decapentaplegic: Biological Overview | Evolutionary Homologs | Transcriptional regulation | Targets of activity | Protein Interactions | Post-transcriptional Regulation | Developmental Biology | Effect of mutation | References

Gene name - decapentaplegic

Synonyms -

Cytological map position - 22F1-2

Function - secreted morphogen

Keywords - dorsal-ventral patterning, imaginal disc development

Symbol - dpp

FlyBase ID:FBgn0000490

Genetic map position - 2-4.0

Classification - TGF-beta-like and BMP-like

Cellular location - secreted

NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Harmansa, S., Alborelli, I., Bieli, D., Caussinus, E. and Affolter, M. (2017). A nanobody-based toolset to investigate the role of protein localization and dispersal in Drosophila. Elife 6 [Epub ahead of print]. PubMed ID: 28395731
The role of protein localization along the apical-basal axis of polarized cells is difficult to investigate in vivo, partially due to lack of suitable tools. This study presents the GrabFP system, a collection of four nanobody-based GFP-traps that localize to defined positions along the apical-basal axis. The localization preference of the GrabFP traps can impose a novel localization on GFP-tagged target proteins and results in their controlled mislocalization. These new tools were used to mislocalize transmembrane and cytoplasmic GFP fusion proteins in the Drosophila wing disc epithelium and to investigate the effect of protein mislocalization. Furthermore, the GrabFP system was used as a tool to study the extracellular dispersal of the Decapentaplegic (Dpp) protein and showed that the Dpp gradient forming in the lateral plane of the Drosophila wing disc epithelium is essential for patterning of the wing imaginal disc.
Barrio, L. and Milan, M. (2017). Boundary Dpp promotes growth of medial and lateral regions of the Drosophila wing. Elife 6. PubMed ID: 28675372
The gradient of Decapentaplegic (Dpp) in the Drosophila wing has served as a paradigm to characterize the role of morphogens in regulating patterning. However, the role of this gradient in regulating tissue size is a topic of intense debate as proliferative growth is homogenous. This study combined the Gal4/UAS system and a temperature-sensitive Gal80 molecule to induce RNAi-mediated depletion of dpp and characterise the spatial and temporal requirement of Dpp in promoting growth. Dpp emanating from the AP compartment boundary was shown to be required throughout development to promote growth by regulating cell proliferation and tissue size. Dpp regulates growth and proliferation rates equally in central and lateral regions of the developing wing appendage and reduced levels of Dpp affects similarly the width and length of the resulting wing. Evidence is presented supporting the proposal that graded activity of Dpp is not an absolute requirement for wing growth.
Dahal, G. R., Pradhan, S. J. and Bates, E. A. (2017). Inwardly rectifying potassium channels regulate Dpp release in the Drosophila wing disc. Development [Epub ahead of print]. PubMed ID: 28684627
Loss of embryonic ion channel function leads to morphological defects, but the underlying reason for these defects remains elusive. This study shows that inwardly-rectifying potassium (Irk) channels regulate release of a Drosophila bone morphogenetic protein (BMP/Dpp) in the developing fly wing and this is necessary for developmental signaling. Inhibition of Irk channels decreases the incidence of distinct Dpp-GFP release events above baseline fluorescence while leading to broader distribution of Dpp-GFP. Work by others in different cell types show Irk channels regulate peptide release by modulating membrane potential and calcium levels. This study found calcium transients in the developing wing and inhibition of Irk channels reduces the duration and amplitude of calcium transients. Depolarization with high extracellular potassium evokes Dpp release. Taken together, these data implicate Irk channels as a requirement for regulated release of Dpp, highlighting the importance of the temporal pattern of Dpp presentation for morphogenesis of the wing.
Sanchez Bosch, P., Ziukaite, R., Alexandre, C., Basler, K. and Vincent, J. B. (2017). Dpp controls growth and patterning in Drosophila wing precursors through distinct modes of action. Elife 6 [Epub ahead of print]. PubMed ID: 28675374
Dpp, a member of the BMP family, is a morphogen that specifies positional information in Drosophila wing precursors. In this tissue, Dpp expressed along the anterior-posterior boundary forms a concentration gradient that controls the expression domains of target genes, which in turn specify the position of wing veins. Dpp also promotes growth in this tissue. The relationship between the spatio-temporal profile of Dpp signalling and growth has been the subject of debate, which has intensified recently with the suggestion that the stripe of Dpp is dispensable for growth. With two independent conditional alleles of dpp this study found that the stripe of Dpp is essential for wing growth. It was then shown that this requirement, but not patterning, can be fulfilled by uniform, low level, Dpp expression. Thus, the stripe of Dpp ensures that signalling remains above a pro-growth threshold, while at the same time generating a gradient that patterns cell fates.
Matsuda, S. and Affolter, M. (2017). Dpp from the anterior stripe of cells is crucial for the growth of the Drosophila wing disc. Elife 6. PubMed ID: 28675373
The Dpp morphogen gradient derived from the anterior stripe of cells is thought to control growth and patterning of the Drosophila wing disc. However, the spatial-temporal requirement of dpp for growth and patterning remained largely unknown. Recently, two studies re-addressed this question. By generating a conditional null allele, one study proposed that the dpp stripe is critical for patterning but not for growth. In contrast, using a membrane-anchored nanobody to trap Dpp, the other study proposed that Dpp dispersal from the stripe is required for patterning and also for medial wing disc growth, at least in the posterior compartment. Thus, growth control by the Dpp morphogen gradient remains under debate. By removing dpp from the stripe at different time points, this study shows that the dpp stripe source is indeed required for wing disc growth, also during third instar larval stages.
Ma, M., Cao, X., Dai, J. and Pastor-Pareja, J. C. (2017). Basement membrane manipulation in Drosophila wing discs affects Dpp retention but not growth mechanoregulation. Dev Cell 42(1): 97-106.e104. PubMed ID: 28697337
Basement membranes (BMs) are extracellular matrix polymers basally underlying epithelia, where they regulate cell signaling and tissue mechanics. Constriction by the BM shapes Drosophila wing discs, a well-characterized model of tissue growth. Recently, the hypothesis that mechanical factors govern wing growth has received much attention, but it has not been definitively tested. This study manipulated BM composition to cause dramatic changes in tissue tension. Increased tissue compression was found when perlecan was knocked down did not affect adult wing size. BM elimination, decreasing compression, reduced wing size but did not visibly affect Hippo signaling, widely postulated to mediate growth mechanoregulation. BM elimination, in contrast, attenuated signaling by bone morphogenetic protein/transforming growth factor beta ligand Dpp, which was not efficiently retained within the tissue and escaped to the body cavity. These results challenge mechanoregulation of wing growth, while uncovering a function of BMs in preserving a growth-promoting tissue environment.
Sarkar, A., Gogia, N., Farley, K., Payton, L. and Singh, A. (2018). Characterization of a morphogenetic furrow specific Gal4 driver in the developing Drosophila eye. PLoS One 13(4): e0196365. PubMed ID: 29702674
During Drosophila eye development, a synchronous wave of differentiation called Morphogenetic furrow (MF) initiates at the posterior margin resulting in differentiation of retinal neurons. There are not any Gal4 drivers available to observe the gain- of- function or loss- of- function of a gene specifically along the dynamic MF. The decapentaplegic (dpp) expresses at the posterior margin and then moves with the MF. However, unlike the MF associated pattern of dpp gene expression, the targeted dpp-Gal4 driver expression is restricted to the posterior margin of the developing eye disc. GMR lines harboring regulatory regions of dpp fused with Gal4 coding region were screened to identify MF specific enhancer of dpp using a GFP reporter gene. Immuno-histochemical approaches were used to detect gene expression. The rationale was that GFP reporter expression will correspond to the dpp expression domain in the developing eye. Two new dpp-Gal4 lines, viz., GMR17E04-Gal4 and GMR18D08-Gal4 were identified that carry sequences from first intron region of dpp gene. GMR17E04-Gal4 drives expression along the MF during development and later in the entire pupal retina whereas GMR18D08-Gal4 drives expression of GFP transgene in the entire developing eye disc, which later drives expression only in the ventral half of the pupal retina. Thus, GMR18D08-Gal4 will serve as a new reagent for targeting gene expression in the ventral half of the pupal retina. Misexpression phenotypes were compared of Wg, a negative regulator of eye development, using GMR17E04-Gal4, GMR18D08-Gal4 with existing dpp-Gal4 driver. The eye phenotypes generated by using the newly identified MF specific driver are not similar to the ones generated by existing dpp-Gal4 driver. It suggests that misexpression studies along MF needs revisiting using the new Gal4 drivers generated in these studies.
Apitz, H. and Salecker, I. (2018). Spatio-temporal relays control layer identity of direction-selective neuron subtypes in Drosophila. Nat Commun 9(1): 2295. PubMed ID: 29895891
Visual motion detection in sighted animals is essential to guide behavioral actions ensuring their survival. In Drosophila, motion direction is first detected by T4/T5 neurons. Their axons innervate one of the four lobula plate layers. How T4/T5 neurons with layer-specific representation of motion-direction preferences are specified during development is unknown. This study shows that diffusible Wingless (Wg) between adjacent neuroepithelia induces its own expression to form secondary signaling centers. These activate Decapentaplegic (Dpp) signaling in adjacent lateral tertiary neuroepithelial domains dedicated to producing layer 3/4-specific T4/T5 neurons. T4/T5 neurons derived from the core domain devoid of Dpp signaling adopt the default layer 1/2 fate. Dpp signaling induces the expression of the T-box transcription factor Optomotor-blind (Omb), serving as a relay to postmitotic neurons. Omb-mediated repression of Dachshund transforms layer 1/2- into layer 3/4-specific neurons. Hence, spatio-temporal relay mechanisms, bridging the distances between neuroepithelial domains and their postmitotic progeny, implement T4/T5 neuron-subtype identity.
Kane, N. S., Vora, M., Padgett, R. W. and Li, Y. (2018). bantam microRNA is a negative regulator of the Drosophila decapentaplegic pathway. Fly (Austin). PubMed ID: 30015555
Decapentaplegic (Dpp), the Drosophila homolog of the vertebrate bone morphogenetic protein (BMP2/4), is crucial for patterning and growth in many developmental contexts. The Dpp pathway is regulated at many different levels to exquisitely control its activity. This study shows that bantam (ban), a microRNA, modulates Dpp signaling activity. Overexpression of ban decreases phosphorylated Mothers against decapentaplegic (Mad) levels and negatively affects Dpp pathway transcriptional target genes, while null mutant clones of ban upregulate the pathway. Evidence is provided that dpp upregulates ban in the wing imaginal disc, and attenuation of Dpp signaling results in a reduction of ban expression, showing that they function in a feedback loop. Furthermore, this feedback loop is shown to be important for maintaining anterior-posterior compartment boundary stability in the wing disc through regulation of optomotor blind (omb), a known target of the pathway. These results support a model that ban functions with dpp in a negative feedback loop.
Denton, D., Xu, T., Dayan, S., Nicolson, S. and Kumar, S. (2019). Crosstalk between Dpp and Tor signaling coordinates autophagy-dependent midgut degradation. Cell Death Dis 10(2): 111. PubMed ID: 30737370
The majority of developmentally programmed cell death (PCD) is mediated by caspase-dependent apoptosis; however, additional modalities, including autophagy-dependent cell death, have important spatiotemporally restricted functions. Autophagy involves the engulfment of cytoplasmic components in a double membrane vesicle for delivery to the lysosome. An established model for autophagy-dependent PCD is Drosophila larval midgut removal during metamorphosis. Previous work demonstrated that growth arrest is required to initiate autophagy-dependent midgut degradation and Target of rapamycin (Tor) limits autophagy induction. This study uncovered a role for Decapentaplegic (Dpp) in coordinating midgut degradation. This study provides new data to show that Dpp interacts with Tor during midgut degradation. Inhibiting Tor rescued the block in midgut degradation due to Dpp signaling. It is proposed that Dpp is upstream of Tor and down-regulation promotes growth arrest and autophagy-dependent midgut degradation. These findings underscore a relationship between Dpp and Tor signaling in the regulation of cell growth and tissue removal.
Setiawan, L., Pan, X., Woods, A. L., O'Connor, M. B. and Hariharan, I. K. (2018). The BMP2/4 ortholog Dpp can function as an inter-organ signal that regulates developmental timing. Life Sci Alliance 1(6): e201800216. PubMed ID: 30515478
Developmental transitions are often triggered by a neuroendocrine axis and can be contingent upon multiple organs achieving sufficient growth and maturation. How the neurodendocrine axis senses the size and maturity of peripheral organs is not known. In Drosophila larvae, metamorphosis is triggered by a sharp increase in the level of the steroid hormone ecdysone, secreted by the prothoracic gland (PG). This study shows that the BMP2/4 ortholog Dpp can function as a systemic signal to regulate developmental timing. Dpp from peripheral tissues, mostly imaginal discs, can reach the PG and inhibit ecdysone biosynthesis. As the discs grow, reduced Dpp signaling in the PG is observed, consistent with the possibility that Dpp functions in a checkpoint mechanism that prevents metamorphosis when growth is insufficient. Indeed, upon starvation early in the third larval instar, reducing Dpp signaling in the PG abrogates the critical-weight checkpoint which normally prevents pupariation under these conditions. It is suggested that increased local trapping of morphogen within tissues as they grow would reduce circulating levels and hence provide a systemic readout of their growth status.
Neal, S. J., Dolezal, D., Jusic, N. and Pignoni, F. (2019). Drosophila ML-DmD17-c3 cells respond robustly to Dpp and exhibit complex transcriptional feedback on BMP signaling components. BMC Dev Biol 19(1): 1. PubMed ID: 30669963
BMP signaling is involved in myriad metazoan developmental processes, and study of this pathway in Drosophila has contributed greatly to understanding of its molecular and genetic mechanisms. These studies have benefited not only from Drosophila's advanced genetic tools, but from complimentary in vitro culture systems. However, the commonly-used S2 cell line is not intrinsically sensitive to the major BMP ligand Dpp and must therefore be augmented with exogenous pathway components for most experiments. This study identified and characterized the responses of Drosophila ML-DmD17-c3 cells, which are sensitive to Dpp stimulation and exhibit characteristic regulation of BMP target genes including Dad and brk. Dpp signaling in ML-DmD17-c3 cells is primarily mediated by the receptors Put and Tkv, with additional contributions from Wit and Sax. Furthermore, this study reports complex regulatory feedback on core pathway genes in this system. It is concluded that native ML-DmD17-c3 cells exhibit robust transcriptional responses to BMP pathway induction. It is proposed that ML-DmD17-c3 cells are well-suited for future BMP pathway analyses.
Gui, J., Huang, Y., Montanari, M., Toddie-Moore, D., Kikushima, K., Nix, S., Ishimoto, Y. and Shimmi, O. (2019). Coupling between dynamic 3D tissue architecture and BMP morphogen signaling during Drosophila wing morphogenesis. Proc Natl Acad Sci U S A. PubMed ID: 30760594
At the level of organ formation, tissue morphogenesis drives developmental processes in animals, often involving the rearrangement of two-dimensional (2D) structures into more complex three-dimensional (3D) tissues. These processes can be directed by growth factor signaling pathways. However, little is known about how such morphological changes affect the spatiotemporal distribution of growth factor signaling. Using the Drosophila pupal wing, this study addresses how Decapentaplegic (Dpp)/bone morphogenetic protein (BMP) signaling and 3D wing morphogenesis are coordinated. Dpp, expressed in the longitudinal veins (LVs) of the pupal wing, initially diffuses laterally within both dorsal and ventral wing epithelia during the inflation stage to regulate cell proliferation. Dpp localization is then refined to the LVs within each epithelial plane, but with active interplanar signaling for vein patterning/differentiation, as the two epithelia appose. These data further suggest that the 3D architecture of the wing epithelia and the spatial distribution of BMP signaling are tightly coupled, revealing that 3D morphogenesis is an emergent property of the interactions between extracellular signaling and tissue shape changes.
Lu, Y., Yao, Y. and Li, Z. (2019). Ectopic Dpp signaling promotes stem cell competition through EGFR signaling in the Drosophila testis. Sci Rep 9(1): 6118. PubMed ID: 30992503
Stem cell competition could select the fittest stem cells and potentially control tumorigenesis. However, little is known about the underlying molecular mechanisms. This study finds that that ectopic Decapentaplegic (Dpp) signal activation by expressing a constitutively active form of Thickveins (Tkv(CA)) in cyst stem cells (CySCs) leads to competition between CySCs and germline stem cells (GSCs) for niche occupancy and GSC loss. GSCs are displaced from the niche and undergo differentiation. Interestingly, it was found that induction of Tkv(CA) results in elevated expression of vein, which further activates Epidermal Growth Factor Receptor (EGFR) signaling in CySCs to promote their proliferation and compete GSCs out of the niche. These findings elucidate the important role of Dpp signaling in regulating stem cell competition and tumorigenesis, which could be shed light on tumorigenesis and cancer treatment in mammals.
Huang, H., Liu, S. and Kornberg, T. B. (2019). Glutamate signaling at cytoneme synapses. Science 363(6430): 948-955. PubMed ID: 30819957
This study investigated the roles of components of neuronal synapses for development of the Drosophila air sac primordium (ASP). The ASP, an epithelial tube, extends specialized signaling filopodia called cytonemes that take up signals such as Dpp (Decapentaplegic, a homolog of the vertebrate bone morphogenetic protein) from the wing imaginal disc. Dpp signaling in the ASP was compromised if disc cells lacked Synaptobrevin and Synaptotagmin-1 (which function in vesicle transport at neuronal synapses), the glutamate transporter, and a voltage-gated calcium channel, or if ASP cells lacked Synaptotagmin-4 or the glutamate receptor GluRII. Transient elevations of intracellular calcium in ASP cytonemes correlate with signaling activity. Calcium transients in ASP cells depend on GluRII, are activated by l-glutamate and by stimulation of an optogenetic ion channel expressed in the wing disc, and are inhibited by EGTA and by the GluR inhibitor NASPM (1-naphthylacetyl spermine trihydrochloride). Activation of GluRII is essential but not sufficient for signaling. Cytoneme-mediated signaling is glutamatergic.

In a first phase of its developmentally crucial expression, decapentaplegic is responsible for dorsal/ventral polarity in the fly. In a second phase of its activity, as segments appear, dpp functions in the definition of boundaries between segmental compartments. As part of this process, dpp, along with wingless and hedgehog, defines the position of future limbs, including wings, legs and antenna. Dpp also has an independent role in the structuring of the mesoderm. Later, during the final process of appendage development, and acting downstream of engrailed and hedgehog, dpp defines boundaries between appendage compartments assuring correct anterior/posterior polarity. dpp has an analogous function in the development of the eye, where it is primarily responsible for the progression of the morphogenetic furrow, the induction site of the Drosophila retina.

In the first process, the structuring of dorsal/ventral polarity, dpp is repressed in the ventral portion of the trunk by the dorsal gene product. Receptors for the secreted Dpp protein, Saxophone, Thick veins and Punt, are found in both the ectoderm and the underlying mesoderm. They mediate the transduction of the dpp signal to the interior of the cell through a phosphorylation cascade activating gene transcription (for review, see Raftery, 1999). Phosphorylation, the major mechanism of the cell for transduction of signals from protein to protein, involves the attachment of phosphate residues to appropriate target molecules.

The effects on the dorsal-most region of the fly are regulated by Saxophone in conjuction with Thick veins and Punt. Without Dpp signals through Saxophone, the amnioserosa, the most dorsal ectodermal tissue, does not develop properly and dorsal closure, the sealing of a dorsal "hole" in the developing embryo does not take place. The dorsal region is ventralized, and it develops characteristics of the ventral neuroectoderm. In a sense, the Saxophone-defective fly develops upside down, and the dorsal region starts to resemble ventral tissue.

The initial effects of Dpp on the heart and on visceral mesoderm (gut muscles) are mediated by Thick veins and Punt. An early and important event in the subdivision of the mesoderm is the restriction of tinman expression to dorsal mesodermal cells, the precursors of heart cells.

Whereas initial Dpp signals eminate from the ectoderm, later Dpp takes on a life of its own in the visceral mesoderm. It is activated by Ultrabithorax and repressed by abdominal-A. In the mesodermal midgut, the cluster of homeotic genes Sex combs reduced, Antennapedia, and abd-A are expressed in non-overlapping anterior/posterior domains. They are responsible for drawing string-like constrictions in three parts of the midgut and the outgrowth of pockets, or as they are termed, caeca. Dpp with its ability to define boundaries has a role in this process. Dpp secreted by the mesoderm also leads to the induction of labial in the endoderm. Thus Dpp also induces local differentiation in the endoderm (Manak, 1995). The actions of dpp on gene activation are not always positive. dpp actively suppresses the development of the proventriculus, confining it to the foregut.

The importance of Dpp expression cannot be overstated, both early in allocation of cells to appendages and later in compartment subdivision of these appendages. Secreted in an anterior to posterior stripe in the trunk, Dpp intersects wingless expressing cells under the control of hedgehog in segmentally repeated dorsal/ventral stripes. All three proteins are needed to allocate cells for the formation of imaginal discs, which will ultimately develop into appendages. This process involves induction of distal-less and aristaless, both of which are needed to specify the tips of appendages (Campbell, 1993 and Diaz-Benjumea, 1994).

The imaginal disk expression of Dpp in a narrow stripe of cells along the anterior-posterior compartment boundary is essential for proper growth and patterning of the Drosophila appendages. Dpp receptor function was examined in the formation of the wing to understand how this localized Dpp expression produces its global effects in appendage development. This work depicts in a very clear fashion how Dpp functions as a morphogen. Clones of saxophone (sax) or thick veins (tkv) mutant cells, defective in one of the two type I receptors for DPP, show shifts in cell fate along the anterior-posterior axis. In the adult wing, clones that are homozygous for a null allele of sax or a hypomorphic allele of tkv show shifts to more anterior fates when the clone is in the anterior compartment and to more posterior fates when the clone is in the posterior compartment. The effect of these clones on the expression pattern of the downstream gene spalt-major also correlates with these specific shifts in cell fate. The shift in cell fate is explained by assuming that the cells in mutant clones act as though they see a lower than normal Dpp concentration. Thus cell fate along the A/P axis is directly related to the perceived Dpp level. It is concluded that cell fate is directly related to the distance of cells from the source of Dpp at the A/P axis and that Dpp is responsible for patterning of the entire wing blade in direct response to the long-range Dpp signal. The similar effects of sax null and tkv hypomorphic clones indicate that the primary difference in the function of these two receptors during wing patterning is that Tkv transmits more of the Dpp signal than does Sax. These results are consistent with a model in which a gradient of Dpp reaches all cells in the developing wing blade to direct anterior-posterior pattern. While current evidence suggests that Tkv is absolutely required for Dpp signaling, there appears to be no such absolute requirement for Sax. Thus Dpp receptor complexes that lack a Tkv subunit cannot transmit a sufficient level of Dpp signal to trigger a biological response in the receiving cell. In contrast, receptor complexes lacking Sax subunits are still capable of significant signal reception and downstream signaling (Singer, 1997).

Dpp function in the wing imaginal disc

Cell proliferation and patterning must be coordinated for the development of properly proportioned organs. If the same molecules were to control both processes, such coordination would be ensured. This possibility has been investigated in the Drosophila wing using the Dpp signaling pathway. Previous studies have shown that Dpp forms a gradient along the AP axis that patterns the wing, that Dpp receptors are autonomously required for wing cell proliferation, and that ectopic expression of either Dpp or an activated Dpp receptor, TkvQ253D, causes overgrowth. These findings are extended with a detailed analysis of the effects of Dpp signaling on wing cell growth and proliferation. Increasing Dpp signaling by expressing TkvQ253D accelerates wing cell growth and cell cycle progression in a coordinate and cell-autonomous manner. Conversely, autonomously inhibiting Dpp signaling using a pathway specific inhibitor, Dad, or a mutation in tkv, slows wing cell growth and division, also in a coordinate fashion. Stimulation of cell cycle progression by TkvQ253D is blocked by the cell cycle inhibitor RBF, and requires normal activity of the growth effector, PI3K. Among the known Dpp targets, vestigial was the only one tested that was required for TkvQ253D-induced growth. The growth response to altering Dpp signaling varies regionally and temporally in the wing disc, indicating that other patterned factors modify the response (Martín-Castellanos, 2002).

To address the cell autonomous effects of the Dpp signaling pathway, the Flp/Gal4 method was used to activate or suppress Dpp signaling in clones of cells marked with GFP. First, a mutant version of the Dpp type I receptor Thick veins, TkvQ253D, containing a point mutation in the glycine/serine rich domain (GS) was expressed. This mutation mimics the receptor phosphorylation that occurs upon ligand binding, and therefore renders the receptor constitutively active and ligand independent. TkvQ253D expression strongly activates the Dpp signaling pathway, inducing high levels of the phospho-Mad transducer and expression of two Dpp targets, omb and spalt. Initially, clones of cells that expressed TkvQ253D were induced in early second instar larvae (at 48 hours AED) and the cells were allowed to proliferate until the end of larval development (120 hours AED). Wing cell clones expressing TkvQ253D showed smooth borders compared with control clones, which showed jagged borders, and were also larger than control clones. This phenotype is stronger in lateral areas of the disc, far from the endogenous Dpp source. Approximately half of the lateral clones were completely round and bulged out of the disc epithelium, which generated extra folds around the clones. This phenotype was not seen when TkvQ253D was expressed throughout the disc, indicating that the round bulging clonal phenotype is a consequence of abnormal heterotypic interactions between TkvQ253D-expressing cells and wild-type cells (Martín-Castellanos, 2002).

Induction of clones by heat shock allowed the age of the clones to be controlled, and also allowed the inference of cell proliferation rates from the number of cells per clone. Since cell death was observed by Acridine Orange staining in TkvQ253D-expressing clones, the apoptotic inhibitor p35 was expressed to block cell death. This was necessary to obtain accurate proliferation rate measurements, which are confounded by cell death. Clones were induced and allowed to proliferate for a short time in the period of larval development when imaginal wing cells proliferate exponentially. Because of the regional phenotype described above, the number of cells per clone was counted in lateral and medial areas, as well as in the entire presumptive wing region. Cells over-expressing the activated Dpp receptor proliferate faster than control cells. This phenotype is stronger in lateral areas, where TkvQ253D-expressing cells proliferate 20% faster than controls. TkvQ253D-expressing cells proliferate 10% faster than controls in the medial region. This regional phenotype reflects the graded activity of endogenous Dpp signaling; lateral areas normally low in Dpp are more sensitive to signaling activation (Martín-Castellanos, 2002).

To further analyze the cellular phenotype, flow cytometry (FACS) was performed using co-expressed GFP to identify TkvQ253D-expressing cells. The GFP-negative cell population from the same discs was used as an internal control. TkvQ253D overexpression shifts the distribution of cells in the different phases of the cell cycle. A smaller proportion of the TkvQ253D-expressing cells are in the G1 phase and greater proportion in G2. These data, together with the shorter doubling time of these cells, suggests that TkvQ253D preferentially promotes G1/S progression. This cell cycle phenotype is more severe if the activated receptor is expressed for a longer period of time (Martín-Castellanos, 2002).

To address more carefully the autonomy of the effects of TkvQ253D, the expression patterns of String and Cyclin E protein were analyzed in discs containing TkvQ253D-expressing clones. String and Cyclin E limit progression of the imaginal disc cell cycle through G2/M and G1/S transitions, respectively. S-phase progression in TkvQ253D-expressing clones was also assessed using BrdU incorporation, and mitosis by phospho-Histone H3 detection. The BrdU incorporation assay yielded a result consistent with increased proliferation within TkvQ253D-expressing clones in lateral regions of the discs: these clones show a uniform increase in BrdU uptake. Increased BrdU incorporation is limited to within the TkvQ253D-expressing clones, and no non-autonomous effects were detected. This result implies that TkvQ253D stimulates cell proliferation cell-autonomously. No changes were detected in Cyclin E, String or phospho-Histone H3 expression levels in TkvQ253D-expressing clones or surrounding cells (Martín-Castellanos, 2002).

Although raising the levels of Dpp signaling increases rates of cell proliferation, it does not appear to bypass the developmentally programmed proliferation arrest that occurs at the end of larval development. TkvQ253D-expressing clones induced late in larval development (96 hours AED) contain the same number of cells as control clones. The same result was obtained when p35 was co-expressed. In addition, TkvQ253D-expressing clones induced early (48 hours AED) and analyzed in pupae (168 hours AED) do not contain mitotic cells. This suggests that a dominant, developmentally programmed signal prevents TkvQ253D-expressing cells from continuing to divide beyond the normal proliferation stage (Martín-Castellanos, 2002).

Induction of cell proliferation does not necessarily indicate increased growth. To more directly assess the ability of TkvQ253D to induce growth, areas of the disc epithelium encompassed by TkvQ253D-expressing clones were measured. Clones were induced early in larval development and analyzed at the end of the larval period. The average area of TkvQ253D-expressing clones was 2.5 times larger than that of control clones, indicating that TkvQ253D-expressing cells grow faster than wild-type cells. This phenotype depends on the position of the clone in the anterior-posterior axis. Clones in the lateral areas, far from the source of endogenous Dpp, showed the strongest phenotype. Fifty percent of these lateral clones were larger than the largest control clone. On average, lateral clones expressing TkvQ253D were 3.7 times larger than lateral control clones (Martín-Castellanos, 2002).

The cellular growth effects of TkvQ253D were further assessed using FACS analysis to measure cell size. The ratio of the mean forward light scatter (FSC) of GFP+ cells versus GFP- cells was measured as a cell size indicator. GFP expression did not cause a significant change in cell size. TkvQ253D-expressing cells analyzed by FACS generally showed a size that was not significantly different from wild-type cells. In some experiments, however, these cells were slightly larger than controls. The fact that TkvQ253D-expressing clones are much larger than controls, but consist of cells of roughly normal size, confirms that TkvQ253D accelerates cell cycle progression. Taking the in situ and FACS analyses together, it is concluded that activation of Dpp signaling coordinately increases both rates of cell proliferation and cell growth (Martín-Castellanos, 2002).

To complement these experiments, the effects of autonomously inhibiting Dpp signaling were analyzed by overexpressing the pathway-specific inhibitor Dad, or by generating cell clones mutant for tkv. Dad is an inhibitory Smad protein that, when overexpressed, blocks omb expression and the adult wing phenotypes induced by ectopic Dpp signaling. It is normally activated by Dpp signaling and expressed in a broad domain centered on the AP axis. When Dad was overexpressed using the Flp/Gal4 method, clones were not recovered in the dorsomedial area of wing blade. However, Dad-expressing clones were recovered in medial areas when the apoptotic inhibitor p35 was co-expressed. These clones contained fewer cells than controls, indicating that Dad overexpression impairs proliferation of cells at medial positions. The cell doubling time of Dad overexpressing medial cells was more than 3 hours (22%) longer than the control doubling time. Slow-growing cells are eliminated by a mechanism known as cell competition when normal growing cells surround them. Because Dad overexpressing cells proliferate slowly, this may explain why they are not recovered unless the apoptotic inhibitor p35 is co-expressed (Martín-Castellanos, 2002).

To better understand the basis of this proliferative defect, tkv- clones were generated by mitotic recombination. A recessive lethal allele, tkv7, was used that carries a point mutation in a conserved glutamate residue in the kinase domain and results in loss of expression of Dpp targets. In the medial wing pouch, tkv7 clones survive for 36 hours but are lost within 48 hours of induction (in the 72-120 hours AED interval). In lateral areas, tkv7 mutant clone survival is greater, however mutant clones are still small compared with wild-type twin spots, and show round morphology. This lateral-medial survival phenotype reflects the lower requirement for Dpp signaling in lateral areas of the wing imaginal disc (Martín-Castellanos, 2002).

Flow cytometry was used to analyze tkv7 cells. To counteract cell competition and enrich the population of mutant cells, a cell lethal Minute mutation, M(2)32A1, was used that carries a lesion in ribosomal protein S13, and slows growth when heterozygous. Since M-/- cells are not viable, only M+/+ cells were recovered after mitotic recombination. These M+/+ cells were tkv7 homozygous. In the Minute background, tkv7 cells survive at least 4 days and colonize more tissue than in a wild-type background. However, they are still growth impaired relative to wild-type cells growing in the same Minute+/- background, and they still appear mainly in lateral areas. Approximately 30% of the tkv7 discs showed an aberrant morphology, probably caused by abnormal adhesive interactions between mutant and wild-type cells. tkv7 cells show a cell cycle profile consistent with a proliferation defect; the S phase fraction is extremely reduced and the G1 fraction is increased. This phenotype is opposite that of cells overexpressing TkvQ253D, which has a shortened G1. FACS analysis also showed that tkv7 cells are not detectably different in size from control cells. Previous studies indicate that when cell cycle progression is specifically delayed, cell size increases since cells continue to grow at the normal rates. Since tkv7 cells proliferate very slowly while maintaining a normal cell size, evidently they are impaired for growth as well as cell cycle progression (Martín-Castellanos, 2002).

Interestingly, M(2)32A1/+ cells are larger than wild-type cells. This suggests that these cells divide more slowly than they grow, and thus that the growth defect caused by the Minute mutation affects cell cycle progression preferentially. In fact, in both budding and fission yeast cell cycle control genes are sensitive to translational conditions. Studies using another Minute mutation that encodes a ribosomal protein, M(3)95A, detected no size alteration in M/+ cells, and thus this effect may be gene specific (Martín-Castellanos, 2002).

Using a third approach to avoid the effects of cell competition, Dad was induced ubiquitously throughout the wing disc using the A9-Gal4 driver. This causes a reduction of disc size. This size reduction is especially pronounced along the AP axis and thus is opposite that of the phenotype resulting from TkvQ253D expression using the same driver, which enlarges the wing disc preferentially along the AP axis. These results show that inhibition of Dpp signaling reduces growth and impairs proliferation, whereas activation of Dpp signaling increases growth and accelerates proliferation (Martín-Castellanos, 2002).

If growth and cell cycle progression are independently regulated by Tkv, one would expect to detect the proliferative effect of TkvQ253D even in growth-impaired cells. Alternatively, if TkvQ253D were to promote cell cycle progression indirectly via stimulating cellular growth, the proliferative effect of TkvQ253D should be inhibited when cell growth is impaired (Martín-Castellanos, 2002).

To suppress cell growth a truncated version of p60, Deltap60, was expressed. This is an adaptor molecule for the class I Phosphoinositide 3-Kinase (PI3K/Dp110 in Drosophila. Dp110 signaling is a potent growth inducer. Adaptor molecules, such as p60, bind to the Dp110 kinase and recruit it to the Insulin Receptor, allowing full activation of the enzyme. Deltap60 binds the Insulin Receptor but cannot bind Dp110, and thus inhibits Dp110 signaling in a dominant-negative manner. When expressed in wing cells, Deltap60 reduces cell size and strongly delays G1 progression. Flp/Gal4 clones expressing Deltap60 contain very few cells compared with controls. Overexpressed Deltap60 also dominantly blocks the growth and proliferation effects of TkvQ253D. Clones of cells that co-express Deltap60 and TkvQ253D contain as few cells as those expressing Deltap60 alone, and these cells are just slightly larger than those expressing Deltap60 alone. Thus, loss of growth resulting from loss of PI3K activity cannot be rescued by hyperactivating Dpp signaling, and cell proliferation induced by Dpp probably requires Dp110 activity. These results are consistent with the model in which Dpp-driven cell growth indirectly promotes cell cycle progression (Martín-Castellanos, 2002).

Although clonal growth is blocked by co-expressing Deltap60 and TkvQ253D, cells that co-express Deltap60 and TkvQ253D do not show the G1 delay characteristic of cells expressing Deltap60 alone. Thus, TkvQ253D appears to be able to promote G1/S progression even in the presence of Deltap60. This suggests that some aspects of cell cycle progression induced by TkvQ253D may be Dp110 independent. However, the slight increase in size observed in cells co-expressing Deltap60 and TkvQ253D makes it difficult to rule out the possibility that this effect on G1/S progression also occurs indirectly, as a consequence of increased growth (Martín-Castellanos, 2002).

In the wing imaginal disc, omb, spalt and vestigial (vg) have been reported to respond to Dpp signaling. It was of interest to know which if any of these genes was involved in controlling tissue growth effected by TkvQ253D. spalt is probably not required, since Spalt protein is not induced by TkvQ253D expression in the lateral areas of the wing disc, where the strongest overgrowth effects are observed. In the case of omb and vg, null alleles were used as a genetic background in which the expression of the activated Dpp receptor was induced. TkvQ253D can promote growth in the absence of Omb (Martín-Castellanos, 2002).

By contrast, TkvQ253D is not able to promote tissue growth in a null vg83b27R background. This result points to Vg as a possible effector of growth induced by Dpp signaling. Consistently, ectopic Vg expression induces wing-like outgrowths in imaginal discs. However, it was surprising to find that clones expressing TkvQ253D do not show increased levels of Vg protein, regardless of their position in the disc. Some lateral clones express Vg, but these most probably originate in the Vg expression domain. In fact, clones in lateral positions where Vg is expressed over-grow better than in other regions. These results suggest that activation of Dpp signaling is not sufficient to induce Vg expression, but that TkvQ253D and Vg might synergize to effect tissue growth (Martín-Castellanos, 2002).

Thus cell growth and cell cycle progression are coordinately regulated. These findings extend earlier studies that indicated a role for Dpp signaling in tissue growth. The 'balanced' effects on cell growth and cell proliferation caused by TkvQ253D differ markedly from results obtained when other growth stimulatory factors are manipulated in the developing wing. Ras, Myc and PI3K stimulate wing cell growth. Growth mediated by ectopic expression of these factors leads to a truncated G1 phase, which in the case of Ras and Myc has been attributed to post-transcriptional upregulation of the G1/S regulator Cyclin E. However, hyperactivation of Ras, Myc or PI3K signaling does not increase overall rates of wing cell proliferation, apparently because of a failure to stimulate G2/M progression. Consequently, these factors drive 'unbalanced' growth characterized by substantial increases in cell size. By contrast, ectopic TkvQ253D causes an increase in overall rates of cell division. Thus, TkvQ253D must induce G2/M as well as G1/S progression. Although no changes in Cyclin E or String levels have been detected by immunofluorescence, it is possible that small differences not detectable by antibody staining are responsible for G1/S and G2/M promotion (Martín-Castellanos, 2002).

Although early studies of wing development suggested that gradients of signaling might be the driving force that promotes cell growth in the wing, recent work has suggesting that Dpp signaling need not be employed in a gradient to stimulate growth. Dpp signaling in TkvQ253D-expressing clones is intense and homogenous, as assayed by anti-phospho-Mad staining, even in lateral areas. This suggests that gradients of Dpp signaling within these clones have been obliterated. Nevertheless, a variety of assays indicate that cell proliferation is promoted uniformly and autonomously throughout the clones, rather than at their edges, where sharp differentials of signaling intensity occur. Gradient models also predict non-autonomous effects on growth in regions bordering TkvQ253D-expressing clones. Although cell growth rates were not directly analyzed in these regions, inspection of markers for cell cycle progression did not detect major non-autonomous effects on cell proliferation. Thus, all these observations suggest that absolute intracellular levels of Dpp signaling, rather than gradients, are important for growth (Martín-Castellanos, 2002).

Survival of tkv-cells is better in regions of the wing that experience low level Dpp signaling. However, even in lateral regions far from the Dpp source, tkv- cells have a growth and proliferation defect. This suggests that all cells in the wing disc, including lateral cells, receive and require at least low levels of a Tkv ligand for normal growth. This led to the suggestion that some of the Dpp targets that mediate its growth effects might not have regionalized, nested expression patterns like two well-characterized Dpp targets, spalt and omb (which appear not to be mediators of TkvQ253D-induced growth). Instead, it seems plausible that some of the Dpp targets that mediate cell growth and proliferation are more uniformly expressed in regions where Dpp is required (Martín-Castellanos, 2002).

How might Dpp, expressed in a gradient, drive expression of growth regulatory targets more uniformly? It has been proposed that induction of target genes in cells receiving low levels of Dpp must overcome the activity of the transcriptional repressor, Brinker. brinker mutant clones in lateral areas of the wing disc exhibit a round morphology and over-growth phenotypes that are similar to TkvQ253D-expressing clones. brinker mutant discs also exhibit a dramatic over-growth phenotype along the AP axis similar to discs that overexpress TkvQ253D ubiquitously. Thus, it seems plausible that all wing cells require a threshold level of Dpp activity to grow, and that in lateral regions this threshold is equal to the amount of signaling activity needed to overcome repression of Dpp growth targets by Brinker. When Brinker is lost or TkvQ253D is expressed in lateral regions, this threshold level of signaling may be greatly surpassed, causing increased expression of growth regulators and acceleration of cell growth rates beyond normal levels (Martín-Castellanos, 2002).

The growth response of a cell to altered Dpp signaling varies according to its location in the disc. Ectopic TkvQ253D causes the strongest over-growth phenotypes in lateral regions, far from the source of endogenous Dpp, whereas inhibition of Dpp signaling has the strongest phenotypes in medial areas of the disc, where Dpp levels are normally high. Similar region-specific responses have been observed in experiments in which Notch or Wingless signaling is activated ectopically using cell autonomous effectors, or ligands. What is the significance of these region-specific responses? Without knowing the pertinent growth regulatory targets of these signaling systems, it is only possible to speculate. Perhaps the differential responses reflect cooperation between several regionally expressed signals that affect tissue growth, both positively and negatively, in a combinatorial fashion. Observations relating to vg seem consistent with this possibility. vg is required by TkvQ253D to promote tissue growth, yet Vg protein is not up-regulated by ectopic TkvQ253D, and TkvQ253D is capable of promoting overgrowth in wing regions where Vg is not detectable. The complex growth responses of cells to Dpp signaling illustrate how much is unknown about mechanisms of growth control. New, more global, approaches to studies of growth modulation will be required before its regulation by patterning signals can be understood. Important tasks for future studies include identifying the Dpp targets that stimulate cellular metabolism to effect growth, and determining how these targets integrate input from other patterning signals such as Wingless, Notch, Hedgehog and the Egfr ligands (Martín-Castellanos, 2002).

Specificity of Drosophila cytonemes for distinct signaling pathways

Cytonemes are types of filopodia in the Drosophila wing imaginal disc that are proposed to serve as conduits in which morphogen signaling proteins move between producing and target cells. The specificity was investigated of cytonemes that are made by target cells. Cells in wing discs made cytonemes that responded specifically to Decapentaplegic (Dpp) and cells in eye discs made cytonemes that responded specifically to Spitz (the Drosophila epidermal growth factor protein). Tracheal cells had at least two types: one made in response to Branchless (a Drosophila fibroblast growth factor protein, Bnl), to which they segregate the Bnl receptor, and another to which they segregate the Dpp receptor. It is concluded that cells can make several types of cytonemes, each of which responds specifically to a signaling pathway by means of the selective presence of a particular signaling protein receptor that has been localized to that cytoneme (Roy, 2011).

Cells in developing tissues are influenced by multiple signals that they process and integrate to control cell fate, proliferation, and patterning. An example is in the Drosophila wing imaginal disc, where cells depend on several signaling systems that are intrinsic to the disc. Dpp, Wingless (Wg), Hedgehog (Hh), and epidermal growth factor (EGF) are produced and released by different sets of disc cells, and receipt of these signaling proteins programs their neighbors to develop and grow. The mechanisms by which morphogen signaling proteins influence target cells must ensure both specificity and accuracy, and one possibility is that these proteins transfer at points of direct contact. Imaginal discs are flattened sacs that have a monolayer of columnar cells on one side and squamous peripodial cells on the other. Many cells in wing discs make filopodial extensions that lie along the surfaces of the monolayers, oriented toward morphogen-producing cells. These extensions have been termed cytonemes to denote their appearance as cytoplasmic threads and to distinguish them as specialized structures that polarize toward morphogen-producing regions (Roy, 2011).

In wing discs dissected from third instar larvae, cytonemes can be seen as filaments extending from randomly generated somatic clones engineered to express a fluorescent protein such as soluble, cytoplasmic green fluorescent protein (GFP) or a membrane-bound form such as mCD8:GFP (the extracellular and transmembrane domains of the mouse lymphocyte protein CD8 fused to GFP). To image disc cytonemes, unfixed discs were placed peripodial side down on a coverslip, covered with a 1-mm-square glass, and mounted over a depression slide with the disc hanging from the coverslip. Because fluorescence levels in cytonemes were low relative to background, recorded images were processed to increase intensity and were subjected to de-convolution. Expression of CD8:GFP in wing disc clones revealed cytonemes emanating from both the apical and basal surfaces of columnar cells, as well as from peripodial cells (whose apical and basal surfaces could not be distinguished). Most cytonemes were perpendicular to the anterior/posterior (A/P) axis of the disc and oriented toward the cells that produce Dpp at the A/P compartment border; others were oriented toward the cells that produce Wingless at the dorsal/ventral (D/V) compartment border. Disc-associated myoblasts also had filopodia (Roy, 2011).

In the eye disc, cells in the columnar layer organize into ommatidial clusters as a wave of differentiation [the morphogenetic furrow (MF)] passes from posterior to anterior. A second axis, centered at the equator, is orthogonal to the MF and defines a line of mirror-image symmetry where dorsal and ventral ommatidia are juxtaposed. The columnar cells divide during the third instar period but stop or divide only once after the MF passes. CD8:GFP expression was induced in somatic clones and the columnar cells were examined. Whereas clones of six to eight cells were present on both sides of the MF, only cells anterior to the MF had visible cytonemes. Cytonemes emanating from these clones oriented either toward the axis defined by the MF or toward the axis defined by the equator. Single clones with cytonemes oriented both toward the MF and toward the equator were not observed, and there was no apparent correlation between clone position and cytoneme orientation or cytoneme length. Cells in the peripodial layer of the eye disc also had cytonemes (Roy, 2011).

The EGF pathway is a key signaling system for eye development, and cells in the MF express the EGF protein Spitz (Spi). Because one of the two types of anterior cell cytonemes extended toward the MF and to explore the distribution of membrane-bound receptor proteins, clones were induced that expressed an epidermal growth factor receptor:GFP (EGFR:GFP) fusion protein. Anterior cells expressing EGFR:GFP had cytonemes that oriented toward the MF, and most of these cytonemes had fluorescent puncta; no cytonemes that were marked by EGFR:GFP oriented toward the equator. Other than their 'furrow-only' orientation, the cytonemes marked by EGFR:GFP were similar to those marked by CD8:GFP. In contrast, co-expression of CD8:GFP with (nonfluorescent) EGFR marked both furrow-directed and equator-directed cytonemes. Thus, expression of EGFR:GFP does not eliminate the equator-directed cytonemes, suggesting that the specific localization of EGFR:GFP to furrow-directed cytonemes is not a consequence of ectopic (over)expression of this fusion protein (Roy, 2011).

Evidence that the furrow-directed cytonemes depend on Spi/EGF signaling was obtained by expressing a dominant negative form of EGFR. Although EGFR is required for cell proliferation in the disc, small clones expressing EGFRDN were recovered that co-expressed EGFRDN and CD8:GFP; in these clones, only cytonemes that appeared to be randomly oriented were present, indicating that the long, furrow-directed cytonemes may require EGFR signal transduction in the cytoneme-producing cells (Roy, 2011).

Wing disc-associated tracheal cells also make cytonemes. The transverse connective (TC) is a tracheal tube that nestles against the basal surface of the wing disc columnar epithelium and that sprouts a new branch [the air sac primordium (ASP)] during the third instar period in response to Branchless (Bnl) expressed by the wing disc. Tracheal tubes are composed of a monolayer of polarized cells whose apical surfaces line a lumen. Expression of CD8:GFP throughout the trachea (btl-Gal4 UAS-CD8:GFP) made it possible to detect GFP fluorescence in several types of cytonemes emanating from the basal surfaces of the TC and ASP. Cytonemes at the tip of the ASP (length range, 12 to 50 μm; average length of 23 μm) contained the Breathless (Btl); the Drosophila fibrobast growth factor receptor (FGFR) and appeared to contact disc cells that express Bnl. Short cytonemes (length range, 2 to 15 μm; average length of 8.5 μm) extended from the TC cells in the vicinity of the ASP (Roy, 2011).

Tests were carried out to se whether Dpp, Spi, Bnl, and Hh affected wing disc, eye disc, and tracheal cytonemes differentially. Ubiquitous expression of Spi, Bnl, or Hh (induced by heat shock) did not alter the A/P-oriented apical cytonemes in the wing disc, and, in the eye disc, the long cytonemes of the columnar layer were unaltered after ubiquitous expression of Dpp, Bnl, or Hh. In contrast, long oriented cytonemes were absent in wing discs after ubiquitous expression of Dpp, and only short cytonemes that appeared to be randomly oriented were observed. Similarly, 0.5 to 3 hours after cSpi, a constitutively active form of EGF, was expressed ectopically by heat shock induction, clones expressing CD8:GFP in the eye disc had many short cytonemes that lacked apparent directional bias; in contrast to controls, no long cytonemes oriented toward the MF were observed. Cytonemes with normal orientation and length (including MF-directed cytonemes) were present in eye discs that were examined later, 8 hours after a pulse of cSpi expression. To monitor EGFR-containing cytonemes for sensitivity and responsiveness to Spi, cSpi was expressed by heat shock induction, and cells in clones expressing EGFR:GFP were examined. After a pulse of cSpi expression, the extensions oriented outward without apparent directional bias, and the EGFR:GFP puncta were present in all cytonemes (Roy, 2011).

To examine responses of the ASP tip cytonemes, Hh, Spi, Dpp, and Bnl were overexpressed by heat shock and GFP-marked cytonemes at the ASP tip were examined. No differences in number of cytonemes were detected until about 3 hours after heat shock. Four to 5 hours after heat shock, expression of Bnl increased the number of tip cytonemes by ~2.6 times, and although most of the cytonemes were <30 μm, the cytonemes >30 μm also increased (~3.2 times). Most of the long cytonemes in these preparations were oriented in directions other than toward the cells that normally express Bnl. The number of long cytonemes >30 μm did not change after overexpression of Hh, Spi, and Dpp (0.6 to 0.8 times); the number of short cytonemes increased after Dpp overexpression (~1.7 times) but not after overexpression of Hh or Spi (Roy, 2011).

Thus, the responses of apical wing disc cytonemes to overexpressed Dpp, of eye disc cytonemes to ubiquitous Spi, and of ASP tip cytonemes to exogenous Bnl (Drosophila FGF) are similar. These results suggest that the cytonemes detected in the wing discs and eye discs may have orientations and lengths that are dependent specifically on the respective sources of Dpp and Spi, whereas the ASP may extend cytonemes in response to more than one signaling protein. These results are, however, complicated by the heat shock mode of induction because both the cells that expressed GFP (and extended marked cytonemes) as well as the surrounding cells expressed the signaling proteins. To overcome this problem, a method was developed to induce two types of somatic clones in the same tissue, one that expressed GFP and another that expressed Dpp (Roy, 2011).

The GAL4 system was used to label cytonemes with CD8:GFP. Clones of GAL4-expressing cells were generated with heat shock-induced flippase (FLP recombinase). The second type of clone expressed a Dpp:Cherry fusion and was generated with a variant Cre-progesterone receptor recombinase that could be activated with a regime of heat shock and RU486. By adjusting the timing and strength of induction, wing discs were produced with small, independent, and relatively infrequent clones. In discs with clones that expressed ectopic Dpp as well as clones that expressed CD8:GFP, apical cytonemes tagged with GFP were detected that oriented toward nearby Dpp:Cherry-expressing cells and not toward either the A/P or D/V signaling centers. Such 'abnormally directed' cytonemes were never observed in control discs. The abnormally oriented cytonemes suggest that apical cytonemes in the wing blade respond directly to sources of Dpp and that their orientation reflects extant sources of signaling protein (Roy, 2011).

To characterize the relationship between tracheal ASP tip cytonemes and FGF signaling from the wing disc, the distribution of Btl (FGFR) was examined in ASP cells and in ASP cytonemes. In preparations from larvae with tracheal expression of both CD8:GFP and Btl:Cherry (btl-GAL4 UAS-CD8:GFP;UAS-Btl:Cherry), cytonemes were marked by CD8:GFP, some of which had fluorescent Btl:Cherry puncta. Each ASP had only a few long (>30 μm) cytonemes, most of which contained Btl:Cherry puncta. Few of the more numerous short cytonemes (<30 μm) contained Btl:Cherry puncta. To characterize Btl:Cherry after overexpression of Bnl, focus was placed on preparations obtained 1 to 2 hours post-induction (genotype btl-GAL4 UAS-CD8:GFP/HS-Bnl;UAS-Btl:Cherry/Gal80ts), because during this time interval the ASP morphology was close to normal but cytonemes had changed. ASPs were ignored after longer postinduction intervals because of major malformations to ASP morphology after 3 to 4 hours. Long cytonemes with Btl:Cherry puncta were present 1 hour after a pulse of Bnl expression; but 2 hours after the pulse, most ASPs had no long cytonemes, and the number of short puncta-containing cytonemes increased at the tip and along the shaft of the ASPs. After control heat shock or heat shock-induced expression of Dpp, the distribution of Btl:Cherry puncta in the ASP tip cytonemes was similar to normal controls: Long cytonemes had Btl:Cherry puncta, but most short cytonemes did not (Roy, 2011).

Because the number of small cytonemes at the ASP tip may have increased after ectopic Dpp expression, whether the thickveins (tkv) gene, which encodes a subunit of the Dpp receptor, is expressed in the ASP was investigated. Expression of the tkv reporter, tkv-lacZ (P{lacW}tkv16713), was detected in the ASP. When Tkv:GFP and Btl:Cherry were expressed together, Tkv:GFP and Btl:Cherry segregated to separate tip cytonemes at the ASP tip. Whereas Tkv-containing cytonemes were short (<30 μm), most of the Btl-containing cytonemes were longer (three of four of the Btl:Cherry-containing cytonemes were longer than 30 μm), and they lay in focal planes closer to the disc. These properties were consistent in all preparations examined in which both green Tkv and red Btl cytonemes were intact. Imaging these marked ASPs revealed that overexpressed Tkv:GFP and Btl:Cherry were present not only in the plasma membranes (as expected) but also in separate puncta in the cell bodies. This shows that Tkv and Btl receptors also segregated to separate locations in the ASP cell bodies (Roy, 2011).

These findings suggest that the ASP has long cytonemes that are specific to Bnl and specifically harbor Btl-containing puncta and that the ASP also has cytonemes that are specific to Dpp and specifically harbor Tkv. Similarly in the eye disc, the presence of EGFR:GFP in furrow-oriented cytonemes and not in equator-oriented cytonemes suggests that cytonemes in the eye disc also selectively localize receptors. And as was previously shown, apical cytonemes in the wing disc selectively localize Tkv. The apparent ligand specificities and contrasting makeup of these cytonemes suggest a diversity of functionally distinct subtypes: Cells appear to make cytonemes that respond specifically to the Dpp, EGF, or Bnl signaling proteins. The basal filopodia implicated in Delta-Notch signaling in the wing disc may represent yet another type (Roy, 2011).

The mechanism that endows cytonemes with specificity for a particular signaling protein cannot be based solely on tissue-specific expression of a receptor. Spi, Dpp, and Hh are active in eye discs, but only changes in Spi signaling affected the furrow-directed cytonemes. And in the wing disc, both the Hh and EGF signal transduction pathways are active in cells at the A/P compartment border, but the apical cytonemes only responded to overexpressed Dpp. The findings that tracheal cells in the ASP respond to both Dpp and Bnl and that the Tkv and Btl receptors are present in different cytonemes that the ASP cells extend suggest that specificity may be a consequence of the constitution of the cytoneme, not on which receptors the cells make. The mechanism that localizes receptors to different cytonemes is not known, but because the marked receptors that were expressed also segregated to different intracellular puncta, the processes that concentrate these receptors in separate locations may not be exclusive to cytonemes. There is a precedent for segregation of proteins to different cellular extensions, neurons segregate proteins to dendrites or axons, so extending projections with specific and distinct attributes may be a general property of cells (Roy, 2011).

Identification of target genes regulated by the Drosophila histone methyltransferase Eggless reveals a role of Decapentaplegic in apoptotic signaling

Epigenetic gene regulation is essential for developmental processes. Eggless (Egg), the Drosophila orthologue of the mammalian histone methyltransferase, SETDB1, is known to be involved in the survival and differentiation of germline stem cells and piRNA cluster transcription during Drosophila oogenesis; however the detailed mechanisms remain to be determined. Using high-throughput RNA sequencing this study investigated target genes regulated by Egg in an unbiased manner. Egg was shown to play diverse roles in particular piRNA pathway gene expression, some long non-coding RNA expression, apoptosis-related gene regulation, and Decapentaplegic (Dpp) signaling during Drosophila oogenesis. Furthermore, using genetic and cell biological approaches, this study demonstrate that ectopic upregulation of dpp caused by loss of Egg in the germarium can trigger apoptotic cell death through activation of two pro-apoptotic genes, reaper and head involution defective. A model is proposed in which Egg regulates germ cell differentiation and apoptosis through canonical and noncanonical Dpp pathways in Drosophila oogenesis (Kang, 2018).

This study used RNA-seq data analysis and qRT-PCR validation to demonstrate that Egg plays diverse roles in the regulation of piRNA production, lncRNA expression, apoptosis-related gene expression, and Dpp signaling during Drosophila oogenesis. Furthermore, using genetic and cell biological approaches, it was demonstrated that ectopic upregulation of dpp caused by loss of Egg in the germarium can trigger apoptosis in vivo (Kang, 2018).

Regarding piRNA production, the results revealed that among the known piRNA machinery components, ago3, krimp, mael, and zuc genes are the major piRNA-related targets of Egg. Consistent with the previously suggested role of Egg for promoting piRNA production, all of the putative target genes were downregulated in egg mutant ovaries. In Drosophila, two piRNA processing pathways, primary processing and secondary processing, have been proposed; the primary processing pathway functions in both germline and somatic cells by processing precursor piRNAs into piRNAs whereas the secondary processing pathway functions only in germline cells in which piRNAs are amplified by the ping-pong cycle. Given the involvement of Egg in both germline and somatic piRNA production, tests were performed to see whether decreased expression of zuc is responsible for the ovarian phenotypes caused by loss of Egg. Phenotypic changes were investigated after introduction of wild-type zuc transgenes under the control of the actin5C-Gal4 driver into egg mutant background, but the ovaries of the genotypes (egg2138/Df(2R)Dll-Mp; HA-tagged zuc (or EGFP-tagged zuc)/actin5C-Gal4) did not show any significant phenotypic changes compared with those of egg mutants. Ago3 is essentially involved in the secondary processing pathway along with Aub. The nuage, which surrounds the nuclei of nurse cells, has been proposed as a site for the ping-pong cycle. Various types of proteins, including Ago3, Krimp, and Mael, have been identified as nuage components. The decreased expression levels of the particular nuage components in egg mutant ovaries suggest that the reduction of germline piRNAs in egg mutants may result from not only a reduction of precursor piRNA transcription, as proposed previously, but also from a failure of the piRNA amplification pathway (Kang, 2018).

The data also revealed a previously unknown role of Egg as an lncRNA regulator. In Drosophila, the existence of lncRNAs has long been known, but only a few lncRNAs have been investigated. This study revealed that Egg is involved in regulating the expression of 100 potential lncRNA genes, and appears to play a repressive role in the expression of these lncRNAs. Among the upregulated lncRNAs in egg mutant ovaries, two well-known heat-inducible lncRNAs, αγ-element and hsr-ω are located in genomic regions where numerous TEs are found. Given the involvement of Egg in regulating piRNA production and thus TE mobilization, this raises an intriguing possibility of a link between the upregulation of αγ-element and hsr-ω in egg mutant ovaries and their genomic locations as TE hotspots. A strong upregulation of pncr003:2L and pncr004:X by loss of Egg is noteworthy, but the functional significance of these lncRNAs in the Drosophila ovary has not been determined. Although attempts were made to knock down pncr003:2L and pncr004:X using transgenic flies containing dsRNA for RNAi of pncr003:2L or pncr004:X under the control of the act5C-Gal4 driver, no phenotypic changes during oogenesis were detected. Interestingly, pncr003:2L was originally annotated as an lncRNA, but it was recently reported to encode two small functional peptides that are involved in regulating calcium transport in the Drosophila heart. Determination of the function of pncr003:2L in the Drosophila ovary is an interesting issue that needs to be addressed (Kang, 2018).

This study has demonstrated that dpp and dally were strongly upregulated in egg mutant ovaries and that dpp knockdown in ECs and early follicle cells in egg mutant background resulted in an increase in the size of the ovaries. Previously, by analysis of egg-RNAi knockdown in ECs, an EC-specific requirement for egg in controlling germ cell differentiation was suggested. Moreover, germ cell differentiation defects caused by egg knockdown in ECs was attributed to an increase in Dpp signaling because removal of one copy of dpp partially suppressed the tumorous phenotype caused by egg knockdown in ECs. The expression level of dally, an enhancer of Dpp signaling, may be maintained at a high level in the dpp knockdown in egg mutant background; therefore, the enhancement of Dpp signaling caused by loss of Egg may be maintained in the dpp knockdown in egg mutant germarium, thereby exhibiting similar differentiation defects as those observed in the egg mutant germarium. However, the defective cells in the dpp-knockdown in egg mutant background could be maintained for a longer time, which may be attributed to a reduction of the enhanced apoptosis in egg mutant ovaries because the increased expression levels of rpr and hid in egg mutant ovaries were significantly reduced in the dpp-knockdown in egg mutant background. Given that Egg is broadly expressed in germ cells and somatic cells in the ovary during oogenesis, dpp knockdown only in ECs and early follicle cells may not be sufficient to consistently counteract the overall apoptosis-promoting effect caused by dpp upregulation in egg mutant ovaries, which may explain the relatively low occurrence of the effect of the dpp knockdown in ECs and early follicle cells on the increase in ovary size (Kang, 2018).

A model is proposed in which loss of Egg may initially cause a relatively mild level of ectopic dpp and dally overexpression that may be sufficient to cause germ cell differentiation defects through repression of bam. Apoptosis may then be initiated when the level of ectopic dpp overexpression reaches a certain threshold level capable of inducing rpr and hid upregulation perhaps through a dpp positive-feedback loop. Alternatively, it cannot be ruled out that Egg represses dpp, dally, rpr and hid separately although the possibilities are not necessarily exclusive. Further studies are needed to determine the exact molecular mechanism by which Dpp signaling is increased in egg mutant ovaries. Given the role of BMPs in many mammalian stem cell systems and the existence of mammalian homologues of Egg and Dpp, the role of Egg in regulating Dpp signaling may provide important insights into their potential roles in mammalian stem cells (Kang, 2018).

CycD/Cdk4 and discontinuities in Dpp signaling activate TORC1 in the Drosophila wing disc

The molecular mechanisms regulating animal tissue size during development are unclear. This question has been extensively studied in the Drosophila wing disc. Although cell growth is regulated by the kinase TORC1, no readout exists to visualize TORC1 activity in situ in Drosophila. Both the cell cycle and the morphogen Dpp are linked to tissue growth, but whether they regulate TORC1 activity is not known. This study developed an anti-phospho-dRpS6 antibody that detects TORC1 activity in situ. Unexpectedly, it was found that TORC1 activity in the wing disc is patchy. This is caused by elevated TORC1 activity at the cell cycle G1/S transition due to CycD/Cdk4 phosphorylating TSC1/2.TORC1 is also activated independently of CycD/Cdk4 when cells with different levels of Dpp signaling or Brinker protein are juxtaposed. This study has thereby characterize the spatial distribution of TORC1 activity in a developing organ (Romero-Pozuelo, 2017).

During animal development, tissues increase tremendously in mass, yet stop growing at very stereotyped sizes in a robust manner. For instance, the Drosophila wing is specified as a cluster of circa 50 cells, which increases in mass ~500-fold before terminating growth. Once growth has ceased, the left and right wings of an individual fly are virtually identical in size, to within 1%, illustrating the robustness of this process. How animal tissue size is regulated is a fundamental open question in developmental biology (Romero-Pozuelo, 2017).

As mitotically growing tissues develop, two independent cellular processes occur in a coordinated manner: proliferation and cell growth. By itself, proliferation -- the division of cells -- does not lead to mass accumulation. This was nicely shown in the Drosophila wing where overexpression of E2F speeds up the cell cycle, but leads to a normally sized tissue containing more, smaller cells. For a tissue to grow, cells need to accumulate biomass. The mechanisms interconnecting cell proliferation and cell growth are not completely understood. In organisms from yeast to humans, growth is in large part regulated by the target of rapamycin complex 1 (TORC1) kinase. TORC1 promotes biomass accumulation by promoting anabolic metabolic pathways such as protein, lipid, and nucleotide biosynthesis, while repressing catabolic processes such as autophagy. Hence, to understand tissue growth it would be of interest to study the spatial distribution of TORC1 activity in a developing tissue. This line of investigation has been hampered, however, by the lack of readouts for TORC1 activity that can be used in situ (Romero-Pozuelo, 2017).

One signaling pathway that strongly affects tissue size is the Dpp pathway. Dpp is expressed and secreted by a stripe of cells in the medial region of the wing imaginal disc, and forms an extracellular morphogen gradient that both helps to pattern the wing and affects its size. In the absence of Dpp signaling during development, only small rudimentary wings are formed. In contrast, overexpression of Dpp leads to strong tissue overgrowth, in particular along the axis of the morphogen gradient. Several models have been proposed for how Dpp signaling regulates wing size. The exact mechanism by which Dpp regulates tissue size, however, is an unresolved issue. Dpp signaling acts to repress expression of a transcription factor called Brinker. Brinker appears to mediate most of the size effects of Dpp signaling. When Brinker is genetically removed, Dpp signaling becomes dispensable for wing growth. Given that Dpp signaling promotes tissue growth, an open question is whether Dpp signaling promotes TORC1 activity (Romero-Pozuelo, 2017).

Thia study examined whether Dpp signaling promotes TORC1 activity in the Drosophila wing disc. To this end, a phospho-RpS6 (pS6) antibody was developed that allows TORC1 activity to be assayed in situ in tissue. This reagent reveals unexpectedly that TORC1 activity in the growing wing disc is neither uniform nor graded, but is instead patchy. This patchiness is mediated via CycD/Cdk4 and the tuberous sclerosis 1 (TSC1)-TSC2 complex in response to cell cycle stage. Using this pS6 antibody, this study found that TORC1 activity is also induced by discontinuities in Dpp signaling or discontinuities in Brinker levels. It is proposed that these discontinuous conditions may be analogous to regenerative conditions that happen in the wing disc in response to tissue damage. In sum, this work reveals the pattern of TORC1 activity in the context of a developing organ (Romero-Pozuelo, 2017).

TORC1 activity in the wing disc is modulated by the cell cycle, with cells in early S phase showing the highest TORC1 activity. Interestingly, an accompanying paper finds similar results in the Drosophila eye disc (Kim, 2017). This might reflect a metabolic requirement by early S-phase cells for large amounts of nucleotide biosynthesis, an anabolic process promoted by TORC1. Indeed, in various contexts S6K and TORC1 activity were found to be required for the transition from G1 to S. Connections between mechanistic TOR (mTOR) and the cell cycle have previously been found in cultured cells. In human fibroblasts, mTOR shuttles in and out of the nucleus in a cell cycle-dependent manner, peaking in the nucleus shortly before S phase. The relevance of this subcellular relocalization to what is observe in this study, however, is unclear. In fibroblasts, S6K1 activity was found to be highest during early G1, whereas in HeLa cells it was found to be highest during M phase. In sum, it is unclear to what extent cells in culture recapitulate endogenous development, or whether the influence of the cell cycle on TORC1 activity is very context dependent. The TSC1/2 complex has been reported to be phosphorylated by cell cycle-dependent kinases.TSC1 is phosphorylated on Thr417 by Cdk1 during the G2/M transition. This inhibitory phosphorylation would lead to elevated TORC1 activity during G2/M, which does not fit with what was observe here, and thus might be relevant in a different developmental context. Instead, this study found that TSC2 can be phosphorylated by the CycD/Cdk4 complex on Ser1046, and possibly other sites as well, and that this leads to activation of TORC1. This fits with several observations in the literature. Firstly, in U2OS cells the TSC complex was also found to bind cyclin D, leading to its phosphorylation at unknown sites. In U2OS cells, this causes destabilization of the Tsc1 and Tsc2 proteins, which was not observed in this study. Secondly, Tsc1/2 and CycD/Cdk4 were previously found to interact genetically in Drosophila: The reduced tissue growth caused by Tsc1 + Tsc2 overexpression was found to be fully suppressed by expression of CycD + Cdk4. This fits well with the current data suggesting that CycD/Cdk4 directly inhibits the TSC complex via phosphorylation. Thirdly, Cyclin D and Cdk4 were previously reported in Drosophila to promote cell and tissue growth, fitting with activation of the TORC1 complex by CycD/Cdk4. It is worth noting that some patchy TORC1 activity is still seen in CycD- or Cdk4-null discs and in discs with the single phospho-site mutations in TSC2. Hence it is possible that Cdk4 may not be the only factor regulating TORC1 activity in response to the cell cycle, and that Cdk4 might phosphorylate TSC2 on additional sites (Romero-Pozuelo, 2017).

What are the roles of CycD/Cdk4 in cell cycle progression and cell growth? Whereas mammals have three cyclin D genes, CycD1-3, and two CycD binding kinases, Cdk4 and Cdk6, Drosophila has a single CycD, a single Cdk4, and no Cdk6. Hence Drosophila provides an opportunity to elucidate the function of the CycD/Cdk4 complex without difficulties arising from redundancy. Indeed, results in Drosophila clearly show that CycD/Cdk4 promotes cell growth and not cell cycle progression. Both CycD- and Cdk4-null animals are viable, and fluorescence-activated cell sorting (FACS) analysis of null cells revealed that they have a normal cell cycle profile, indicating that they are dispensable for normal cell cycle progression. Instead, Cdk4- and CycD-null animals are 10%-20% smaller than controls, indicating that they promote cell growth. The finding that CycD/Cdk4 activates TORC1 during the G1/S transition can provide one mechanism by which the CycD/Cdk4 complex promotes growth. Hence, from these data it is proposed that in Drosophila the CycD/Cdk4 complex is not part of the core machinery required for cell cycling, but is rather an effector 'side branch' activated at G1/S to promote cell growth. Data from the mouse suggest something similar. CycD1, CycD2, and CycD3 knockout mice are all viable. One could imagine this to be due to redundancy between these three genes, but actually CycD1, CycD2, CycD3 triple-knockout mice survive to mid-gestation, and the triple-knockout mouse embryonic fibroblasts proliferate relatively normally. The mid-gestation lethality of the triple knockouts appears to be due to specific effects in hematopoietic and myocardial cells. Hence, cyclins D1-D3 are also dispensable for cell cycle progression in mice. Interestingly, CycD1 knockout mice and CycD1, CycD2 double-knockout mice are viable but have reduced body size, reminiscent of the size phenotype observed in CycD knockout flies. In sum, despite CycD/Cdk4 being claimed in most reviews on the cell cycle as playing an important role in G1/S progression, it appears that this complex may function rather to promote cell growth in a cell cycle-dependent manner (Romero-Pozuelo, 2017).

Does Dpp control growth in the wing? When discontinuities in Dpp activity or in Brinker levels were genetically induce, activation was observed of TORC1 at the site of discontinuity. Hence, Dpp signaling per se does not appear to activate TORC1; rather, the comparison between high Dpp signaling and low Dpp signaling cells does. In an unperturbed disc, no pattern of pS6 staining was observed that correlates with the Dpp activity gradient, which is highest medially and drops toward the anterior and posterior extremities. This might be due to the fact that in an unperturbed disc the Dpp and Brinker gradients are smooth and do not have such discontinuities. A similar effect of Dpp was previously observed on cell prolife ration, except that in this case the effect of the Dpp discontinuity was very transient, lasting only a few hours after clone induction, whereas the effect seen on growth is sustained. Dpp signaling is, nonetheless, required for growth, because in the absence of Dpp, small vestigial wings are formed. Hence one interpretation might be that low levels of Dpp signaling are continuously required for growth, but that Dpp signaling becomes instructive for tissue growth only when discontinuities in the gradient arise, perhaps as a result of tissue damage or cell delamination, to initiate a regenerative response (Romero-Pozuelo, 2017).

One additional interesting non-autonomous phenomenon observed is that sometimes when a region of the wing disc has high pS6 levels, the rest of the disc loses its typically patchy pS6 pattern and becomes pS6 negative. This phenomenon is not understood, and future work will be necessary to understand it molecularly (Romero-Pozuelo, 2017).


cDNA clone length - There are 3 major transcripts ( 3.3, 3.8 and 4.3 kb). Transcript A is present early and reaches a peak at between 4 and 12 hours. Transcript B is detected from 8 to 12 hours and remains high throughout development. Transcript C accumulates early and remains at high levels. Minor transcripts are detected as well. Major transcripts differ in the first exon, which makes up most of the 3'UTR. Thus each of these different transcripts have different proximal promoters and each shares a second and third exon. The common second and third exons code for the single protein species of Dpp (St. Johnston, 1990).

Bases in 5' UTR - variable

Exons - three

Bases in 3' UTR - 1041 and variable


Amino Acids - 588

Structural Domains

The carboxy-terminal 100 amino acids have 25-40% homology to human and porcine TGFbeta, Inhibin A and Inhibin B. The homologous region is preceded by three arginine dimers that each function as proleolytic cleavage sites (Padget, 1987).

decapentaplegic :
Evolutionary Homologs | Transcriptional regulation | Targets of activity | Protein Interactions | Post-transcriptional Regulation | Developmental Biology | Effect of mutation | References
date revised: 26 Dec 96 

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