (Marchetti, 2019).

Evidence derived from vertebrate models indicates that the temporal competence of neuronal precursors to generate different neuronal subtypes is governed by the combination of cell-intrinsic programs and extrinsic cues. In contrast, fate determination in the Drosophila nervous system appeared to be mainly determined by intrinsic cascades. Only recently, first reports started indicating that extrinsic factors can modulate fate decisions in the nervous system of the fly. Thus, fate decisions in the fly nervous system might follow principles that are more relatable to the ones utilised in vertebrate lineages than previously expected. Along these lines, the current data revealed a central role of TGF-β signalling in temporal fate specification during MB development. In the rodent hindbrain, midbrain and spinal cord, TGF-β signalling constrains the neural progenitor potency to promote fate transition from early to late born cell types, acting as a temporal switch signal regulating the expression of intrinsic identity factors in young progenitors. These similarities suggest that TGF-β might represent an evolutionary conserved extrinsic signal modulating temporal fate specification (Marchetti, 2019).

The present data suggest that TGF-β signalling links the temporal neuronal fate program to developmental progression. Re-examination of the EcR-β1 expression in dSmad21 mutant MB clones at late larval stages revealed a 12 hours delay in the onset of EcR-β1 expression leading to inability of MB neurons to respond to the prepupal ecdysone peak. Thus, TGF-β signalling might help to synchronize the production of distinct MB neuron subtypes coordinating diverse developmental programs. Accordingly, this study found that the glial Myo ligand mediates the TGF-β-dependent MB fate transition. Given that the prepupal ecdsyone peak is triggered after the larva reaches the critical weight point, it was hypothesise that glia serve as nutrition sensors in the brain during larval development and could be coordinating developmental timing of the fate specification program (Marchetti, 2019).

Although α'β' neurons are born during the larval stage, based on their immature dendrites and axons, and on the absence of functional response in appetitive olfactory learning behaviour, it appears that they are not fully differentiated at the end of larval life. Therefore, the initial state of these immature α'β' neurons could be labile. Their immature neurite trajectories might possess a certain degree of morphological plasticity, since at early pupal stages the axonal lobes are primarily made of α'β' axons, after γ axons have completely pruned. Indeed, the data provide strong support for the presence of an active consolidation signal required to maintain α'β' fate at adult stage. In fact, after impairment of TGF-β signalling, neurons born in the time window corresponding to the production phase of α'β' displayed the expected axonal pattern for α'β' neurons and expressed an α'β' marker before metamorphosis. Taken these data together, the alternative hypothesis that TGF-β signalling could be involved in the initial specification of α'β' MB neurons at mid-third instar appears much more unlikely. Notably, studies on fate specification in vertebrate systems have described a postmitotic fate consolidation event for developing motor and cortical neurons. In particular, the homeobox gene HB9 has an essential function in maintaining the fate of the motor neurons by actively suppressing the alternative V5 interneuron genetic program. Indeed, mice lacking HB9 function showed a normal number of motor neurons that acquired, though, molecular features of V5 interneurons. Interestingly, in absence of HB9 motor neurons are initially specified and they retain their characteristic axonal projection. Similarly, the expression of the retinoic acid receptor (RAR) is required to maintain the fate of layer V-III cortical neurons, and when the expression of RAR is abolished these neurons acquire the identity of layer II cortical neurons. These similarities in fate consolidation programs might reflect a common strategy in both invertebrates and vertebrates to first specify and then refine neuronal fate, according to the appropriate context (Marchetti, 2019).

Recently, RNA profiling analysis of MB neurons at different developmental time points uncovered a complex feedback regulation network that governs EcR expression. This combination of positive as well as negative feedback loops is required to coordinate EcR expression levels and its temporal regulation during brain development. FISH analysis suggested that TGF-β signalling promotes the transcription of the EcR-β1 gene in MB neurons at late wandering larval stage. Although detectable EcR-β1 protein is restricted to postmitotic MB neurons, genetic data revealed that TGF-β signalling is necessary in the MB progenitors to allow the expression of EcR-β1. This evidence raises the possibility that TGF-β signalling promotes the transcription of EcR gene in neuronal progenitors and potentially post-transcriptional mechanisms are involved to narrow down the translation of the EcR-β1 receptor only postmitotically. However, the data are against this hypothesis, since expression of EcR-β1 specifically in MB progenitors did not rescue the TGF-β signalling-dependent fate defects. Moreover, given that TGF-β signalling is required to consolidate the fate of the larval-born α'β' neurons at the end of larval stage, suggests that the TGF-β pathway regulates a consolidation fate process independently of cell division. In this scenario, the expression of EcR-β1 in the newly born neurons could be promoted via a cell-to-cell communication signalling cascade initiated in neuronal progenitors by the activity of TGF-β signalling. Examples of this type of signal transmission are represented by the juxtacrine signalling mediated by Notch, Semaphorin or Ephrin pathways. In particular, the intercellular interaction between Notch and its ligand Delta in neighbouring cells is fundamental to direct cell fate decisions (Marchetti, 2019).

In addition to an upstream regulation of ecdysone signalling, this study uncovered the intrinsic factor Kr-h1 as a downstream modulator of the ecdysone-dependent fate consolidation program. Interestingly, the transition from larval stage to metamorphosis is regulated by the balance of the two major hormones, the juvenile hormone (JH) and ecdysone. JH prevents metamorphosis by the induction of the transcription factor Kr-h1 within the ring gland, which in turn suppresses the up-regulation of the ecdysone-dependent metamorphic genes E93 and Broad Complex. The TGF-β/Activin pathway contributes to decreasing Kr-h1 expression via E93 allowing the beginning of metamorphosis. Along these lines, the antagonism between ecdysone and JH through Kr-h1 could potentially regulate the MB temporal fate cascade at the onset of metamorphosis (Marchetti, 2019).

In conclusion, this work shed light on the intrinsic and extrinsic mechanisms regulating the consolidation of the terminal fate. Understanding these processes will help gain insights into their dysregulation in neurodevelopmental disorders and into their role in stem cell reprogramming (Marchetti, 2019).

Myostatin-like proteins regulate synaptic function and neuronal morphology

Growth factors of the TGF-beta superfamily play key roles in regulating neuronal and muscle function. Myostatin (or GDF8) and GDF11 are potent negative regulators of skeletal muscle mass. However, expression of both Myostatin and its cognate receptors in other tissues, including brain and peripheral nerves, suggests a potential wider biological role. This study shows that Myoglianin (MYO), the Drosophila homolog of Myostatin and GDF11, regulates not only body weight and muscle size, but also inhibits neuromuscular synapse strength and composition in a Smad2-dependent manner. Both Myostatin and GDF11 affected synapse formation in isolated rat cortical neuron cultures, suggesting an effect on synaptogenesis beyond neuromuscular junctions. This study also shows that Myoglianin acts in vivo to inhibit synaptic transmission between neurons in the escape response neural circuit of adult flies. Thus, these anti-myogenic proteins act as important inhibitors of synapse function and neuronal growth (Augustin, 2017).

Growth factors regulate many aspects of tissue development, growth and metabolism. Myostatin and GDF11 are highly homologous members of the TGF-β superfamily of growth factors. While GDF11 plays a role in a variety of systems, the role of Myostatin appears to be confined to skeletal and cardiac muscles (Augustin, 2017).

Despite the previously described roles of MYO in neural remodelling and synapse refinement (Awasaki, 2011; Yu, 2013) very little is known about the impact of Myoglianin on synaptic physiology. This study first established muscle-derived MYO as a negative regulator of both spontaneous and evoked response at the NMJ, demonstrating its role as a broad regulator of synaptic transmission. The highly coordinated apposition of active zones and glutamate receptors underlies their ability to regulate synaptic strength and plasticity of the larval NMJ. This study has shown that muscle expression of myo inversely affects the NMJ quantity of Brp and GluRIIA, critical pre-and post-synaptic proteins, and determinants of evoked neurotransmitter release and quantal size (i.e., postsynaptic sensitivity to presynaptically released transmitter), respectively. While it is possible that MYO exerts its influence on synaptic strength through other mediators, GluRIIA and Brp are their likely downstream effectors. The electrophysiological results, obtained using the GAL4-UAS system for targeted manipulation of myo, differ from the ones obtained recently using a genetic null myo mutant showing slightly reduced miniature amplitudes (Kim, 2014). The likely explanation is that compensatory effects happen in other tissues in the tissue-specific knockdown animals that cannot occur in genetic nulls, especially for systemic type factors. The other possible explanation is differential cross regulation between different (MYO-like) ligands in genetic null vs tissue knockdown animals. These results thus indicate the relevance of tissue specificity of MYO action, and of myo expression levels, in regulating synaptic function, and emphasize the need for caution when interpreting results from different types of gene manipulations (Augustin, 2017).

myo expression was detected in the glial cells of the larval neuromuscular junction. While Drosophila NMJ contains at least 2 subtypes of glia, myo expression appears confined to the 'repo-positive' subtype both in the central (Awasaki, 2011) and peripheral nervous system. The dual muscle and glial presence makes MYO ideally positioned for regulating NMJ function. Due to the small size of the compartment, however, glia-derived MYO likely has a modulatory role at the neuromuscular junction (Augustin, 2017).

This study also found that muscle suppression of Myoglianin, a Drosophila homolog of Myostatin and GDF11, promotes increased larval weight and body-wall muscle size in developing larvae, resembling the effect of Myostatin knockdown in mammals. Interestingly, pan-glial expression of myo negatively affected larval wet weight, but not the size of somatic myofibers, suggesting previously unsuspected systemic roles for glial cells (Augustin, 2017).

Smad2 is a mediator of MYO action on both evoked response and postsynaptic sensitivity, with MAD having a minor effect on the latter. While MAD primarily functions as a cytoplasmic transducer of BMP signalling, it has been demonstrated that, under certain conditions, MAD can be phosphorylated in response to Activin pathway activation (Peterson, 2012; Augustin, 2017 and references therein).

This study detected elevated levels of phosphorylated Akt and GSK-3/Shaggy in larval somatic muscles of animals with reduced myo expression in this tissue. In flies and mammals, the Akt- mTOR axis promotes skeletal muscle growth, and phosphorylation-induced inhibition of GSK-3/Shaggy induces hypertrophy in skeletal myotube. The effects of attenuated myo expression on larval tissue size, however, do not appear to be mediated by Smad2 (or MAD) activation as their overexpression does not reverse the weight phenotype in 'low myo' background. Indeed, 'non-Smad' signalling pathways have been demonstrated for various TGF-β ligands in vertebrates and Drosophila. In addition to its role as an inhibitor of the NMJ growth and active zone formation in developing Drosophila larvae, GSK-3β is also a critical promoter of synaptic plasticity, possibly through regulation of glutamate receptor function or trafficking. This work has revealed Shaggy as a mediator of reduced MYO action, and as a negative regulator of synaptic strength at the larval NMJ. While MYO likely affects both sides of the synapse directly, an unlikely but possible scenario is that presynaptic motoneuron responds to a retrograde signal released from muscle/glial cells at the NMJ in response to an induction by MYO. An attractive hypothesis is that MYO negatively regulates presynaptic release directly, in conjunction with muscle-secreted Gbb, a positive regulator of neuromuscular synapse development and growth. The effects of MYO could also be mediated through the transmembrane protein Plum, previously proposed to regulate connectivity at the larval NMJ by sequestrating Myoglianin (Yu, 2013; Augustin, 2017 and references therein).

Myostatin negatively regulates synaptic function and neuronal morphology This study found that injections of Myostatin into rapidly growing larvae abolish the positive effect of myo down-regulation on NMJ strength and composition, and reverse the elevated muscle p-Akt levels. Furthermore, both Myostatin and GDF11 surpressed the growth of neuronal processes and perturbed the formation of synapses in cultured brain neurons, suggesting a direct action on neurons and regulation of synaptogenesis beyond neuromuscular junctions. Recently, Myostatin transcript and protein were detected in the mouse hippocampus and olfactory system neurons, respectively, and Myostatin type I (Alk4/5) and type II (ActIIB) receptors were found to be expressed in the mammalian nervous system. The current results therefore expand on these findings, suggesting functional relevance for Myostatin in both peripheral and central nervous system, and beyond its action as a canonical regulator of skeletal muscle growth. These novel roles remain to be further explored (Augustin, 2017).

This study expanded analysis of the functional relevance of MYO in the nervous system by demonstrating its importance in a non-NMJ synapse. Specifically, Myoglianin plays a role in the development of a mixed electro-chemical synapse in the Drosophila escape response pathway, likely by regulating the density of shakB-encoded gap junctions at the GF-TTMn synapse (Blagburn, 1999). These findings implicate MYO as a broad negative regulator of neuronal function across the nervous system and developmental stages (Augustin, 2017).

This work thus reveals broad and novel roles for anti-myogenic TGF-β superfamily of proteins in the nervous system and suggests new targets for interventions into synaptic function across species (Augustin, 2017).

Extrinsic activin signaling cooperates with an intrinsic temporal program to increase mushroom body neuronal diversity

Temporal patterning of neural progenitors leads to the sequential production of diverse neurons. To understand how extrinsic cues influence intrinsic temporal programs, Drosophila mushroom body progenitors (neuroblasts) were studied that sequentially produce only three neuronal types: γ, then α'β', followed by αβ. Opposing gradients of two RNA-binding proteins Imp and Syp comprise the intrinsic temporal program. Extrinsic activin signaling regulates the production of α'β' neurons but whether it affects the intrinsic temporal program was not known. This study shows that the activin ligand Myoglianin from glia regulates the temporal factor Imp in mushroom body neuroblasts. Neuroblasts missing the activin receptor Baboon have a delayed intrinsic program as Imp is higher than normal during the α'β' temporal window, causing the loss of α'β' neurons, a decrease in αβ neurons, and a likely increase in γ neurons, without affecting the overall number of neurons produced. These results illustrate that an extrinsic cue modifies an intrinsic temporal program to increase neuronal diversity (Rossi, 2020).

The building of intricate neural networks during development is controlled by highly coordinated patterning programs that regulate the generation of different neuronal types in the correct number, place and time. The sequential production of different neuronal types from individual progenitors, i.e. temporal patterning, is a conserved feature of neurogenesis. For instance, individual radial glia progenitors in the vertebrate cortex sequentially give rise to neurons that occupy the different cortical layers in an inside-out manner. In Drosophila, neural progenitors (called neuroblasts) also give rise to different neuronal types sequentially. For example, projection neurons in the antennal lobe are born in a stereotyped temporal order and innervate specific glomeruli. In both of these examples, individual progenitors age concomitantly with the developing animal (e.g., from embryonic stages 11-17 in mouse and from the first larval stage (L1) to the end of the final larva stage (L3) in Drosophila). Thus, these progenitors are exposed to changing environments that could alter their neuronal output. Indeed, classic heterochronic transplantation experiments demonstrated that young cortical progenitors placed in an old host environment alter their output to match the host environment and produce upper-layer neurons (Rossi, 2020).

The adult Drosophila central brain is built from ~100 neuroblasts that divide continuously from L1 to L3. Each asymmetric division regenerates the neuroblast and produces an intermediate progenitor called ganglion mother cell (GMC) that divides only once, typically producing two different cell types. Thus, during larval life central brain neuroblasts divide 50-60 times, sequentially producing many different neuronal types. All central brain neuroblasts progress through opposing temporal gradients of two RNA-binding proteins as they age: IGF-II mRNA binding protein (Imp) when they are young and Syncrip (Syp) when they are old. Loss of Imp or Syp in antennal lobe or Type II neuroblasts affects the ratio of young to old neuronal types. Imp and Syp also affect neuroblast lifespan. Thus, a single temporal program can affect both the diversity of neuronal types produced and their numbers (Rossi, 2020).

Since central brain neuroblasts produce different neuronal types through developmental time, roles for extrinsic cues have recently garnered attention. Ecdysone triggers all the major developmental transitions including progression into the different larval stages and entry in pupation. The majority of central brain neuroblasts are not responsive to ecdysone until mid-larval life when they begin to express the Ecdysone Receptor (EcR). Expressing a dominant-negative version of EcR (EcR-DN) in Type II neuroblasts delays the Imp to Syp transition that normally occurs ~60 hr after larval hatching (ALH). This leads to many more cells that express the early-born marker gene Repo and fewer cells that express the late-born marker gene Bsh (Rossi, 2020).

To further understand how extrinsic signals contribute to temporal patterning, Drosophila mushroom body neuroblasts were studied because of the deep understanding of their development. The mushroom body is comprised of ~2000 neurons (Kenyon cells) that belong to only three main neuronal types that have unique morphologies and play distinct roles in learning and memory. They receive input mainly from ~200 projection neurons that each relays odor information from olfactory receptor neurons. Each projection neuron connects to a random subset of Kenyon cells and each Kenyon cell receives input from ~7 different projection neurons. This connectivity pattern requires a large number of mushroom body neurons (~2,000) to represent complex odors. To produce this very large number of neurons, mushroom body development is unique in many respects. Mushroom body neurons are born from four identical neuroblasts that divide continuously (unlike any other neuroblast) from the late embryonic stages until the end of pupation (~9 days for ~250 divisions each). Furthermore, the two neurons born from each mushroom body GMC are identical. The neuronal simplicity of the adult mushroom body makes it ideal to study how extrinsic cues might affect diversity since the loss of any single neuronal type is obvious given that each is represented hundreds of times (Rossi, 2020).

The three main neuronal types that make up the adult mushroom body are produced sequentially during neurogenesis: first γ, followed by α'β', and then αβ neurons (see α'β' neurons are not generated from babo mutant neuroblasts), representing the simplest lineage in the central brain. The γ temporal window extends from L1 (the first larval stage) until mid-L3 (the final larval stage) when animals attain critical weight and are committed to metamorphosis; the α'β' window from mid-L3 to the beginning of pupation, and the αβ window from pupation until eclosion (the end of development). Like all other central brain neuroblasts Imp and Syp are expressed by mushroom body neuroblasts, but in much shallower gradients through time, which accounts for their extended lifespan. Imp and Syp are inherited by newborn neurons where they instruct temporal identity. Imp positively and Syp negatively regulate the translation of chronologically inappropriate morphogenesis (chinmo), a gene encoding a transcription factor that acts as a temporal morphogen in neurons. The first-born γ neurons are produced for the first ~85 cell divisions, when Imp levels in neuroblasts, and thus Chinmo in neurons, are high. α'β' neurons are produced for the next ~40 divisions, when Imp and Syp are at similar low levels that translate into lower Chinmo levels in neurons. Low Chinmo then regulates the expression in neurons of maternal gene required for meiosis (mamo), which encodes a transcription factor that specifies the α'β' fate and whose mRNA is stabilized by Syp. αβ neurons are generated for the final ~125 neuroblast divisions, when Syp levels are high, Imp is absent in neuroblasts, and thus Chinmo and Mamo are no longer expressed in neurons (Rossi, 2020).

Extrinsic cues are known to have important roles in regulating neuronal differentiation during mushroom body neurogenesis. The ecdysone peak that controls entry into pupation regulates γ neuron axonal remodeling. Ecdysone was also proposed to be required for the final differentiation of α'β' neurons. EcR expression in γ neurons is timed by activin signaling, a member of the TGFβ family, from local glia. Activin signaling from glia is also required for the α'β' fate (Marchetti, 2019): Knocking-down the activin pathway receptor Baboon (Babo) leads to the loss of α'β' neurons. It was proposed that activin signaling in mushroom body neuroblasts regulates the expression of EcR in prospective α'β' neurons and that when the activin pathway is inhibited, it leads to the transformation of α'β' neurons into later-born pioneer-αβ neurons (a subclass of the αβ class) (Marchetti and Tavosanis, 2019) (Rossi, 2020).

Although there is strong evidence that extrinsic cues have important functions in neuronal patterning in the Drosophila central brain, it remains unknown how extrinsic temporal cues interface with the Imp and Syp intrinsic temporal program to regulate neuronal specification. This question was addressed using the developing mushroom bodies. Activin signaling from glia was shown to be required for α'β' specification. However, this study also showed that activin signaling lowers the levels of the intrinsic factor Imp in mushroom body neuroblasts to define the mid-α'β' temporal identity window. Removing the activin receptor Babo in mutant clones leads to the loss of α'β' neurons, to fewer last-born αβ neurons, and to the likely generation of additional first-born γ neurons without affecting overall clone size. This appears to be caused by a delayed decrease in Imp levels, although the intrinsic temporal clock still progresses even in the absence of activin signaling. This study also demonstrated that ecdysone signaling is not necessary for the specification of α'β' neurons, although it might still be involved in later α'β' differentiation. These results provide a model for how intrinsic and extrinsic temporal programs operate within individual progenitors to regulate neuronal specification (Rossi, 2020).

Mushroom body neurogenesis is unique and programmed to generate many copies of a few neuronal types. During the early stages of mushroom body development, high Imp levels in mushroom body neuroblasts are inherited by newborn neurons and translated into high Chinmo levels to specify γ identity. As in other central brain neuroblasts, as development proceeds, inhibitory interactions between Imp and Syp help create a slow decrease of Imp and a corresponding increase of Syp. However, at the end of the γ temporal window (mid-L3), activin signaling from glia acts to rapidly reduce Imp levels in mushroom body neuroblasts without significantly affecting Syp, establishing a period of low Imp (and thus low Chinmo in neurons) and also low Syp. This is required for activating effector genes in prospective α'β' neurons, including Mamo, whose translation is promoted by Syp (Liu, 2019). The production of αβ identity begins when Imp is further decreased and Syp levels are high during pupation (see Model of how activin signaling defines the α'β' temporal identity window.). Low Chinmo in αβ neurons is also partly regulated by ecdysone signaling through the activation of Let-7-C, which targets chinmo for degradation. Based on this model, α'β' neurons could not be rescued by knocking-down Imp in babo clones, since low Imp is required for α'β' specification while the knockdown reduces its level below this requirement. It would be expected to rescue α'β' neurons if Imp levels were specifically reduced to the appropriate levels at L3. However, reducing Imp levels might not be the only function of activin signaling, which may explain why α'β' neurons are not simply made earlier (e.g., during L1-L2) when Imp is knocked-down (Rossi, 2020).

In babo mutant clones, it is speculated that additional γ neurons are produced at the expense of α'β' neurons since Imp levels in neuroblasts (as well as Chinmo in neurons) are higher for a longer time during development; There was also a significant decrease in the total number of αβ neurons in babo mutant clones that contrasts with a recent report by Marchetti (2019) that instead concluded that additional pioneer-αβ neurons are produced. It is believed that there is both an increase in the number of γ neurons and of the pioneer-αβ neuron subclass because pioneer-αβ neurons are the first of the αβ class to be specified (when Imp is still present at very low levels) during pupation. It is speculated that pioneer-αβ neurons are produced during the extended low Imp window that was detected during pupation in babo clones. However, this does not leave the time for the remaining population of αβ neurons to be formed, which explains why their number is reduced (Rossi, 2020).

This study has focused on the three main classes of mushroom body neurons although at least seven subtypes exist: 2 γ, 2 α'β' and 3 αβ. The subtypes are specified sequentially suggesting that each of the three broad mushroom body temporal windows can be subdivided further, either by fine-scale reading of the changing Imp and Syp gradients, by additional extrinsic cues, or perhaps by a tTF series as in other neuroblasts (Rossi, 2020).

Postembryonic central brain neuroblasts are long-lived and divide on average ~50 times. Unlike in other regions of the developing Drosophila brain, rapidly progressing series of tTFs have not yet been described in these neuroblasts. Instead, they express Imp and Syp in opposing temporal gradients. Conceptually, how Imp and Syp gradients translate into different neuronal identities through time has been compared to how morphogen gradients pattern tissues in space. During patterning of the anterior-posterior axis of the Drosophila embryo, the anterior gradient of the Bicoid morphogen and the posterior Nanos gradient are converted into discrete spatial domains that define cell fates. Since gradients contain unlimited information, differences in Imp and Syp levels through time could translate into different neuronal types. Another intriguing possibility is that tTF series could act downstream of Imp and Syp, similarly to how the gap genes in the Drosophila embryo act downstream of the anterior-posterior morphogens. This study has shown that another possibility is that temporal extrinsic cues can be incorporated by individual progenitors to increase neuronal diversity. In mushroom body neuroblasts activin signaling acts directly on the intrinsic program, effectively converting two broad temporal windows into three to help define an additional neuronal type. It is proposed that subdividing the broad Imp and Syp temporal windows by extrinsic cues may be a simple way to increase neuronal diversity in other central brain neuroblasts (Rossi, 2020).

This study has also shown that activin signaling times the Imp to Syp transition for mushroom body neuroblasts, similar to the function of ecdysone for other central brain neuroblasts. In both cases however, the switch still occurs, indicating that a separate independent clock continues to tick. This role for extrinsic cues during Drosophila neurogenesis is reminiscent of their roles on individual vertebrate progenitors. For example, hindbrain neural stem cells progressively produce motor neurons followed by serotonergic neurons before switching to producing glia. The motor neuron to serotonergic neuron switch is fine-tuned by TGFβ signaling. It would be interesting to determine if hindbrain neuronal subtypes are lost in TGFβ mutants, similar to how α'β' identity is lost in the mushroom bodies in babo mutants (Rossi, 2020).

The specification of α'β' neurons begins at mid-L3 with the onset of Mamo expression. In contrast, high levels of EcR are detected in mature mushroom body neurons starting at late L3. At this stage, both γ and α'β' neurons already exist and new α'β' neurons are still being generated. Thus, Mamo expression precedes EcR expression. These non-overlapping expression patterns suggest that ecdysone signaling does not regulate Mamo and therefore cannot control the specification of α'β' neurons. Furthermore, expression of UAS-EcR-RNAi or mutants for usp do not lead to the loss of α'β' neurons. It is noted that usp results contradict the loss of α'β' neuron reported by Marchetti (2017) in usp clones. However, α'β' neurons were seen in these clones based on the morphology of these neurons but the remodeling defect of γ neurons makes α'β' neurons difficult to identify. Nevertheless, ecdysone might still function later during α'β' differentiation, particularly during pupation when all mushroom body neurons express EcR (Rossi, 2020).

This study and that of Marchetti both show that expression of UAS-EcR-DN leads to the loss of α'β' neurons by acting in mushroom body neurons but not in neuroblasts. However, EcR must be first be expressed in the target cells of interest in order to make any conclusions about ecdysone function using UAS-EcR-DN. Since this study could not detect EcR protein in Mamo+ cells at L3, but expressing UAS-EcR-DN inhibits Mamo in those cells, it is concluded that EcR-DN artifactually represses Mamo and leads to the loss of α'β' neurons. This explains why expressing UAS-EcR-B1 does not rescue α'β' neurons in babo clones. However, Marchetti did rescue babo-RNAi by expressing EcR (Marchetti, 2019). This is likely because the current experiments were performed using babo MARCM clones in which the loss of α'β' neurons is much more severe than with babo-RNAi used in their experiments. Indeed, when attempts were made to eliminate α'β' neurons using a validated UAS-babo-RNAi construct, γ neurons did not remodel but there was only a minor (but significant) decrease in the number of α'β' neurons. This indicates that knocking-down babo with mb-Gal4 that is only weakly expressed in neuroblasts and newborn neurons is not strong enough to inhibit α'β' specification. Thus, it is speculated that the LexA line used by Marchetti (GMR26E01-LexA) may not be a reliable reporter for α'β' neurons upon babo knockdown, and that it might be ecdysone sensitive later in α'β' differentiation. Since EcR expression in all mushroom body neurons at L3 may be dependent on activin signaling directly in neurons, as it is in γ neurons for remodeling, expressing UAS-EcR-B1 together with UAS-babo-RNAi using OK107-Gal4 might both reduce the effectiveness of the RNAi while also allowing for the re-expression of GMR26E01-LexA (Rossi, 2020).

Glia are a source of the activin ligand myo, which is temporally expressed in brain glia starting at L3 to initiate the remodeling of mushroom body γ neurons (Awasaki, 2011) and α'β' specification (this study and Marchetti, 2019). However, knocking-down Myo from glia is not as severe as removing Babo from mushroom body neuroblasts. This might be due to incomplete knockdown of myo or to other sources of Myo, potentially from neurons. For example, in the vertebrate cortex, old neurons signal back to young neurons to control their numbers. It is also possible the Babo is activated by other activin ligands, including Activin and Dawdle. An intriguing hypothesis is that the temporal expression of myo in glia beginning at mid-L3 is induced by the attainment of critical weight and rising ecdysone levels. It would be interesting to determine whether blocking ecdysone signaling in glia leads to the loss of α'β' specification, similar to how blocking ecdysone reception in astrocytes prevents γ neuron remodeling (Rossi, 2020).

It is well established that extrinsic cues play important roles during vertebrate neurogenesis, either by regulating temporal competence of neural stem cells or by controlling the timing of temporal identity transitions. Competence changes mediated by extrinsic cues were demonstrated in classic heterochronic transplantation studies that showed that young donor progenitors produce old neuronal types when placed in older host brains. Recent studies show that the reverse is also true when old progenitors are placed in a young environment (Rossi, 2020).

Mechanisms of intrinsic temporal patterning are also conserved. For example, vertebrate retinal progenitor cells use an intrinsic tTF cascade to bias young, middle, and old retinal fates. Two of the factors (Ikaros and Casz1) used for intrinsic temporal patterning are orthologs to the Drosophila tTFs Hb and Cas. tTF series might also exist in cortical radial glia progenitors and even in the spinal cord. Recent results also show the importance of post-transcriptional regulation in defining either young or old cortical fates, which can be compared to the use of post-transcriptional regulators that are a hallmark of neuronal temporal patterning in Drosophila central brain neuroblasts. These studies highlight that the mechanisms driving the diversification of neuronal types are conserved (Rossi, 2020).

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).

Intertissue control of the nucleolus via a myokine-dependent longevity pathway

Recent evidence indicates that skeletal muscle influences systemic aging, but little is known about the signaling pathways and muscle-released cytokines (myokines) responsible for this intertissue communication. This study shows that muscle-specific overexpression of the transcription factor Mnt decreases age-related climbing defects and extends lifespan in Drosophila. Mnt overexpression in muscle autonomously decreases the expression of nucleolar components and systemically decreases rRNA levels and the size of the nucleolus in adipocytes. This nonautonomous control of the nucleolus, a regulator of ribosome biogenesis and lifespan, relies on Myoglianin<, a myokine induced by Mnt and orthologous to human GDF11 and Myostatin. Myoglianin overexpression in muscle extends lifespan and decreases nucleolar size in adipocytes by activating p38 mitogen-activated protein kinase (MAPK), whereas Myoglianin RNAi in muscle has converse effects. Altogether, these findings highlight a key role for myokine signaling in the integration of signaling events in muscle and distant tissues during aging (Demontis, 2014. PubMed ID: 24882005).

Distinct signaling of Drosophila Activin/TGF-β family members

Growth factors of the TGF-beta family signal through type I/II receptor complexes that phosphorylate SMAD transcription factors. This study analyzed signaling of all seven TGF-beta members to identify those that mediate growth through the Drosophila type I receptor BABO. It was found that two potential ligands of BABO, Myoglianin (MYO) and Maverick (MAV), do not activate dSMAD2. Only Drosophila Activin (dACT) and the Activin-like ligand Dawdle (DAW) signal through BABO in combination with the type II receptor PUNT and activate dSMAD2. Surprisingly, it was found that activation of BABO can also lead to the phosphorylation of the 'BMP-specific' MAD. In wing discs, expression of an activated form of dSMAD2 promotes growth similar to dACT and activated BABO. By itself, activated dSMAD2 does not affect DPP/GBB target genes. However, coexpression of activated forms of dSMAD2 and MAD additively induces the expression of spalt. In contrast to dACT, it was found that DAW does not promote growth when expressed in wings. In fact, coexpression of DAW with MAD or dSMAD2 decreases growth. daw mutants die primarily during larval stages and exhibit anal pad phenotypes reminiscent of babo mutants. The rescue of daw mutants by restricted expression in neuroendocrine cells indicates that Activin-type ligands are likely distributed through the endocrine system. The distinct signaling of dACT, DAW and MYO through BABO suggests the existence of co-receptors that modulate the canonical SMAD pathway (Gesualdi, 2007; full text of article).

The Drosophila type II receptor, Wishful thinking, binds BMP and myoglianin to activate multiple TGFβfamily signaling pathways

Wishful thinking (Wit) is a Drosophila transforming growth factor-β (TGFβ) superfamily type II receptor most related to the mammalian bone morphogenetic protein (BMP) type II receptor, BMPRII. To better understand its function, a biochemical approach was undertaken to establish the ligand binding repertoire and downstream signaling pathway. It was observed that BMP4 and BMP7, bound to receptor complexes comprised of Wit and the type I receptor Thickveins and Saxophone to activate a BMP-like signaling pathway. Further it was demonstrated that both Myoglianin and its most closely related mammalian ligand, Myostatin, interacted with a Wit and Baboon (Babo) type II-type I receptor complex to activate TGFβ/activin-like signaling pathways. These results thereby demonstrate that Wit binds multiple ligands to activate both BMP and TGFβ-like signaling pathways. Given that Myoglianin is expressed in muscle and glial-derived cells, these results also suggest that Wit may mediate Myoglianin-dependent signals in the nervous system (Lee-Hoeflich, 2005).

To provide insight into the molecular mechanisms of Wit that contribute to the biological functions of Wit, this study has characterized the Wit interacting ligands, their compatible type I receptor partners, and their downstream signaling pathways. The binding of BMP7, the mammalian ligand most related to Gbb, to Wit is in agreement with and gives biochemical evidence for results obtained from genetic analysis indicating that Wit mediates Gbb-activated BMP signaling in collaboration with the type I receptors, Tkv and Sax. The demonstration of the binding of BMP4, a functional ortholog of Dpp, to the receptor complex also suggests the possibility of Wit mediating Dpp signals. Dpp is not expressed in muscle or motoneurons but Wit is widely expressed in the central nervous system from embryonic stages suggesting that this putative Dpp signaling might regulate early developmental processes other than NMJ formation. The data showing that a receptor complex comprised of Wit and Tkv can activate MAD phosphorylation is also in agreement with the observation of impaired phosphorylation of MAD in Wit deficient flies and provides further support for a role of Wit in mediating BMP signaling (Lee-Hoeflich, 2005).

While the expression of dActivin in the developing nervous system and its proposed function in neuronal remodeling downstream of Wit or Punt have led to a suggestion that dActivin might induce Wit-mediated activin signaling, this study observed that mammalian activin, which is most closely related to dActivin, does not bind to Wit. One possible explanation for this discrepancy is that Wit might bind ligands other than dActivin and that this indirectly compensates for the lack of Punt-dActivin interaction. An attempt to produce dActivin in mammalian cells using a heterologous system was unsuccessful, thus the possibility cannot be eliminated that mammalian and Drosophila activins have different binding specificities. Generation of dActivin null flies or cell clones and testing for functional equivalence in rescue experiments should help resolve this issue (Lee-Hoeflich, 2005).

Alternatively, it is speculated that Wit might mediate activin signaling via Myoglianin since myostatin, the mammalian ligand most closely related to Myoglianin, activates a TGFβ/activin-like pathway. Accordingly, it was found that both myoglianin and myostatin bind to the Wit and Babo receptor complex. Furthermore, it was observed that coexpression of Wit and Babo induces dSmad2 phosphorylation and mediates myostatin-induced transcriptional activation of a TGFβ/activin-responsive reporter. In agreement to these observations, ectopic expression of Wit induces dSmad2 phosphorylation in insect S2 cells. Retrograde signaling between target-derived factors and the presynaptic terminal is crucial for NMJ development. Since Drosophila Myoglianin is abundantly expressed in muscle at late developmental stages and since Wit-mediated retrograde signaling had been identified previously, it is postulated that Myoglianin might activate a novel retrograde Wit signaling pathway. Interestingly, myostatin inhibits the BMP7 signaling response by competitive binding to type II receptor, ActRIIB, thus the binding of Myoglianin to Wit might also affect Gbb-mediated signaling and thus contribute to NMJ formation. Generation of flies harboring myoglianin loss-of-function mutations will shed light on these issues. These observations underscore the diverse mechanisms controlling Wit signaling and add impetus to further experiments in the context of the Wit receptor (Lee-Hoeflich, 2005).

Sequence and expression of myoglianin, a novel Drosophila gene of the TGF-beta superfamily

Various members of the TGF-beta superfamily of signaling molecules are known to have important roles in mesoderm patterning and differentiation during vertebrate and invertebrate embryogenesis. This study has characterize a new TGF-beta member from Drosophila, Myoglianin, that is most closely related to the vertebrate muscle differentiation factor Myostatin and to vertebrate BMP-11. Northern analysis shows that myoglianin is expressed throughout the Drosophila life cycle. In situ hybridization detects maternally-derived transcripts that are enriched in the pole plasm and later become enclosed in the pole cells. Between stages 11 and 14, myoglianin mRNA is exclusively detected in glial cells and their precursors. Following stage 14, high levels of myoglianin expression are observed in the developing somatic muscles as well as in visceral muscles and cardioblasts. It was also shown that the zygotic expression of the recently described Drosophila activinβ, which maps to the same interval on chromosome 4 as myoglianin, is restricted to the developing central and peripheral nervous system (Lo, 1999).

Using in situ hybridization, the pattern of myoglianin expression was examined during Drosophila development. In preblastoderm embryos, a high level of maternally-deposited myoglianin transcripts is uniformly distributed throughout the embryo, with an even higher localization at the very posterior end. This posterior concentration of message appears to be in the pole plasm since embryos laid by females homozygous for the oskar6 mutation lack pole plasm and do not show localization of myoglianin transcripts at the posterior. In stage 6 gastrulation embryos, the localized pole plasm message appears to be incorporated into pole cells, while the overall level of maternal transcript in the rest of the embryo has decreased. Zygotic expression of myoglianin is first observed in stage 11 embryos in what appear to be glial precursor cells. By stage 14, strong expression is evident in glial cells (CNS and exit/peripheral glia). This glial expression of myoglianin is completely lost in stage 15 embryos and expression is now detected in the somatic, visceral, and heart musculature, which then persists through late embryogenesis. In third instar larvae, the only specific pattern of expression observed is in the brain and ventral nerve cord, where a particular subset of cells, presumably glial cells, express myoglianin (Lo, 1999).


Functions of Myoglianin orthologs in other species

Sexually dimorphic control of gene expression in sensory neurons regulates decision-making behavior in C. elegans

Animal behavior is directed by the integration of sensory information from internal states and the environment. Neuroendocrine regulation of diverse behaviors of Caenorhabditis elegans is under the control of the DAF-7/TGF-β (see Drosophila myo) ligand that is secreted from sensory neurons. This study shows that C. elegans males exhibit an altered, male-specific expression pattern (see Drosophila sex determination) of daf-7 in the ASJ sensory neuron pair with the onset of reproductive maturity, which functions to promote male-specific mate-searching behavior. Molecular genetic analysis of the switch-like regulation of daf-7 expression in the ASJ neuron pair reveals a hierarchy of regulation among multiple inputs-sex, age, nutritional status, and microbial environment-which function in the modulation of behavior. These results suggest that regulation of gene expression in sensory neurons can function in the integration of a wide array of sensory information and facilitate decision-making behaviors in C. elegans (Hilbert, 2017).


REFERENCES

Search PubMed for articles about Drosophila Myoglianin

Augustin, H., McGourty, K., Steinert, J. R., Cocheme, H. M., Adcott, J., Cabecinha, M., Vincent, A., Halff, E. F., Kittler, J. T., Boucrot, E. and Partridge, L. (2017). Myostatin-like proteins regulate synaptic function and neuronal morphology. Development 144(13):2445-2455. PubMed ID: 28533206

Awasaki, T., Huang, Y., O'Connor, M. B. and Lee, T. (2011). Glia instruct developmental neuronal remodeling through TGF-β signaling. Nat. Neurosci. 14(7):821-3. PubMed ID: 21685919

Blagburn, J. M., Alexopoulos, H., Davies, J. A. and Bacon, J. P. (1999). Null mutation in shaking-B eliminates electrical, but not chemical, synapses in the Drosophila giant fiber system: a structural study. J Comp Neurol 404(4): 449-458. PubMed ID: 9987990

Demontis, F., Patel, V. K., Swindell, W. R. and Perrimon, N. (2014). Intertissue control of the nucleolus via a myokine-dependent longevity pathway. Cell Rep 7(5):1481-94. PubMed ID: 24882005

Gesualdi, S. C. and Haerry, T. E. (2007). Distinct signaling of Drosophila Activin/TGF-beta family members. Fly (Austin). 1(4): 212-21. PubMed ID: 18820452

Hilbert, Z.A. and Kim, D.H. (2017). Sexually dimorphic control of gene expression in sensory neurons regulates decision-making behavior in C. elegans. Elife 6. PubMed ID: 28117661

Jensen, P. A., Zheng, X., Lee, T. and O'Connor, M. B. (2009). The Drosophila Activin-like ligand Dawdle signals preferentially through one isoform of the Type-I receptor Baboon. Mech. Dev. 126: 950-957. PubMed ID: 19766717

Kim, M. J. and O'Connor, M. B. (2014). Anterograde Activin signaling regulates postsynaptic membrane potential and GluRIIA/B abundance at the Drosophila neuromuscular junction. PLoS One 9(9): e107443. PubMed ID: 25255438

Lee-Hoeflich, S. T., Zhao, X., Mehra, A., Attisano, L.T. (2005). The Drosophila type II receptor, Wishful thinking, binds BMP and myoglianin to activate multiple TGFbeta family signaling pathways FEBS Lett. 579: 4615-21. PubMed ID: 16098524

Liu, L. Y., Long, X., Yang, C. P., Miyares, R. L., Sugino, K., Singer, R. H. and Lee, T. (2019). Mamo decodes hierarchical temporal gradients into terminal neuronal fate. Elife 8. PubMed ID: 31545163

Lo, P. C. and Frasch, M. (1999). Sequence and expression of myoglianin, a novel Drosophila gene of the TGF-beta superfamily. Mech. Dev. 86(1-2): 171-5. PubMed ID: 10446278

Marchetti, G. and Tavosanis, G. (2017). Steroid hormone ecdysone signaling specifies mushroom body neuron sequential fate via Chinmo. Curr Biol 27(19): 3017-3024 e3014. PubMed ID: 28966087

Marchetti, G. and Tavosanis, G. (2019). Modulators of hormonal response regulate temporal fate specification in the Drosophila brain. PLoS Genet 15(12): e1008491. PubMed ID: 31809495

Marin, E.C., Watts, R.J., Tanaka, N.K., Ito, K. and Luo, L. (2005). Developmentally programmed remodeling of the Drosophila olfactory circuit. Development 132: 725-737. PubMed ID: 15659487

Peterson, A. J., Jensen, P. A., Shimell, M., Stefancsik, R., Wijayatonge, R., Herder, R., Raftery, L. A. and O'Connor, M. B. (2012). R-Smad competition controls activin receptor output in Drosophila. PLoS One 7(5): e36548. PubMed ID: 22563507

Rossi, A. M. and Desplan, C. (2020). Extrinsic activin signaling cooperates with an intrinsic temporal program to increase mushroom body neuronal diversity. Elife 9. PubMed ID: 32628110

Yu, X. M., Gutman, I., Mosca, T. J., Iram, T., Ozkan, E., Garcia, K. C., Luo, L. and Schuldiner, O. (2013). Plum, an immunoglobulin superfamily protein, regulates axon pruning by facilitating TGF-beta signaling. Neuron 78(3): 456-468. PubMed ID: 23664613

Zheng, X., et al. (2003). TGF-ß signaling activates steroid hormone receptor expression during neuronal remodeling in the Drosophila brain. Cell 112: 303-315. 12581521

Zhu, C. C., et al. (2008). Drosophila Activin-β and the Activin-like product Dawdle function redundantly to regulate proliferation in the larval brain. Development 135(3): 513-21. PubMed ID: 18171686


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date revised: 15 December 2020

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