Mothers against dpp



The single 2.6-kb MAD mRNA is detected in all developmental stages, although it appear most abundant in early embryos, pupae, and adult females. In 0 to 4 hour embryos, which contain maternally loaded mRNA as well as early zygotic transcripts, a minor transcript that is slightly smaller than 2.6 kb is also detected. The abundance of the MAD transcript decreases throughout embryonic and larval development and then returns to high levels in pupae and adult females. A larger transcript is detected in total RNA from early pupal stages and from adult females (Sekeley, 1995)

Biphasic activation of the BMP pathway patterns the Drosophila embryonic dorsal region

The BMP pathway patterns the dorsal region of the Drosophila embryo. Using an antibody recognizing phosphorylated Mad (pMad), signaling was followed directly. In wild-type embryos, a biphasic activation pattern is observed. At the cellular blastoderm stage, high pMad levels are detected only in the dorsal-most cell rows that give rise to amnioserosa. This accumulation of pMad requires the ligand Screw (Scw), the Short gastrulation (Sog) protein, and cleavage of their complex by Tolloid (Tld). When the inhibitory activity of Sog is removed, Mad phosphorylation is expanded. In spite of the uniform expression of Scw, pMad expansion is restricted to the dorsal domain of the embryo where Dpp is expressed. This demonstrates that Mad phosphorylation requires simultaneous activation by Scw and Dpp. Indeed, the early pMad pattern is abolished when either the Scw receptor Saxophone (Sax), the Dpp receptor Thickveins (Tkv), or Dpp are removed. After germ band extension, a uniform accumulation of pMad is observed in the entire dorsal domain of the embryo, with a sharp border at the junction with the neuroectoderm. From this stage onward, activation by Scw is no longer required, and Dpp suffices to induce high levels of pMad. In these subsequent phases pMad accumulates normally in the presence of ectopic Sog, in contrast to the early phase, indicating that Sog is only capable of blocking activation by Scw and not by Dpp (Dorfman, 2001).

Thus two distinct phases of pMad activation have been identified. The early phase requires activation by both Scw and Dpp ligands, while the second phase depends only on Dpp. Signaling is first detected in the cellular blastoderm embryo. While activation is observed within the dorsal-most 8-10 cell rows, the sensitivity of the detection method fails to monitor signaling in the rest of the dorsal domain. High signaling levels are induced by Scw, and give rise to amnioserosa. Within the domain where pMad is observed, graded activation is detected, which may have the capacity to induce more than one cell fate in the region (Dorfman, 2001).

The cardinal players in the generation of the early pMad gradient are Scw, Tld and Sog. Tld has been suggested to generate a sink for the active ligand, by cleaving the Sog/ligand complex. The similarity between the pMad pattern of scw and tld mutants suggests that Tld is primarily involved in the release of Scw from the complex with Sog. Absence of Scw, Tld or Sax abolished the early pMad pattern while retaining the second phase, indicating that the second phase relies only on Dpp signaling. Similarly, overexpression of Sog eliminated only the early but not the subsequent pMad patterns. This suggests that Sog preferentially associates with Scw, in agreement with previous biological assays of Sog activity. Generation of graded patterning in the dorsal region does not rely on restricted gene expression within this domain. Rather, expression of genes confined to the neuroectoderm may lead to graded distribution of their gene products within the dorsal domain. The essential component for generation of graded patterning appears to be Sog, which is produced only in the neuroectoderm, but is capable of diffusing to the dorsal region. Disruption of the normal distribution of Sog by uniform misexpression, abolishes the early pMad activation profile (Dorfman, 2001).

This suggests that normally Sog may form a graded distribution in the dorsal region, which is essential for patterning. When the Sog/Scw complex is cleaved by Tld, Scw is released and can bind either Sog or Sax. The data suggest that in regions closer to the neuroectoderm, the levels of Sog are high and titrate the free ligand. In the dorsal-most region however, where Sog levels are low, the released Scw has a greater probability of binding and activating the Sax receptor, rather than being trapped again by Sog. Thus, the graded distribution of Sog is critical for generating the reciprocal distribution of Scw, and the ensuing activation profile (Dorfman, 2001).

In sog mutant embryos an expansion of the early pMad pattern is observed. In the absence of Sog, a uniform distribution of Scw is expected, and hence the activation level should be lower than the maximal level in wild-type embryos. The staining levels in wild-type and sog mutant embryos have been quantitated. While the pattern of staining is reproducible in all wild-type embryos, variations in the absolute levels of up to threefold between embryos were observed in any given staining reaction. It is thus difficult to compare reliably the wild-type level to the absolute staining levels of sog mutants. Nevertheless, the impression is that the expanded pMad in sog mutant embryos is comparable in levels to the maximal signaling levels in wild-type embryos. In spite of this expanded pMad activation pattern, amnioserosa cell fates are abolished in sog mutants. This result suggests that in addition to the role of Sog in determining the graded distribution of Scw, Sog or its cleavage products may provide an additional signal facilitating the induction of amnioserosa cell fates (Dorfman, 2001).

Activation of Tkv by Dpp is essential for the appearance of the early pMad pattern, corresponding to the future amnioserosa cells. At this stage, distinct cell fates are also induced in the dorsolateral cells, as reflected by expression of pnr and repression of msh expression. It is assumed that low levels of activation that may be induced by Dpp alone, but not detected by pMad antibodies, are responsible for these fates. Elimination of Dpp or Tkv leads to complete absence of early, as well as late, pMad patterns. Thus, Scw is not sufficient for the early activation phase, and the presence of Dpp is crucial. Cooperativity between Scw and Dpp occurs at the level of receptor activation. One possibility is that the observed pMad levels reflect only an additive effect of Scw and Dpp signaling. Indeed, the number of dpp copies has a profound effect on signaling levels and the shape of the early pMad distribution. Alternatively, it is possible that there is a synergistic interaction between Scw and Dpp signaling. In this case, the requirement of both ligands for the production of the early pMad pattern may indicate that synergy occurs at the level of receptor activation. Phosphorylation of Mad may require the formation of heterotetrameric receptors, containing both Sax/Put and Tkv/Put pairs. Cross linking experiments of the vertebrate receptors support this model (Dorfman, 2001).

Scw is required for generating the pMad pattern only in the early phase. All subsequent patterns rely only on Dpp. This feature may be explained differently by each of the above two models. If Scw and Dpp are required additively in the early phase, higher levels of Dpp may suffice to induce the pMad pattern at later stages. The autoregulatory effects of Dpp on its transcription may account for the elevation in Dpp levels. Alternatively, if Scw and Dpp signaling is synergistic, why is such a synergism necessary only in the early phase? In the early embryo, a maternal transcript encoding an inhibitor of BMP signaling may be translated, to block signaling by Sax/Put or Tkv/Put dimers. Such inhibitor(s) may be displaced only in ligand-bound heterotetrameric receptor complexes. The maternal transcripts of the inhibitor(s) may diminish by stage 9, to allow pMad production by activation of Tkv/Put alone (Dorfman, 2001).

By stage 8/9, Dpp/Tkv activation is sufficient to induce detectable levels of phosphorylated Mad. The second phase of activation does not rely on execution of the early phase, and is detected in scw, tld or sax mutants. A uniform pattern of pMad is observed at this stage within the entire dorsal domain, in accordance with the pattern of autoregulated dpp expression. In the neuroectoderm, brinker (brk) is expressed to suppress Dpp autoregulation. The uniform pMad pattern corresponds to the resulting expression pattern of genes like pannier (pnr) at stage 9, indicating that this second phase of activation is indeed instructive for induction of target genes in the entire dorsal domain. Once cell intercalation leading to germ band extension has been completed, it may be necessary to induce, within the dorsal region, such a uniform activation of Dpp target genes (Dorfman, 2001).

In the second phase, sharp borders of pMad localization are observed, with no detectable activation in the neuroectoderm. Dpp is a diffusible ligand, as indicated by the induction of pMad several cell rows away from the dorsal row of cells expressing Dpp at stage 11. Direct visualization of Dpp in the wing disc has also demonstrated its diffusion capacity over many cell rows. How are the sharp pMad borders generated at stage 9, in view of the diffusability of Dpp? It is suggested that the neuroectoderm cells may produce an inhibitor that prevents activation of the pathway by Dpp molecules that could diffuse from the adjacent dorsal region. Alternatively, the neuroectoderm cells may express cell surface proteins that would block the diffusion of Dpp into the neuroectoderm. When Dpp is expressed ectopically at physiological levels in perpendicular stripes, no pMad activation is observed in the neuroectoderm outside the stripes of Dpp expression. Thus, lower levels of Dpp are not capable of activating the pathway in the neuroectoderm at stage 9 (Dorfman, 2001).

EGF receptor attenuates Dpp signaling and helps to distinguish the wing and leg cell fates in Drosophila

Wing and leg precursors of Drosophila are recruited from a common pool of ectodermal cells expressing the homeobox gene Dll. Induction by Dpp promotes this cell fate decision toward the wing and proximal leg. The receptor tyrosine kinase Egfr antagonizes the wing-promoting function of Dpp and allows recruitment of leg precursor cells from uncommitted ectodermal cells. By monitoring the spatial distribution of cells responding to Dpp and Egfr, it has been shown that nuclear transduction of the two signals peaks at different positions along the dorsoventral axis when the fates of wing and leg discs are specified and that the balance of the two signals assessed within the nucleus determines the number of cells recruited to the wing. Differential activation of the two signals and the cross talk between them critically affect this cell fate choice (Kubota, 2000).

The spatial distribution of cells responding to Dpp and its relationship to Egfr signals was studied. To this end, an antibody specific to phosphorylated C-terminal sequence of Mad was produced. The phosphorylated sequence corresponds to the site at which the type I BMP receptor phosphorylates SMad1. The antibody detects an antigen distributed in a pattern similar to, but broader than, that of DPP mRNA. This immunoreactivity is dependent on Dpp signaling, as it is absent in stage 11 mutants of thick veins encoding type I Dpp receptor and in dpp mutants. This indicates that other extant TGFbeta-related signaling molecules present in Drosophila embryos do not substitute for Dpp to induce this immunoreactivity. Conversely, ectopic expression of Dpp results in high accumulation of this immunoreactivity. These results suggest that the antibody detects a Dpp-specific signaling event, most likely the phosphorylation and nuclear transport of Mad. Hereafter, the immunoreactivity detected by this antibody is called pSSVS (Kubota, 2000).

pSSVS is found mainly localized in the nucleus and distributed in regions a few cells wider in diameter than those of dpp-expressing cells. These properties are consistent with the previous findings that Mad transduces the Dpp signal to the nucleus. Double labeling of pSSVS and DLL mRNA shows that pSSVS expression is higher in the dorsal region of Dll-expressing cells. Combined with the double-labeling results of dpMAPK and Dll or dpp, it is concluded that cells responding to Dpp and Egfr overlap, but the peak of the responses are shifted. Such differential distribution of the two signals results in an arrangement of cells responding to a different strength of Dpp and Egfr along the dorsoventral axis (Kubota, 2000).

The increase in the number of wing disc cells in rho mutants resembles the overexpression phenotype of Dpp and raises a possibility that Egfr might prevent wing disc development by negatively regulating Dpp signaling. Such a cross talk could occur at several levels including the following: (1) regulation of dpp transcription, (2) signal transduction from Dpp receptors to the nucleus, and (3) transcriptional regulation of downstream target genes. The analyses excluded the first two possibilities for two reasons. (1) The expression pattern of DPP mRNA is unaffected by the mutation of rho. A previous report showing an expansion of dpp expression in Egfr mutants probably reflects the global patterning role of Egfr in the earlier stage. (2) pSSVS expression around limb primordia does not change in rho mutants. Conversely, the expression pattern of dpMAPK is not changed by a null mutation of tkv. These results suggest that the differential distribution of cells responding to Dpp and Egfr is set up independently of each other's activity (Kubota, 2000).

dad is an immediate transcriptional target gene of Dpp, the expression of which closely parallels that of pSSVS expression in embryos and is inducible by Dpp. dad expression is not affected in Egfr or rho mutants. Furthermore, elevated dad expression induced by Dpp is not affected by sSpi, suggesting that at least one of the immediate transcriptional responses to Dpp is unaffected by elevated Egfr signaling (Kubota, 2000).

The antagonism between Dpp and Egfr during wing disc development raises a question: what is the default state of the wing and leg primordia in the absence of the two signals? Double mutant phenotypes of Dpp and Egfr signaling were examined. tkv mutants lack wing discs and their leg discs are malformed. This phenotype reflects a disc cell autonomous requirement for Dpp signaling, because the phenotype is reproduced by the disc-specific inhibition of Dpp signaling by dad, which inhibits Mad. The phenotype of either tkv;rho or tkv;Egfr double mutants is a simple addition of each mutation, in which wing discs are lost completely and leg discs are severely reduced. Since Dll-expressing limb primordial cells are present in tkv;Egfr double mutants in stage 11, it has been concluded that these cells fail to differentiate as wing discs and their ability to differentiate as leg discs is also compromised. A few Esg-positive cells remain at the position of the leg, and it is speculated that this reflects the presence of a second leg-inducing signal. These results suggest that Dpp is absolutely required for wing disc development irrespective of the activity of Egfr (Kubota, 2000).

The nuclear transduction of the Dpp signal, as visualized by the distribution of pSSVS and expression of dad, is unaffected by Egfr. The results suggest that the antagonistic effect of Egfr on Dpp signaling occurs after transduction into the nucleus. Therefore, the mechanism of SMad inhibition by direct phosphorylation by MAP kinase does not play a major role in this case (Kubota, 2000).

The finding that Egfr is activated in the limb primordium and prevents wing disc formation suggests that Egfr is a key factor in the diversification of the wing and leg fate. It is proposed that the differential activation of Dpp and Egfr, and the dorsal cell migration brings a subset of limb primordial cells out of the range of Egfr signaling, and thereby allows Dpp to induce wing development. It follows that dorsally migrating cells acquire the wing cell identity only after the separation from leg-promoting signals. Consistent with this idea, expression of wing-specific markers Vg and Sna, start only after the separation of the two primordia. Mechanisms that promote the dorsal cell migration remain to be identified. Given that the basic genetic components for the induction of the wing and leg have been identified in the model organism Drosophila, it can now be asked how the genetic mechanism of wing and leg specification has evolved by comparing the expression and function of these genes in limb primordial cells of primitive insects (Kubota, 2000).

Stepwise formation of a SMAD activity gradient during dorsal-ventral patterning of the Drosophila embryo

Genetic evidence suggests that the Drosophila ectoderm is patterned by a spatial gradient of bone morphogenetic protein (BMP). Patterns have been compared of two related cellular responses - signal-dependent phosphorylation of the BMP-regulated R-SMAD, MAD, and signal-dependent changes in levels and sub-cellular distribution of the co-SMAD Medea. Nuclear accumulation of Medea requires a BMP signal during blastoderm and gastrula stages. During this period, nuclear co-SMAD responses occur in three distinct patterns. At the end of blastoderm, a broad dorsal domain of weak SMAD response is detected. During early gastrulation, this domain narrows to a thin stripe of strong SMAD response at the dorsal midline. SMAD response levels continue to rise in the dorsal midline region during gastrulation, and flanking plateaus of weak responses are detected in dorsolateral cells. Thus, the thresholds for gene expression responses are implicit in the levels of SMAD responses during gastrulation. Both BMP ligands, DPP and Screw, are required for nuclear co-SMAD responses during these stages. The BMP antagonist Short gastrulation (Sog) is required to elevate peak responses at the dorsal midline as well as to depress responses in dorsolateral cells. The midline SMAD response gradient can form in embryos with reduced dpp gene dosage, but the peak level is reduced. These data support a model in which weak BMP activity during blastoderm defines the boundary between ventral neurogenic ectoderm and dorsal ectoderm. Subsequently, BMP activity creates a step gradient of SMAD responses that patterns the amnioserosa and dorsomedial ectoderm (Sutherland, 2003).

These in vivo studies validate the molecular model for signal-dependent nuclear accumulation of Medea. Nuclear accumulation of Medea requires both competence to oligomerize and MAD. Nuclear accumulation is signal dependent, requiring both BMP ligands, Dpp and Scw. Conversely, all cells accumulated nuclear Medea in the presence of constitutively active Tkv receptor. At these stages, any independent contribution from activin-like signals is below the detection limit (Sutherland, 2003).

In wild-type embryos, two transitions in the distribution of BMP activity are evident. Many cellular blastoderm embryos lack detectable levels of nuclear Medea, but a few have low levels of nuclear Medea in a broad dorsal domain, with little gradation. From the proportion of cellular blastoderm embryos with this pattern, the duration of nuclear Medea appears to be brief. These data parallel reports of broad, weak PMad staining during mid-cellularization, except that nuclear Medea is detected later and in a broader pattern. The time lag between the earliest reported detection of PMad and detection of nuclear Medea probably stems from a combination of technical differences and the time necessary for nuclear accumulation. In sum, initial BMP activity is weak and distributed broadly in dorsal regions. Low BMP activity at this phase is required to maintain the early phase of zen expression (Sutherland, 2003).

Onset of gastrulation is associated with a dramatic change in the domain of nuclear Medea, which narrows to a tight midline stripe of cells while staining levels intensify. PMad shows a similar transition to a narrower domain, but earlier. Thus, lateral SMAD responses became undetectable just as a steep activity gradient forms along the dorsal midline (Sutherland, 2003).

A third response pattern arises during mid-gastrulation: dorsolateral domains of cells exhibit low levels of nuclear Medea. Response levels remain high in the dorsal-most cells, even as they move laterally during gastrulation. Levels fall off rapidly over a few cells on either side, with a sharp transition to flanking plateaus of weak responses. The subcellular distribution of Medea is unchanging in ventral and ventrolateral cells. The full BMP response domain does not extend as far ventrally as it does during blastoderm, even though many dorsal cells move laterally during germband extension. Thus, the lateral-most cells with responses at blastoderm have decreased responses during gastrulation (Sutherland, 2003).

Mutants with expanded ventral ectoderm show reduced SMAD responses during the first phase of BMP activity. PMad was not detected in blastoderm tld embryos. Homozygotes for moderate dpp alleles have lower PMad levels during blastoderm. Conversely, sog embryos have a slightly expanded PMad response during blastoderm, and a slight expansion of dorsal ectoderm. Thus, BMP activity during blastoderm positions the boundary between dorsal and ventral ectoderm (Sutherland, 2003).

BMP activity in the dorsal ectoderm does not end with germband extension. During stage 9, PMad is detected throughout the dorsal ectoderm and amnioserosa, and might finalize determination of dorsal ectoderm fates. Dpp expression within the dorsal ectoderm contributes to combinatoral regulation of gene expression patterns in subsets of dorsal ectodermal cells. However, the ventral boundary of dpp expression in the stage 9 dorsal ectoderm must be defined by earlier events (Sutherland, 2003).

A genetic network conferring canalization to a bistable patterning system in Drosophila

To achieve the 'constancy of the wild-type,' the developing organism must be buffered against stochastic fluctuations and environmental perturbations. This phenotypic buffering has been theorized to arise from a variety of genetic mechanisms and is widely thought to be adaptive and essential for viability. In the Drosophila blastoderm embryo, staining with antibodies against the active, phosphorylated form of the bone morphogenetic protein (BMP) signal transducer Mad, pMad, or visualization of the spatial pattern of BMP-receptor interactions reveals a spatially bistable pattern of BMP signaling centered on the dorsal midline. This signaling event is essential for the specification of dorsal cell fates, including the extraembryonic amnioserosa. BMP signaling is initiated by facilitated extracellular diffusion that localizes BMP ligands dorsally. BMP signaling then activates an intracellular positive feedback circuit that promotes future BMP-receptor interactions. This study identified a genetic network comprising three genes that canalizes this BMP signaling event. The BMP target eiger (egr) acts in the positive feedback circuit to promote signaling, while the BMP binding protein encoded by crossveinless-2 (cv-2) antagonizes signaling. Expression of both genes requires the early activity of the homeobox gene zerknullt (zen). Two Drosophila species lacking early zen expression have high variability in BMP signaling. These data both detail a new mechanism that generates developmental canalization and identify an example of a species with noncanalized axial patterning (Gavin-Smith, 2013).

This study has identified a genetic network that acts as a phenotypic stabilizer of a spatially bistable patterning process. The minimal bistable systems allowed by theory require a nonlinear activation rate and a linear degradation rate. It is believed that the identified network defined in this study represents the minimal genetic components required for bistability of BMP signaling in D. melanogaster. In turn, bistability canalizes dorsal patterning. During amnioserosa specification, egr provides positive feedback, conferring nonlinearity, while cv-2> acts as a linear negative regulator of the signaling pathway. The loss of both components reveals the inherent noise of facilitated extracellular diffusion of BMP ligands, as without egr and cv-2, embryos manifest a huge range of signaling domain breadth and intensity. The data also reveal that amnioserosa specification in D. melanogaster is robust on multiple levels, with different mechanisms ensuring robustness in various Drosophila species (Gavin-Smith, 2013).

First, egr or bsk RNAi embryos have normal amounts of amnioserosa and minimal embryonic lethality despite the 2-fold reduction in signaling intensity. This demonstrates that amnioserosa specification is robust to decreases of BMP signaling and the wild-type level of BMP signaling in D. melanogaster is much higher than necessary. Second, the D. melanogaster embryo can tolerate at least a 250% increase or a 20% decrease in amnioserosa cell number without compromising viability. Lastly, the variability in amnioserosa cell number in D. yakuba embryos is equivalent to that in D. melanogaster embryos, indicating that amnioserosa specification in D. yakuba is robust against variable BMP signaling intensity. Therefore, in D. yakuba embryos, either less BMP signaling is required to direct amnioserosa specification or a second mechanism downstream of BMP signaling intensity maintains robust amnioserosa specification (Gavin-Smith, 2013).

Finally, as a counterpoint to the predicted ubiquity and selective maintenance of developmental canalization, D. santomea has been identified as a noncanalized wildtype species. D. santomea both has highly variable cell fate specification and is not robust to genetic variants found in its wild population. The identification of this noncanalized species may permit further investigation of the evolutionary factors allowing for this diversity in developmental trajectories (Gavin-Smith, 2013).

Transgenic analysis of the Smad family of TGF-ß signal transducers

Smad signal transducers are required for transforming growth factor-ß-mediated developmental events in many organisms including humans. However, the roles of individual human Smad genes (hSmads) in development are largely unknown. It was hypothesized that an hSmad performs developmental roles analogous to those of the most similar Drosophila Smad gene (dSmad). Six hSmad and four dSmad transgenes were expressed in Drosophila using the Gal4/UAS system and their phenotypes were compared. Phylogenetically related human and Drosophila Smads induce similar phenotypes supporting the hypothesis. In contrast, two nearly identical hSmads generate distinct phenotypes. When expressed in wing imaginal discs, hSmad2 induces oversize wings while hSmad3 induces cell death. This observation suggests that a very small number of amino acid differences, between Smads in the same species, confer distinct developmental roles. These observations also suggest new roles for the dSmads, Medea and Dad, in Drosophila Activin signaling (see Drosophila Activins Activin-ß and Activin Like Protein at 23B; the Drosophila Activin receptor is Baboon) and in potential interactions between these family members. Overall, the study demonstrates that transgenic methods in Drosophila can provide new information about non-Drosophila members of developmentally important multigene families (Marquez, 2001).

hSma1 and Mad can transduce Dpp/BMP signals. hSmad4 and possibly Med can transduce signals for both TGF-ß subfamilies. hSmad4 forms complexes with hSmad1 and Med forms complexes with Mad. These relationships suggest that these Smads will produce similar phenotypes. One copy of UAS.Mad or UAS.hSmad1 does not generate many phenotypes. Strains containing two copies of these transgenes were then used. UAS.Mad and UAS.hSmad1 induced similar wing and leg phenotypes. For example, UAS.Mad/ptc.Gal4 genotypes have ectopic vein tissue between L3 and L5. This vein phenotype is consistent with two previous results: clonal analysis shows a role for Mad in vein formation and ptc.Gal4 expression in wing disc cells that eventually reside between L3 and L4. In UAS.Mad/ptc.Gal4 wings the distance between L3 and L5 appears reduced. This may be due to the smaller size of vein cells vs. intervein cells. A comparison of wing surface areas shows that UAS.Mad/ptc.Gal4 wings are 22% smaller than wild type. UAS.hSmad1/ptc.Gal4 wings have ectopic vein tissue in roughly the same region. UAS.hSmad1/ptc.Gal4 wings are 15% smaller than wild type. UAS.Mad and UAS.hSmad1 expression appears to mimic dpp's role in vein formation. UAS.Mad and UAS.hSmad1 expression does not appear to mimic dpp's other roles in wing development (cell proliferation and/or cell survival and anterior/posterior patterning) (Marquez, 2001).

UAS.Medea/ptc.Gal4 and UAS.hSmad4/ptc.Gal4 wings have ectopic vein tissue in the same region as UAS.Mad/ptc.Gal4 and UAS.hSmad1/ptc.Gal4 wings. This suggests that Med forms complexes with Mad during vein formation. UAS.Med/ptc.Gal4 wings are 11% larger and UAS.hSmad4/ptc.Gal4 wings are 6% larger than wild type. The larger, rather than smaller, size of UAS.Med/ptc.Gal4 and UAS.hSmad4/ptc.Gal4 wings suggests that multi-subfamily signaling Smads can influence wing size and vein formation (Marquez, 2001).

UAS.Mad/ptc.Gal4 flies have an ectopic leg on the ventral side of a normal limb. The ectopic leg has several segments and terminates in a set of tarsal claws. During leg development, wingless normally represses dpp expression on the ventral side of the limb. In limbs expressing ectopic dpp an additional leg develops on the ventral side of the limb. The similarity between phenotypes that result from ectopic dpp expression and UAS.Mad expression suggests that UAS.Mad is capable of simulating Dpp signals in leg patterning. UAS.hSmad1/ptc.Gal4 flies also have an ectopic leg on the ventral side of a normal limb. The ectopic leg consists of a single segment. UAS.Med/ptc.Gal4 and UAS.hSmad4/ptc.Gal4 flies have ectopic legs of a different type. Legs from these genotypes have short, abnormally wide tibia that lead to duplicated tarsi of one or more segments. The abnormal tibia of UAS.Med/ptc.Gal4 flies has an additional patterning defect: severe bristle overgrowth (Marquez, 2001).

Overall, these four Smads generated comparable vein and leg phenotypes. The phenotypes of phylogenetically related Smads show the greatest similarity. The phenotypes suggest that UAS.hSmad1 and UAS.hSmad4 can also simulate Dpp signaling in Drosophila limb development. These findings are consistent with cell culture studies noted above and further support the view that these hSmads transduce BMP signals during human development. The size of UAS.Med and UAS.hSmad4 wings suggests a role for these multi-subfamily signaling Smads not shared with the Dpp/BMP signaling Smads UAS.Mad and UAS.hSmad1 (Marquez, 2001).

In summary, this analysis of hSmad and dSmad transgenes supports the hypothesis that phylogenetically related Smads fulfill developmental roles that are conserved between humans and Drosophila. The results also suggest a number of new hypotheses regarding roles for human and Drosophila Smads in pattern formation, cell proliferation, and cell death. The data suggest that a small number of amino acid differences between two very similar Smads in the same species can confer distinct activities. Overall, this study demonstrates that transgenic methods in Drosophila can provide new information about mammalian members of developmentally important multigene families (Marquez, 2001).

Organism-scale modeling of early Drosophila patterning via bone morphogenetic proteins

Advances in image acquisition and informatics technology have led to organism-scale spatiotemporal atlases of gene expression and protein distributions. To maximize the utility of this information for the study of developmental processes, a new generation of mathematical models is needed for discovery and hypothesis testing. A data-driven, geometrically accurate model has been developed of early Drosophila embryonic bone morphogenetic protein (BMP)-mediated patterning. Nine different mechanisms for signal transduction with feedback, eight combinations of geometry and gene expression prepatterns, and two scale-invariance mechanisms were tested for their ability to reproduce proper BMP signaling output in wild-type and mutant embryos. It was found that a model based on positive feedback of a secreted BMP-binding protein, coupled with the experimentally measured embryo geometry, provides the best agreement with population mean image data. The results demonstrate that using bioimages to build and optimize a three-dimensional model provides significant insights into mechanisms that guide tissue patterning (Umulis, 2010).

In many systems, spatially patterned cellular differentiation is regulated by signaling molecules called morphogens, which initiate spatiotemporal patterns of gene expression in a concentration-dependant manner. In early Drosophila embryos, a morphogen composed of a heterodimer of Decapentaplegic (Dpp) and Screw (Scw), two members of the bone morphogenetic protein (BMP) family. Unlike classical morphogen systems that rely on the slow spreading of a molecule from a localized source to establish a gradient, BMPs in the early Drosophila embryo are secreted from a broad region making up the dorsal-most 40% of the embryo circumference. Subsequently, they are dynamically concentrated into a narrow region centered about the dorsal midline that makes up only 10% of the embryo circumference (Umulis, 2010).

A number of extracellular regulators contribute to the dynamics and localization of BMP signaling. Laterally secreted Short gastrulation (Sog) and dorsally secreted Twisted gastrulation (Tsg) diffuse from their regions of expression and form a heterodimer inhibitor (Sog/Tsg) that binds to Dpp-Scw, preventing it from binding to receptors. The cell matrix may mediate the formation of this complex, as it has recently been shown that collagen can bind both BMPs and Sog, thereby facilitating their association (Wang, 2008). The extracellular binding reactions lead to a gradient of inhibitor-bound Dpp-Scw that is high laterally and low at the dorsal midline, and an opposing gradient of free Dpp-Scw that is high at the dorsal midline. The dorsally secreted metalloprotease Tolloid (Tld) processes Sog only when Sog is bound to BMP ligands, and the degradation of Sog by Tld further enhances both the gradient of inhibitor-bound Dpp-Scw and of free Dpp-Scw. Thus, extracellular Dpp-Scw is redistributed by a combination of binding to inhibitor, processing of this complex, and diffusion (Umulis, 2010).

Simultaneously, receptors and other surface-localized binding proteins compete with Sog to bind the available Dpp-Scw. Dpp-Scw activates signaling by binding to and recruiting the Drosophila type I receptors, Thickveins (Tkv) and Saxophone (Sax), into a high-order complex containing two subunits of the type II receptor Punt. The receptor complex phosphorylates Mad (pMad), a member of the Smad family of signal transducers, and phosphorlyated Mad binds to the co-Smad Medea, forming a complex that then accumulates in the nucleus, where it regulates gene expression in a concentration-dependent manner (Umulis, 2010).

Although complex formation and transport favor a net movement of ligand toward the dorsal midline of the embryo, positive feedback in response to pMad signaling is needed to further concentrate the surface-localized Dpp-Scw at the dorsal midline. A loss of extracellular BMP regulators or positive feedback impedes the attenuation of pMad laterally as well as the accumulation of pMad signaling at the dorsal midline. Although feedback, extracellular transport, and signal transduction each provide a specific mode of Dpp-Scw signal regulation, it is the dynamic interaction of these regulatory mechanisms that patterns the dorsal surface of Drosophila embryos. Not only does the mechanism work under optimal laboratory conditions, but dorsal surface patterning appears to be remarkably resilient to nonideal conditions such as temperature fluctuations, reductions in the level of regulatory factors such as Tsg, ectopic gene expression, and other perturbations. These issues illustrate the complexity of the problem and suggest that it is not possilbe to rely solely on genetic and biochemical data to fully explain this rather simple patterning problem (Umulis, 2010).

To address a number of unanswered questions about Dpp-Scw-mediated patterning and to take full advantage of the available data on Drosophila development, a methodology was developed that seamlessly integrates biological information in the form of prepatterns, geometry, mechanisms, and training data into an organism-scale model of the blastoderm embryo that is based on a reaction-diffusion description of patterning. The mathematical model is simulated by using the widely available computational frameworks Comsol and Matlab, which makes extensive use of the model and methodology feasible (Umulis, 2010).

An image analysis protocol was developed to obtain model training and initial condition data and to calculate population statistics for patterns of pMad signaling in wild-type (wt) and mutant D. melanogaster. Both the mean and variability of pMad signaling along the dorsal-ventral (DV) axis depends on anterior-posterior (AP) position and the specific choice of threshold. Using mutations previously considered robust, differences could be detected between mutant and wild-type pMad signaling patterns, which provided an information-rich data set for model training and for testing the contributions of diverse positive-feedback mechanisms and of proteins that concentrate BMPs at the cell surface. Unexpectedly, it was found that geometry also has a large impact on the predicted patterns of BMP-bound receptors, whereas the prepatterned expression of receptors and other modulators of signaling did not greatly affect model-data correspondence. It was found that if the embryo geometry is perturbed slightly in the model, then including the prepattern information greatly enhanced the model's ability to fit the observed pMad patterns, which suggests that the prepatterns may mitigate the effects of slightly misshapen embryos. Conditions in the model were identified that improve the scale invariance of patterning and tested the model predictions by staining for pMad in different species of Drosophila. These studies demonstrate that building a model based on image data and training the three-dimensional (3D) model against multidimensional expression data provide insights into the properties of several important developmental principles, including positive feedback, biological robustness, and scale invariance (Umulis, 2010).

Specification of neuropeptide cell identity by the integration of retrograde BMP signaling and a combinatorial transcription factor code

Individual neurons express only one or a few of the many identified neurotransmitters and neuropeptides, but the molecular mechanisms controlling their selection are poorly understood. In the Drosophila ventral nerve cord (VNC), the six Tv neurons express the neuropeptide gene FMRFamide (FMRFa). Each Tv neuron resides within a neuronal cell group specified by the LIM-homeodomain (LIM-HD) gene apterous (ap). The zinc-finger gene squeeze acts in Tv cells to promote their unique axon pathfinding to a peripheral target. There, the BMP ligand Glass bottom boat activates the Wishful thinking receptor, initiating a retrograde BMP signal in the Tv neuron. This signal acts together with apterous and squeeze to activate FMRFamide expression. Reconstituting this 'code,' by combined BMP activation and apterous/squeeze misexpression, triggers ectopic FMRFamide expression in peptidergic neurons. Thus, an intrinsic transcription factor code integrates with an extrinsic retrograde signal to select a specific neuropeptide identity within peptidergic cells (Allan, 2003).

FMRFa is specifically expressed in the six Tv neuroendocrine neurons located bilaterally in the three thoracic (T1-3) segments of the embryonic and larval VNC. apterous is expressed in three interneurons per VNC hemisegment, as well as in a lateral cluster of four neurons (the ap-cluster) in each of the T1-3 hemisegments. One of the four ap-cluster cells is the FMRFa-expressing Tv neuron. All ap interneurons in the VNC, except for the Tv, join a common ipsilateral axon tract termed the ap-fascicle. The Tv axon instead projects to the midline and exits the VNC dorsally to innervate the dorsal neurohemal organ (DNH). The DNH is a club-like neuroendocrine structure formed by two glial cells protruding from the midline of each thoracic segment. Anteriorly, two additional FMRFa-expressing cells are found, denoted SE2 cells. The SE2 cells do not express, nor depend upon, any regulators described in this study for their FMRFa expression. ap is important for the expression of FMRFa in the Tv neurons, but since most ap neurons do not express FMRFa, other regulators are likely needed for FMRFa regulation (Allan, 2003).

Rotund, a zinc finger protein of the C2H2 Krüppel-type belongs to a conserved subfamily of zinc finger proteins together with Drosophila CG5557, C. elegans Lin-29, and rat CIZ. Squeeze is most closely related to Rotund, with identity greater than 90% throughout the zinc finger region; Squeeze is 78% identical to LIN-29 in the conserved zinc finger region. Both rotund and CG5557 are expressed in subsets of cells in the developing CNS. CG5557 has a larval lethal phase. Mutants eclosed at a low frequency as immotile adults that died within 24 hr. Mutant larvae display a motility defect whereby the body wall musculature over-contract radially during the peristaltic wave typical of insect larval motility, apparent as a 'squeezing' of the intestine. Since this motility phenotype is fully penetrant and scored with 100% accuracy (sqzlacZ/sqzDf), CG5557 was renamed squeeze (sqz) (Allan, 2003).

What is the identity of the retrograde FMRFa-inducing signal? Recently, a Drosophila BMP type-II receptor, wishful thinking (wit), was implicated in mediating a retrograde signal from muscles to motor neurons, responsible for presynaptic maturation. Signaling by the TGF-β/BMP superfamily occurs via activation of a receptor complex, consisting of two type I and two type II receptors, leading to phosphorylation and nuclear translocation of a receptor Smad protein. In Drosophila, BMP signaling leads to the phosphorylation and nuclear translocation of the Smad protein Mothers against dpp (Mad), which can be monitored using antibodies specific to phosphorylated Mad (pMad) (Allan, 2003).

Using antibodies to pMad, BMP activation in peptidergic neurons was assayed. Nuclear pMad was detected not only in motor neurons, but also in the Tv, Va, and Vap neurons, demonstrating that peptidergic neurons projecting out of the VNC also show evidence of BMP activation. Accumulation of pMad in the Tv neurons commences during stage 17, immediately following DNH innervation. These results led to a test of whether Tv innervation of the DNH would be critical for pMad accumulation and consequently for FMRFa expression. Indeed, it was found that the absence of the DNH (in tin mutants), Tv axon pathfinding alterations (in apGAL4/UAS-robo and apGAL4/UAS-racV12) and interference with Tv axonal transport (in apGAL4/UAS-GluedDN and apGAL4/UAS-τ-myc) are all accompanied by loss of pMad staining specifically in Tv neurons. The ectopic ap-cluster FMRFa-expressing cell induced by sqz misexpression is also pMad positive. Given the role of sqz in Tv axon pathfinding, this is interpreted as resulting from sqz dominantly altering the projection of one other ap-cluster cell, forcing it to innervate the DNH. Thus, in all genotypes examined, Tv axonal projection to the DNH is critical for pMad accumulation (Allan, 2003).

Since Wit is expressed in a restricted pattern in the developing VNC, attempts were made to address whether the Tv neurons express Wit. However, single-cell resolution could not be obtained with the Wit antibody and Wit could not be definitely localized in Tv cells. However, the wit-dependent pMad accumulation in Tv neurons, the apGAL4/UAS-tkvA, UAS-saxA-mediated rescue of wit mutants, and the UAS-gbb-mediated 'rescue' of UAS-robo misexpression, provide genetic evidence supporting the expression of wit in Tv cells. Previous studies have shown that gbb is expressed in developing endoderm and visceral mesoderm, but it has not been detected in the VNC. By in situ hybridization, no apparent expression was detected in the DNH. Given that the DNH only contains two cell bodies, low-level gbb expression may be beyond detection. Moreover, since the anterior midgut is positioned in very close proximity to the DNHs, it is possible that Gbb diffuses from the visceral mesoderm to the DNH (Allan, 2003).

The BMP homolog Gbb provides a retrograde signal that regulates synaptic growth at the Drosophila neuromuscular junction

The BMP ortholog Gbb can signal by a retrograde mechanism to regulate synapse growth of the Drosophila neuromuscular junction (NMJ). gbb mutants have a reduced NMJ synapse size, decreased neurotransmitter release, and aberrant presynaptic ultrastructure. These defects are similar to those observed in mutants of BMP receptors and Smad transcription factors. However, whereas these BMP receptors and signaling components are required in the presynaptic motoneuron, Gbb expression is required in large part in postsynaptic muscles; gbb expression in muscle rescues key aspects of the gbb mutant phenotype. Consistent with this notion, blocking retrograde axonal transport by overexpression of dominant-negative p150/Glued in neurons inhibits BMP signaling in motoneurons. These experiments reveal that a muscle-derived BMP retrograde signal participates in coordinating neuromuscular synapse development and growth (McCabe, 2003).

In the case of the BMP signal described in this study, the finding that a high accumulation of P-Mad is detectable in motoneuron nuclei when Gbb is resupplied to nerve terminals from the postsynaptic muscle cell implies that a retrograde signal likely contributes to P-Mad nuclear localization. Consistent with this view is the observation that blocks in the dynein/dynactin motor complex also disrupt P-Mad accumulation similar to what has been reported for transport of activated Trks. Since Mad and Medea mutants also display NMJ defects that are very similar to those exhibited by receptor and ligand mutants, it seems likely that the majority of these defects result from the lack of the retrograde signal itself as opposed to some being caused by the lack of a hypothetical local signal. As is the case for Trks, a signaling endosome consisting of activated heteromeric receptor complexes containing Gbb, Wit, Tkv, and Sax might be transported back to the cell body where these complexes would phosphorylate cytoplasmic Mad, resulting in its translocation to the nucleus. Alternatively, nonphosphorylated Mad may first be transported anterogradely to the nerve. Subsequent to phosphorylation at the NMJ, it may then be selectively transported in a retrograde fashion back to the cell body (McCabe, 2003).

Although loss-of-function and rescue experiments clearly demonstrate that Gbb is required for proper synaptic development at the NMJ, it is not certain that it is the only TGF-β-type ligand or indeed the primary ligand that regulates this process. The electrophysiological and ultrastructure defects observed in gbb mutant synapses are not as severe as those found in wit null mutants. This could simply reflect an inability to produce true null animals that survive to the third instar stage, or it may indicate that another ligand also provides a signal. In support of this view is the observation that P-Mad accumulation is not totally eliminated in gbb1/gbb2 null mutant embryos as it is in wit mutants. In addition, it is noted that while overexpression of Gbb in the CNS only weakly rescued P-Mad accumulation in the CNS, the pattern of accumulation does not appear to change. That is, P-Mad still seems to be found primarily in motoneurons. Thus, other neurons do not appear to be competent to respond to BMP-type ligands, perhaps because a specific cosignal is absent or because they do not express the right combination of receptors. It is interesting to note that in several other developmental contexts in Drosophila, it appears that at least two BMP ligands provide regulatory inputs into a common process (McCabe, 2003).

The and ligand Glass bottom boat and the BMP receptor Wishful thinking, signaling through Mad, are necessary for the rapid induction of synaptic homeostasis

Inhibition of postsynaptic glutamate receptors at the Drosophila NMJ initiates a compensatory increase in presynaptic release termed synaptic homeostasis. BMP signaling is necessary for normal synaptic growth and stability. It remains unknown whether BMPs have a specific role during synaptic homeostasis and, if so, whether BMP signaling functions as an instructive retrograde signal that directly modulates presynaptic transmitter release. This study demonstrates that the BMP receptor (Wit) and ligand (Gbb) are necessary for the rapid induction of synaptic homeostasis. Evidence is provided that both Wit and Gbb have functions during synaptic homeostasis that are separable from NMJ growth. However, further genetic experiments demonstrate that Gbb does not function as an instructive retrograde signal during synaptic homeostasis. Rather, the data indicate that Wit and Gbb function via the downstream transcription factor Mad and that Mad-mediated signaling is continuously required during development to confer competence of motoneurons to express synaptic homeostasis (Goold, 2007).

These data advance understanding of BMP signaling at the Drosophila NMJ in several important ways. First, it was demonstrated that BMP signaling is essential for the rapid, protein-synthesis-independent, induction of synaptic homeostasis identified at this NMJ. Because expression of UAS-wit in motoneurons restores synaptic homeostasis in the wit mutant and because suppression of Mad-mediated signaling in neurons blocks synaptic homeostasis, it is concluded that BMP signaling acts upon the motoneuron to enable the rapid induction of synaptic homeostasis. Next, it was shown that the requirement for BMP signaling during synaptic homeostasis is separable from BMP-dependent support of synaptic growth and baseline neurotransmission. Finally, the temporal and spatial requirements for BMP signaling was dissected. The data support the conclusion that Mad-mediated signaling is required constitutively, downstream of the Wit receptor, in order to maintain the competence of motoneurons to express homeostatic plasticity. Further, the data argue that Gbb is not the retrograde signal that directly acts upon the presynaptic motoneuron terminal to homeostatically modulate presynaptic release (Goold, 2007).

It has been hypothesized that Gbb could function as a homeostatic retrograde signal at the Drosophila NMJ. According to this model, Gbb would be released in proportion to the perturbation of postsynaptic muscle excitation in a glutamate receptor mutant and, thereby, instruct the degree of homeostatic compensation expressed by the presynaptic motoneuron terminal. In favor of this model, homeostatic compensation observed in a glutamate receptor mutant is blocked by the wit mutation. This study present two lines of evidence that are consistent with the necessity of BMP signaling for homeostatic compensation. First, it was confirmed that the rapid induction of homeostatic compensation following application of a use-dependent glutamate receptor antagonist, Philanthotoxin (PhTx) is blocked by null mutations in both wit and gbb. Furthermore, it was shown that muscle-specific rescue of the gbb null mutation is sufficient to restore the rapid induction of homeostatic compensation (Goold, 2007).

Despite these compelling genetic data, several experiments now argue against the possibility that Gbb functions as an instructive, retrograde signal that directly modulates presynaptic release during synaptic homeostasis. First, it was found that although muscle-specific rescue of the gbb null mutation is sufficient to restore synaptic homeostasis, so is neuron-specific rescue of the gbb null mutation. Thus, homeostatic compensation can occur even in the absence of muscle-derived Gbb. These data argue against a model in which Gbb functions as the instructive retrograde signal that directly modulates presynaptic release during synaptic homeostasis (Goold, 2007).

Next, it was demonstrated that homeostatic signaling is blocked by expression of DN-Glued in neurons, which disrupts retrograde axonal transport. In this experiment, Gbb signaling at the NMJ should, in theory, persist. Furthermore, it was established that an intact motor axon is not required for the rapid induction of synaptic homeostasis. Thus, it can be concluded that trans-synaptic Gbb signaling from muscle to nerve is not sufficient for the rapid induction of synaptic homeostasis (Goold, 2007).

Given that Wit and Gbb are necessary for synaptic homeostasis, how do they participate in the process if Gbb is not the instructive retrograde signal? This study demonstrates that Mad is necessary for synaptic homeostasis, and evidence is providied that Mad-mediated signaling is required in the motoneuron. In addition, neuronal expression of UAS-Gbb restores homeostatic compensation in the presence of the DN-Glued transgene. These results suggest that the reason DN-Glued disrupts synaptic homeostasis is because it interferes with the retrograde axonal transport of P-Mad downstream of the Wit receptor. This is consistent with the prior demonstration that neuronal expression of Gbb can restore nuclear P-Mad in the presence of UAS-DN-Glued. Because the induction of synaptic homeostasis does not require the motoneuron soma, it is concluded that Gbb does not function as an acute, retrograde signal. Rather, Gbb may be a muscle-derived signal that acts developmentally to confer the competence of motoneurons to express synaptic homeostasis. Thus, the identity of the homeostatic retrograde signal at the NMJ remains unknown. It remains possible that other TGF-β superfamily signaling molecules could function at the NMJ in this capacity, including myoglianin and maverick, though it has been shown that synaptic homeostasis is intact in the baboon receptor mutant (Goold, 2007).

There are several possible ways in which BMP signaling could confer competence for motoneurons to express homeostatic plasticity. One possibility is that the BMPs control a transcriptional program that is necessary for synaptic homeostasis. For example, BMPs are potent regulators of cell fate during embryonic development. Perhaps the ability of motoneurons to express synaptic homeostasis is related to the maintenance of their cellular or electrical identity. An alternate possibility is that BMPs control the expression of essential presynaptic proteins that are required for synaptic homeostasis. For example, it has been shown in other systems that target-dependent TGF-β signaling can modulate neuronal ion channel expression. It has been demonstrated that CaV2.1 calcium channels are required for synaptic homeostasis at the Drosophila NMJ. However, it is considered unlikely that BMPs control synaptic homeostasis through the regulation of CaV2.1 channel expression because there is not a strong correlation between altered baseline synaptic transmission and the expression of synaptic homeostasis. Furthermore, overexpression of a GFP-tagged CaV2.1 calcium channel (cacophony-GFP) is unable to restore synaptic homeostasis when coexpressed with UAS-dad. Finally, BMP signaling could influence the expression of synaptic homeostasis by targeting the rate of spontaneous miniature release. Spontaneous release events that persist in the absence of evoked neurotransmission are sufficient to induce homeostatic compensation at the Drosophila NMJ. However, no strong correlation is found between baseline mEPSP frequency and whether or not a mutant NMJ is able to express synaptic homeostasis. Although the wit mutants show a severe decrease in mEPSP rate compared to wild-type, the expression of UAS-dad or UAS-DN-Glued both block synaptic homeostasis without severely impairing baseline mEPSP rate. Ultimately, continued forward genetic investigation of homeostatic signaling may be required to identify the BMP-dependent mechanisms that control the expression of synaptic homeostasis (Goold, 2007).

BMP signaling is required for NMJ growth, baseline neurotransmission, and NMJ stability in addition to being required for synaptic homeostasis. It is a challenge, therefore, to determine whether BMP signaling has a specific function during synaptic homeostasis versus a more general role during synapse development. This study presents several lines of evidence that BMP signaling may have a separable function during synaptic growth versus synaptic homeostasis. First, it was demonstrated that synaptic homeostasis can occur at BMP mutant synapses that show severely impaired synaptic growth. For example, the gbb hypomorphic mutant has a decrease in bouton number that is just as severe as the gbb null mutant, but the gbb hypomorphic mutant shows normal homeostatic compensation. As another example, animals in which UAS-gbb and UAS-DN-Glued are coexpressed have a severe decrease in bouton number but normal homeostatic compensation. Thus, it is concluded that normal BMP-dependent synaptic growth is not required for the expression of synaptic homeostasis (Goold, 2007).

It was also possible to dissociate BMP-dependent baseline transmission from both synaptic growth and synaptic homeostasis. (1) Muscle-specific rescue of the gbb null mutation significantly restores synaptic growth and rescues synaptic homeostasis, but baseline transmission remains at levels observed in the null mutant. (2) Motoneuron-specific rescue of the wit mutation (OK371-GAL4) similarly rescues bouton number and synaptic homeostasis, although baseline transmission remains severely impaired. (3) Animals in which UAS-gbb and UAS-DN-Glued are coexpressed have a severe decrease in baseline transmission but normal homeostatic compensation. (4) Results were obtained that show the converse effect. When UAS-dad is expressed for 1.5 days at the end of larval development, both synaptic homeostasis and baseline transmission are significantly impaired, but synaptic bouton numbers remain wild-type. From these data it is concluded that impaired synaptic homeostasis is not a secondary consequence of BMP-dependent functional NMJ development. It also appears that there may be distinct effects of BMP signaling on the anatomical versus functional development of the NMJ. One possibility, consistent with BMPs being a classical morphogen, is that different levels of the ligand could initiate specific transcriptional programs with distinct effects on bouton number, baseline transmission, and homeostatic plasticity. It is also possible that the site of action of BMP signaling will play an important role in specifying signaling outcome (Goold, 2007).

It has been speculated that synaptic homeostasis might function, over the course of development, to ensure that the muscle cell is normally depolarized by the NMJ. How can one explain the observation that csp and syx/+ mutations have decreased baseline neurotransmitter release but normal acute synaptic homeostasis in response to PhTx application, or other genotypes explored in this study that show impaired baseline transmission and normal acute synaptic homeostasis? It has been demonstrated that the acute induction of synaptic homeostasis is independent of evoked neurotransmission. Thus, synaptic homeostasis may not function to modulate the absolute amplitude of evoked neurotransmitter release. Rather, synaptic homeostasis might be a rapid system to offset acute perturbations of postsynaptic receptor function. In this case, developmental programs that specify NMJ anatomy and active zone addition would achieve the reproducible development of the NMJ. Alternatively, the mechanisms of acute homeostatic compensation following PhTx application may be separable, either temporally or molecularly, from the other potential mechanisms that monitor and homeostatically control evoked EPSP amplitudes (Goold, 2007).

The data also suggest a possible link between the expression of homeostatic plasticity and the mechanisms of neuromuscular degenerative disease. Genetic mutations that impair retrograde axonal transport have been shown to cause familial amyotrophic lateral sclerosis. It has also been shown that, in Drosophila and mice, mutations that disrupt dynein-dynactin complex function lead to neuromuscular synapse degeneration. It is hypothesized that impaired retrograde axonal transport deprives motoneurons of muscle-derived trophic support leading to motoneuron degeneration. This study has demonstrated that impaired retrograde axonal transport blocks the expression of homeostatic plasticity at the NMJ. This deficit can be restored by expression of BMPs in the central nervous system, bypassing retrograde axonal transport as the source of BMPs to the motoneuron cell body. It is tempting to speculate that impaired synaptic homeostasis at the NMJ may play a role in the progression of motoneuron disease associated with impaired retrograde axonal transport (Goold, 2007).

Finally, the data could have relevance to the sustained expression of homeostatic plasticity in regions of the adult nervous system. BMPs and downstream signaling proteins such as the Smads continue to be expressed in the adult nervous system. In particular, BMPs are secreted into the cerebral spinal fluid at concentrations that are relevant for neuronal signaling. It is, therefore, interesting to speculate that circulating levels of BMPs might sustain the competence of neurons to express homeostatic plasticity without driving morphological plasticity in the adult nervous system (Goold, 2007).

Retrograde BMP signaling at the synapse: a permissive signal for synapse maturation and activity-dependent plasticity

At the Drosophila neuromuscular junction (NMJ), the loss of retrograde, trans-synaptic BMP signaling causes motoneuron terminals to have fewer synaptic boutons, whereas increased neuronal activity results in a larger synapse with more boutons. This study shows that an early and transient BMP signal is necessary and sufficient for NMJ growth as well as for activity-dependent synaptic plasticity. This early critical period was revealed by the temporally controlled suppression of Mad, the SMAD1 transcriptional regulator. Similar results were found by genetic rescue tests involving the BMP4/5/6 ligand Glass bottom boat (Gbb) in muscle, and alternatively the type II BMP receptor Wishful Thinking (Wit) in the motoneuron. These observations support a model where the muscle signals back to the innervating motoneuron's nucleus to activate presynaptic programs necessary for synaptic growth and activity-dependent plasticity. Molecular genetic gain- and loss-of-function studies show that genes involved in NMJ growth and plasticity, including the adenylyl cyclase Rutabaga, the Ig-CAM Fasciclin II, the transcription factor AP-1 (Fos/Jun), and the adhesion protein Neurexin, all depend critically on the canonical BMP pathway for their effects. By contrast, elevated expression of Lar, a receptor protein tyrosine phosphatase found to be necessary for activity-dependent plasticity, rescued the phenotypes associated with the loss of Mad signaling. Synaptic structure and function develop using genetically separable, BMP-dependent mechanisms. Although synaptic growth depended on Lar and the early, transient BMP signal, the maturation of neurotransmitter release was independent of Lar and required later, ongoing BMP signaling (Burke, 2013).

This study investigated how retrograde BMP signaling by Gbb, Wit, and Mad influences the development of the Drosophila NMJ. The experiments examined the timing of retrograde signaling and the relationship between BMP signaling and the activity-dependent modulation of NMJ development. The results indicate that an early and transient period of BMP signaling, acting through Mad, activates key developmental programs necessary for synapse maturation. Transcriptional regulation by Mad in the first larval instar (L1, 24 h) is necessary and sufficient for robust NMJ growth during the second (L2) and third (L3) instars (72 h). Mad signaling during L1 also allows activity to enhance the growth process. By contrast, Mad signaling in L1 through L3 is required for normal active zone morphology and the developmental increase in quantal content. The results therefore indicate that retrograde BMP signaling 'gates' NMJ development and plasticity by initiating two genetically separable programs for growth and physiology (Burke, 2013).

In the absence of retrograde BMP signaling, the NMJ shows only residual growth, forming weak connections that are insensitive to activity-dependent modulation. BMP signaling mutations do not disrupt axonal guidance, target selection, or the initiation of synaptogenesis. They instead have profound effects on later aspects of synaptic development, affecting NMJ expansion and bouton stabilization. Expression of Mad1, the protein encoded by the strong dominant-negative mad1 allele, phenocopies BMP signaling mutants, affecting both NMJ growth and physiology. By driving Mad1 expression at various times during development, it was found that Mad-dependent signaling during L1 is both necessary and sufficient for subsequent NMJ growth. The results are consistent with an L1 critical period for BMP signaling. Inducing Mad1 expression at all times except L1 produced a WT-sized NMJ, whereas induction only during L1 reduced NMJ size to that seen in mad mutants. The genetic rescue of gbb and wit mutants also revealed the importance of the retrograde BMP pathway signaling during embryogenesis and L1. The timing of retrograde BMP signaling, after synaptogenesis yet before growth commences, suggests a model where the muscle uses BMP signaling to inform the motoneuron nucleus of a successfully formed synapse, activating subsequent growth and plasticity programs. The exact timing of this critical period, however, requires knowing when the Mad1 transgene inhibits transcription of Mad's major effectors of NMJ growth, which are unknown. The data indicate that a 24 h exposure to RU-486 during L1 expresses enough Mad1 to suppress NMJ growth. It also suggests that the perdurance of the dominant negative plus the time needed for its activation must be less than 24 h, as expression during embryogenesis, L2, and L3 led to NMJs that were nearly WT in size. Therefore, although the critical period may be shifted later in development than the data indicate, it is likely to begin as early as 5 h after the onset of L1 (Burke, 2013).

The data provide an entrance into the mechanisms regulating the critical period. Based on its timing between the embryonic and L2 stages, it is possible that molting hormones influence Mad activity. Recent work also indicates that anterograde activin signaling induces Gbb expression in body wall muscles, whereas postsynaptic dCIP4 signaling (Drosophila Cdc42 Interacting Protein 4) and the activity of dRich, a conserved Cdc 42-selective guanosine triphosphatase-activating protein, inhibits Gbb secretion from these muscles (Nahm, 2010a; Nahm, 2010b). Furthermore, the secretion of a TGF-β ligand (Maverick) from peripheral glia strongly regulates Gbb signaling from the postsynaptic muscle, affecting both pMad levels within motoneurons and NMJ growth. It would therefore be interesting to perform phenocritical analyses on activin, dCIP4, dRich, and Maverick and to address whether postsynaptic depolarization influences the activity of dCIP4 or dRich. Genetic interaction experiments found no evidence to suggest that moderate increases in BMP signaling modulate the final size of the NMJ or synergize with presynaptic activity. The findings of an early critical period for NMJ growth differ slightly from a previous study, which overexpressed an inhibitory SMAD. In an effort to reconcile the current results with these earlier data, Dad was transiently expressed during L1 but no alteration was found in NMJ size, perhaps resulting from different mechanisms of BMP pathway inhibition (Burke, 2013).

An unexpected result from this study is that the early BMP signal is necessary and sufficient for subsequent growth and structural plasticity, but not for synaptic function. In the absence of later BMP signaling, the NMJ grows to its structurally normal size but has reduced neurotransmission. The later requirement for BMP signaling is consistent with the observation that pMad levels remain high in motoneuron nuclei throughout larval development. In the absence of continual Mad activity, the number of active zones is reduced and their size is aberrantly enlarged. The separable roles of BMP signaling for NMJ growth and function are also consistent with the distinct actions of the two known targets of retrograde BMP signaling in motoneurons, Trio and Target of wit (Twit) (Burke, 2013).

It is not known whether the active zone phenotype arising from the loss of BMP signaling late in development results from defects in the formation or the maintenance of these structures. The active zone protein Sunday driver (dSyd) and the Teneurin-a adhesion molecule are potentially involved in Mad's physiological program, as their loss causes physiological and ultrastructural phenotypes that overlap with those of BMP mutants. Future studies of how Mad simultaneously and separately regulates NMJ growth and physiology could lead to a deeper understanding of how dSyd and Teneurin-a might act dowstream of Mad. The separable downstream effects of Mad on growth versus physiology may depend on post-translational modifications that can occur at the linker between the MH1 and MH2 domains, changes that can affect the affinity of Mad's binding partners. Unfortunately, Mad's noncanonical binding partners in motoneurons are entirely uncharacterized (Burke, 2013).

Elevated action potential firing in motoneurons resulting from the loss of repolarizing K+ channels or by high-temperature rearing substantially enhances NMJ growth and synaptic transmission. Mutations affecting the BMP signaling pathway block this plasticity without suppressing presynaptic hyperactivity. The results indicate that retrograde BMP signaling allows motoneuron growth to be responsive to increased levels of activity. Indeed, the level of excitability during Mad's critical period for growth strongly affected synaptic size in mature larvae. Understanding how this early activity engages BMP-dependent programs could be very insightful given that cAMP, AP-1, and Fas-2 signaling failed to rescue the effects of Mad1 expression (Burke, 2013).

Instead, the receptor protein tyrosine phosphatase Lar rescued both normal and activity-dependent NMJ growth, and Lar was required for activity-dependent developmental plasticity. Lar family members are essential, well-conserved regulators of synaptogenesis from worms and flies to mammals. Despite the prior identification of extracellular ligands for Lar family RPTPs, there has been no significant insight into the temporal regulation of Lar signaling during synaptogenesis in any system. Current evidence suggests that the Lar pathway regulates cytoskeletal assembly and active zone formation while antagonizing the activity of the highly conserved Abelson (Abl) tyrosine kinase. At the larval stage, Abl negatively regulates NMJ size and abl mutations have phenotypes reciprocal to those of Lar. Lar may relieve the growth-inhibitory action of Abl, promoting synaptic expansion in response to elevated activity or postsynaptic growth. Lar acts via the actin-modulating protein Ena for NMJ growth, whereas Trio and Lar regulate growth and the presynaptic cytoskeleton by interacting with Diaphanous, a member of the formin family of proteins. As Trio levels are reduced in BMP mutants and the expression of Trio only partially rescues NMJ growth, it is possible that Ena or Diaphanous act independently of Mad signaling (Burke, 2013).

In the absence of BMP signaling, the small NMJs can be genetically rescued by increased expression of Lar. This suggests that reduced BMP signaling in some fashion reduces Lar activity or function at the NMJ. qPCR analyses show that Lar transcript levels remain normal despite reduced pMad activity, leaving open the possibility that post-transcriptional mechanisms reduce Lar expression, NMJ localization, or activity, either by changes to the receptor itself, to Lar's binding partners (for e.g., Liprins), or to the heparin sulfate proteoglycan ligands. The observations support a model where retrograde BMP signaling allows synaptic growth to be modulated by neural activity, with Lar acting as the downstream 'gain controller' to establish the specific level of synaptic efficacy. In this model, postsynaptic BMP release initiates competence of the presynaptic terminal to respond to the matrix via Lar. Lar's heparin sulfate proteoglycan ligands and its anchoring proteins (Liprins) might then provide spatial information or couple Lar function to synaptic activity. Heparin sulfate proteoglycans play important roles during critical periods, and they modulate the signaling of BMPs, Wnts, and fibroblast growth factors. It is therefore possible that the extracellular matrix provides a key integrator that coordinates multiple trans-synaptic signals in a developmental and activity-dependent manner (Burke, 2013).

A novel, noncanonical BMP pathway modulates synapse maturation at the Drosophila neuromuscular junction

At the Drosophila NMJ, BMP signaling is critical for synapse growth and homeostasis. Signaling by the BMP7 homolog, Gbb, in motor neurons triggers a canonical pathway-which modulates transcription of BMP target genes, and a noncanonical pathway-which connects local BMP/BMP receptor complexes with the cytoskeleton. This study describes a novel noncanonical BMP pathway characterized by the accumulation of the pathway effector, the phosphorylated Smad (pMad), at synaptic sites. Using genetic epistasis, histology, super resolution microscopy, and electrophysiology approaches, it was demonstrated that this novel pathway is genetically distinguishable from all other known BMP signaling cascades. This novel pathway does not require Gbb, but depends on presynaptic BMP receptors and specific postsynaptic glutamate receptor subtypes, the type-A receptors. Synaptic pMad is coordinated to BMP's role in the transcriptional control of target genes by shared pathway components, but it has no role in the regulation of NMJ growth. Instead, selective disruption of presynaptic pMad accumulation reduces the postsynaptic levels of type-A receptors, revealing a positive feedback loop which appears to function to stabilize active type-A receptors at synaptic sites. Thus, BMP pathway may monitor synapse activity then function to adjust synapse growth and maturation during development (Sulkowski, 2016).

BMPs fulfill multiple functions during NMJ development via canonical and noncanonical pathways. In motor neurons, signaling by Gbb triggers a canonical BMP signaling that regulates transcription of BMP target genes and a noncanonical BMP pathway that connects Wit with LIMK1 and the cytoskeleton. This study describes a novel non-canonical BMP pathway, which induces selective accumulation of pMad at presynaptic sites. This pathway does not require Gbb, but depends on presynaptic BMP receptors Wit and Sax and postsynaptic GluRIIA. This novel pathway does not contribute to the NMJ growth and instead appears to set up a positive feedback loop that modulates the postsynaptic distribution of type-A and type-B receptors as a function of synapse activity (Sulkowski, 2016).

At the Drosophila NMJ, BMP signaling controls NMJ growth and promotes synapse homeostasis. BMP fulfills all these functions via canonical and noncanonical pathways. Canonical BMP signaling activates presynaptic transcriptional programs with distinct roles in the structural and functional development of the NMJ. For example, the BMP pathway effector Trio can rescue NMJ growth in BMP pathway mutants, but does not influence synapse physiology, whereas Target of Wit (Twit) can partially restore the mini frequency but has no effect on NMJ growth. It has been shown that both muscle and neuron derived Gbb are required for the structural and functional integrity of NMJ, and multiple mechanisms that regulate Gbb expression, secretion and extracellular availability have been described. Binding of Gbb to its receptors also triggers a noncanonical, Mad-independent pathway that requires the C-terminal domain of Wit. This domain is conserved among Drosophila Wit and vertebrate BMPRII and functions to recruit and activate cytoskeletal regulators such as LIMK1. In flies, Wit-mediated activation of LIMK1 mediates synapse stability and enables rapid, activity-dependent synaptic growth (Eaton, 2005; Piccioli, 2014; Sulkowski, 2016 and references therein).

This study uncovered a novel, noncanonical BMP pathway that triggers accumulation of presynaptic pMad in response to postsynaptic GluRIIA receptors. This pathway requires Wit and Sax, suggesting that various BMP pathways compete for shared components. Super resolution imaging mapped the pMad domains at active zones, in close proximity to the presynaptic membrane. These domains concentrate the pMad immunoreactivities into thin discs that reside mostly within individual synapse boundaries. The size and shape of pMad domains suggest that pMad could associate with membrane-anchored complexes at the active zone. Since BMP signals are generally short lived, these pMad domains likely represent pMad that, upon phosphorylation, remains associated with the BMP/BMPR kinase complexes at synaptic sites. Alternatively, pMad may accumulate in synaptic aggregates that protect it from dephosphorylation. While the second possibility cannot be excluded, several lines of evidence support the first scenario. First, excess Mad cannot increase the levels of synaptic pMad. Second, neuronal expression of activated Tkv/Sax but not Mad can restore the synaptic pMad at Importin impβ11 mutant NMJs. Finally, during neural tube closure, junctional pSmad1/5/8 and its association with PAR complexes depend on BMPs. Previous studies indicate a reduction of synaptic pMad signals in response to muscle-specific Mad RNAi. This study too has observed such a reduction. In addition, this study found a significant decrease of postsynaptic IIA/IIB ratio in Mad-depleted muscles: GluRIIA and GluRIIB synaptic levels were reduced to 49% and respectively 78% of control. Since GluRIIA is key to the synaptic pMad accumulation it is suspected that the muscle Mad RNAi phenotype is due to perturbation in synaptic GluRIIA levels, perhaps by interference with the Activin signaling pathway (Sulkowski, 2016).

How are the BMP/BMPR complexes stabilized at synaptic sites? Studies on single receptors demonstrate that the confined mobility of BMPRI on the plasma membrane is key to stabilize BMP/BMPR complexes and differentially stimulate canonical versus noncanonical signaling. Direct interactions between phosphorylated Smad5 and the Par3-Par6-aPKC polarity complex occur at the apical junctions. Similarly, synaptic pMad, which remains associated with BMP/BMPR complexes, may engage in interactions that restrict the mobility of BMP/BMPR complexes on the presynaptic membrane. Nemo-mediated phosphorylation of Mad-S25 could disrupt the pMad/BMPR association and expose the BMP/BMPR complexes, so they could dissociate and/or be internalized. The heteromeric BMPR complexes are transient; ligand binding greatly increases their lifespan and stability. Albeit Gbb is not essential for synaptic pMad, it may act redundantly with other ligands to stabilize BMP/BMPR local complexes. Several ligands secreted in the synaptic cleft have been shown to bind and signal via BMPRII; they include glia secreted Maverik, Myoglianin, which could be secreted from muscle and/or glia, and Activins. However, these ligands also appear to signal via a canonical Activin pathway, which regulates the postsynaptic GluRIIA/GluRIIB abundance at the Drosophila NMJ. Alterations in the Activin signaling pathway drastically alter the synaptic recruitment of both iGluR subtypes, in particular the GluRIIA, which controls synaptic pMad, making it difficult to identify the nature and the directionality of the signaling molecule(s) involved in the synaptic pMad accumulation. Interestingly, all of these ligands are substrates for BMP-1/Tolloid-type enzymes, which control their activity and distribution. Treatments that induce long-term stimulation up-regulate a BMP-1/Tolloid homolog in Aplysia neurons (Sulkowski, 2016).

An intriguing aspect of this novel BMP pathway is the dependence on active postsynaptic GluRIIA, which is both required and sufficient for pMad accumulation at active zones. Since pMad and BMP/BMPR complexes cluster at synaptic sites, it is speculated that trans-synaptic complexes may couple postsynaptic type-A glutamate receptors with presynaptic BMP/BMPRs. The synaptic cleft is 200 Å; the iGluR tetramer expands 135 Å in the synaptic cleft, and the BMP/BMPR complexes ~55 Å. The iGluRs auxiliary subunit Neto has extracellular CUB and LDLa domains predicted to expand 120-130 Å in the synaptic cleft, based on related structures. CUB domains are BMP binding motifs that may localize BMP activities and/or facilitate ligand binding to BMPRs. In this model, Neto provides the link between postsynaptic GluRIIA and presynaptic BMP/BMPR complexes. During receptors gating cycle, the iGluRs undergo corkscrew motions that shorten the channels as revealed by cryo-electron microscopy. Such movements may influence the stability of trans-synaptic complexes and allow synaptic pMad to function as a sensor for GluRIIA activity (Sulkowski, 2016).

While more components of this novel pathway remain to be determined, it is clear that this pathway does not contribute to NMJ growth and instead has a critical role in synapse maturation. Unlike canonical BMP signaling, loss of local pMad induces minor reductions in bouton number and does not rescue the NMJ overgrowth of endocytosis mutants. Local pMad accumulates independently of Wit-mediated LIMK1 activation and does not appear to influence synapse stabilization; in fact, nrx mutants have synapse adhesion defects but show increased synaptic pMad levels. The striking correlation between synaptic pMad levels and GluRIIA activity, together with previous findings that GluRIIA activity and gating behavior directly impacts receptor mobility and synaptic stabilization suggest a positive feedback mechanism in which active GluRIIA receptors induce stabilization of BMP/BMPR complexes at synaptic sites which, in turn, promote stabilization of type-A receptors at PSDs. In this scenario, presynaptic pMad marks active BMP/BMPR complexes and acts to maintain the local BMP/BMPR complexes in large clusters that evade endocytosis. Selective disruption of local pMad via a neuronal dominant-negative MadS25D presumably destabilizes the large presynaptic BMP/BMPR clusters and causes a significant reduction in the IIA/IIB ratio and quantal size (Sulkowski, 2016).

This positive feedback couples synaptic activity with synapse development and is controlled by (1) active GluRIIA receptors, (2) presynaptic BMP receptors, Wit, Sax, and likely Tkv, (3) mechanisms regulating BMPR heteromers assembly, endocytosis and turnover, and (4) the ability of pMad to remain associated with its own kinase upon phosphorylation. Perturbations of any of these components trigger variations in local pMad levels accompanied by changes in the IIA/IIB ratio and/or quantal size. For example, nemo mutants have increased synaptic pMad levels and increased mEJCs, while imp mutants have decreased synaptic pMad levels and decreased mEJPs. The assembly and function of these putative trans-synaptic complexes, in particular ligand availability, should be influenced by the composition and organization of the synaptic cleft. Indeed, local pMad and quantal size are increased in mutants lacking heparan sulfate 6-O-endosulfatase (sulf1), or 6-O-sulfotransferase (hs6st). Since this Mad-dependent, noncanonical pathway shares components with the other BMP signaling pathways, the balance among different BMP pathways may coordinate the NMJ development and function (Sulkowski, 2016).

The complexity of BMP signaling at the Drosophila NMJ is reminiscent of the neurotrophin-regulated signaling in vertebrate systems. Neurotrophins were first identified as neuronal survival factors. Like BMPs, they are secreted as pro-proteins that must be processed to form mature ligands. The active dimers bind to transmembrane kinase receptors and induce their activation through trans-phosphorylation. Neurotrophin/receptor complexes are internalized and transported along axons to the cell soma; signaling in the cell soma controls gene expression and promotes neuronal differentiation and growth. In addition, local neurotrophin signaling regulates growth cone motility, enhances the presynaptic release of neurotransmitter and mediates activity-dependent synapse formation and maturation. At the Drosophila NMJ, several neurotrophins have been implicated in neuron survival, axon guidance and synapse growth. It will be interesting to test for the crosstalk between neurotrophin and BMP signaling at these synapses (Sulkowski, 2016).

The novel noncanonical BMP pathway reported in this study is the first example of a BMP pathway triggered by selective neurotransmitter receptors and influencing receptor distribution at PSDs. It is expected that some of these functions will apply to mammalian glutamatergic synapses: First, as indicated in the Allen Brain Atlas, glutamate receptors and Neto proteins are widely expressed in mammalian brain structures where BMPs, BMPRs and Smads are expressed. Second, BMPs have been shown to rapidly potentiate glutamate-mediated currents in human retina neurons, presumably via a noncanonical pathway. Finally, mice lacking Chordin, a BMP antagonist, have enhanced paired-pulse facilitation and LTP and show improved learning in a water maze test. Such changes could not be explained by Smad-dependent transcriptional responses and were not accompanied by structural alterations in synapse morphology. Instead, presynaptic noncanonical BMP pathway may influence the activity of postsynaptic glutamate receptors by modulating their synaptic distribution and stability (Sulkowski, 2016).

The equilibrium between antagonistic signaling pathways determines the number of synapses in Drosophila

Using the Drosophila larval neuromuscular junction, this study shows a PI3K-dependent pathway for synaptogenesis (a pro-syaptogenesis pathway) which is functionally connected with other previously known elements including the Wit receptor, its ligand Gbb, and the MAPkinases cascade. Based on epistasis assays, the functional hierarchy within the pathway was determined. Wit seems to trigger signaling through PI3K, and Ras85D also contributes to the initiation of synaptogenesis. However, contrary to other signaling pathways, PI3K does not require Ras85D binding in the context of synaptogenesis. In addition to the MAPK cascade, Bsk/JNK undergoes regulation by Puc and Ras85D which results in a narrow range of activity of this kinase to determine normalcy of synapse number. The transcriptional readout of the synaptogenesis pathway involves the Fos/Jun complex and the repressor Cic. In addition, an antagonistic pathway (an anti-synaptogenesis pathway) was identified that uses the transcription factors Mad and Medea and the microRNA bantam to down-regulate key elements of the pro-synaptogenesis pathway. Like its counterpart, the anti-synaptogenesis signaling uses small GTPases and MAPKs including Ras64B, Ras-like-a, p38a and Licorne. Bantam downregulates the pro-synaptogenesis factors PI3K, Hiw, Ras85D and Bsk, but not AKT. AKT, however, can suppress Mad which, in conjunction with the reported suppression of Mad by Hiw, closes the mutual regulation between both pathways. Thus, the number of synapses seems to result from the balanced output from these two pathways (Jordan-Alvarez, 2017).

The epistasis assays have determined the in vivo functional links between PI3K and other previously known pro-synaptogenesis factors. Epistasis assays are based on the combined expression of two or more UAS constructs. Several double combinations in this study have produced a phenotype in spite of the apparent ineffectiveness of the single constructs. This type of results underscores the necessity to use epistasis assays in order to reveal functional interactions in vivo, hence, biologically relevant. In addition to the pro-synaptogenesis signaling, the study has revealed an anti-synaptogenesis pathway that composes a signaling equilibrium to determine the actual number of synapses. The magnitude of the synapse number changes elicited by the factors tested here are mostly within the range of 20%-50%. Are these values significant to cause behavioral changes? Reductions in the order of 30% of excitatory or inhibitory synapses in adult Drosophila local olfactory interneurons transform perception of certain odorants from attraction to repulsion and vice versa. In schizophrenia patients, a 16% loss of inhibitory synapses in the brain cortex has been reported. In Rhesus monkeys, the pyramidal neurons in layer III of area 46 in dorsolateral prefrontal cortex show a 33% spine loss, and a significant reduction in learning task performance during normal aging. Thus, it seems that behavior is rather sensitive to small changes in synapse number irrespective of the total brain mass (Jordan-Alvarez, 2017).

The signaling interactions analyzed here were chosen because they were reported in other cellular systems and species previously. Some of these interactions have been confirmed (e.g., Gbb/Wit), while others have proven ineffective in the context of synaptogenesis (e.g., Ras85D/PI3K binding). Likely, the two signaling pathways, pro- and anti-synaptogenesis, are not the only ones relevant for synapse formation. For example, in spite of the null condition of the gbb and wit mutant alleles used here, the resulting synaptic phenotypes are far less extreme than expected if these two factors would be the only source of signaling for synaptogenesis. Although it could be argued that the incomplete absence of synapses in the mutant phenotypes could result from maternal perdurance, Wit is not part of the oocyte endowment while Gbb is. Three alternative possibilities may be considered, additional ligands for Wit, additional receptors for Gbb, and a combination of the previous two. Beyond the identity of these putative additional ligands and receptors, the stoichiometry between ligands and receptors may certainly be relevant. Actually, Gbb levels are titrated by Crimpy. An equivalent quantitative regulation could operate on Wit. The reported data on Wit illustrate already the diversity of the functional repertoire of this receptor. Wit can form heteromeric complexes with Thick veins (Tkv) or Saxophone (Sax) receptors to receive Dpp/BMP4 or Gbb/BMP7 as ligands. However, the same study also showed that Wit could dimerize with another receptor, Baboon, upon binding of Myoglianin to activate a different and antagonistic signaling pathway, TGFβ/activin-like (Jordan-Alvarez, 2017).

The Gbb/Wit/PI3K signaling analyzed in this study is likely not the only pro-synaptogenesis pathway in flies and vertebrates. The ligand Wingless (Wg), member of the Wnt family, and the receptors Frizzled have been widely documented as relevant in neuromuscular junction development, albeit data on synapse number are scant. Interestingly, however, the downstream intermediaries can be as diverse as those mentioned above for Wit. Although generally depicted as linear pathways, a more realistic image would be a network of cross-interacting signaling events whose in situ regulation and cellular compartmentalization remains fully unexplored (Jordan-Alvarez, 2017).

The quantitative regulation of receptors is most relevant to understand their biological effects. In that context, is worth noting that Tkv levels are distinctly regulated from those of Wit and Sax through ubiquitination in the context of neurite growth. On the other hand, although the receptor Wit is considered a RSTK type, the functional link with PI3K is a feature usually associated to the RTK type instead. The link of Wit with a kinase has a precedent with LIMK1 that binds to, and is functionally downstream from, Wit in the context of synapse stabilization. Thus, Wit should be considered a wide spectrum receptor in terms of its ligands, co-receptor partners and, consequently, signaling pathways elicited. Actually, the Wit amino acid sequence shows both, Tyr and Ser/Thr motifs justifying its initial classification as a 'dual' type of receptor. In this report this study did not determine if Wit heterodimerizes with other receptors, as canonical RSTKs do, or if it forms homodimers, as canonical RTKs do. However, the lack of synaptogenesis effects by the putative co-receptors, Tkv and Sax, and the phenotypic similarity with the manipulation of the standard RTK signaling effector Cic, leaves open the possibility that Wit could play RTK-like functions, at least in the context of synaptogenesis (Jordan-Alvarez, 2017).

Consistent with the proposal of a dual mechanism for Wit, its activation seems to be a requirement to elicit two independent signaling steps, PI3K and Ras85D, that could reflect RTK and RSTK mechanisms, respectively. Both steps are independent because the mutated form of PI3K unable to bind Ras85D, PI3KΔRBD, is as effective as the normal PI3K to elicit synaptogenesis. PI3K and Ras85D signaling, however, seem to converge on Bsk revealing a novel feature of this crossroad point. The activity level of Bsk is known to be critical in many signaling processes. The peculiarities of Bsk/JNK activity include its coordinated regulation by p38a and Slpr in the context of stress heat response without interference on the developmental context. Another modulator, Puc, was described as a negative feed-back loop in the context of oxidative stress. The Puc mediated loop is operative also for synaptogenesis, while that of p38a/Slpr is relevant for p38a only, as shown here. Further, Ras85D represents an additional regulator in the neural scenario. The triple regulation of Bsk/JNK by Ras85D, Puc and the MAPKs seems to stablish a narrow range of activity thresholds within which normal number of synapses is determined (Jordan-Alvarez, 2017).

The concept of signaling thresholds is also unveiled in this study by the identification of another signaling pathway that opposes synapse formation. The pro- and anti-synaptogenesis pathways have similar constituents, including small GTPases, MAPKs and transcriptional effectors, Mad/Smad, which are canonical for RSTK receptors. The RSTK type II receptor Put, which can mediate diverse signaling pathways according to the co-receptor bound can be discarded in either the pro- or the anti-synaptogenesis pathways. Thus, the main receptor for the anti-synaptogenesis pathway remains to be identified (Jordan-Alvarez, 2017).

Concerning small GTPases, the pro-synaptogenesis pathway uses Ras85D while its counterpart uses the poorly studied Ras64B. The anti-synaptogenesis pathway includes an additional member of this family of enzymes, Rala. This small GTPase plays a role in the exocyst-mediated growth of the muscle membrane specialization that surrounds the synaptic bouton as a consequence of synapse activity. That is, Rala can influence synapse physiology acting from the postsynaptic side. The experimental expression of a constitutively active form of Rala in the neuron does not seem to affect the overall synaptic terminal branching. However, the null ral mutant shows reduced synapse branching and its vertebrate homolog is expressed in the central nervous system. This study found that Rala under-expression in neurons yields an elevated number of synapses. Thus, it is likely that this small GTPase acts as a break to synaptogenesis, hence its inclusion in the antagonistic pathway (Jordan-Alvarez, 2017).

Synaptogenesis and neuritogenesis are distinct processes since each one can be differentially affected by the same mutant (e.g.: Hiw). Both features, however, share some signals (e.g., Wnd, Hep). This signaling overlap is akin to the case of axon specification versus spine formation for constituents of the apico/basal polarity complex Par3-6/aPKC [127]. These and other examples illustrated in this study underscore the need to discriminate between synapses and boutons. This study is focused on the cell autonomous signaling that takes place in the neuron. Non-cell autonomous signals (e.g., originated in the glia or hemolymph circulating) have not been considered. The active role of glia in axon pruning and bouton number has been the subject of other studies. Considering the reported role of Hiw through the midline glia in the remodeling of the giant fiber interneuron it is not unlikely that the glia-to-neuron signaling may share components with the neuron autonomous signaling addressed here (Jordan-Alvarez, 2017).

The summary scheme (see Summary diagram of antagonistic signaling pathways for synaptogenesis and their interactions) describes the scenario where two signaling pathways mutually regulate each other. Epistasis assays are the only experimental approach for in vivo studies of more than one signaling component, albeit this type of assay is only feasible in Drosophila Thus, it is plausible that vertebrate synaptogenesis will be regulated by a similar antagonistic signaling (Jordan-Alvarez, 2017).

The regulatory equilibrium as a mechanism to determine a biological parameter is the most relevant feature in this scenario for several reasons. First, because this type of mechanism can respond very fast to changes in the physiological status of the cell, and, second because it provides remarkable precision to the trait to be regulated, synapse number in this case. Although bi-stable regulatory mechanisms are known in other contexts, the case of synapse number may seem unexpected because the highly dynamic nature of synapse number has been recognized only recently. Consequently, a molecular signaling mechanism endowed with proper precision and time resolution must sustain this dynamic process. The balanced equilibrium uncovered in this study, although most likely still incomplete in terms of its components, offers such a mechanism (Jordan-Alvarez, 2017).

Dpp of posterior origin patterns the proximal region of the wing

The decapentaplegic (dpp) gene encodes a long-range morphogen that plays a key role in the patterning of the wing imaginal disc of Drosophila. The current view is that dpp is transcriptionally active in a narrow band of anterior compartment cells close to the anterio-posterior (A/P) compartment border. Once the Dpp protein is synthesised, it travels across the A/P border and diffuses forming concentration gradients in the two compartments. A new site of dpp expression has been found in the posterior wing compartment that appears during the third larval period. This source of Dpp signal generates a local gradient of Dpp pathway activity that is independent of that originating in the anterior compartment. This posterior tier of Dpp activity is functionally required for normal wing development: the elimination of dpp expression in the posterior compartment results in defective adult wings in which pattern elements such as the alula and much of the axillary cord are not formed. Moreover, these structures develop normally in the absence of anterior dpp expression. Thus the normal wing pattern requires distinct Dpp organizer activities in the anterior and posterior compartments. It was further shown that, unlike the anterior dpp expression domain, the posterior one is not dependent on Hedgehog activity but is dependant on the activity of the IRO complex gene mirror. Since there is a similar expression in the haltere disc, it is suggested that this late appearing posterior Dpp activity may be an attribute of dorsal thoracic discs (Foronda, 2009).

This study was triggered by a consistent observation of a small region in the P compartment of the wing disc that appeared to be active in dpp transcription. This P compartment expression of dpp has not been properly analysed in previous works about Dpp function in wing disc. dpp expression was carefully examined in third instar wing and haltere discs by in situ hybridization and also with a P-element insertion (P10638) at the disk region of the dpp gene. Transcriptional activity was identified close to and anterior to the A/P border. In addition, dpp transcripts were found in a proximal region of the posterior compartment. A homologous zone of dpp expression was also found in the posterior region of the haltere disc. This dpp posterior transcriptional domain appears during the third larval period; wing discs from early 3rd larval instar do not show it (Foronda, 2009).

According to the fate map, the posterior region containing dpp expression gives rise to proximal adult wing structures, including the alula and the axillary cord. This domain in adult wings was delimited by X-gal staining freshly emerged flies carrying the dpp-lacZ insertion. The area with lacZ activity corresponds mostly to the axillary cord (Foronda, 2009).

To test whether the posterior dpp expression activates the Dpp transduction pathway, use was made of an antibody raised against the phosphorylated (active) form of Mad, an indicator of Dpp pathway activity. As expected from previous work there are high levels of pMad in the centre of the disc, but in addition a domain of pMad activity was observed in the posterior compartment, which includes the dpp expression domain. The zone expressing pMad is bigger than that expressing dpp, consistent with the formation of a diffusion gradient of Dpp activity (Foronda, 2009).

In addition to pMad levels, whether other elements of the Dpp pathway are expressed in the posterior dpp domain was also examined. The gene daughters against dpp (dad) is a target that requires moderate levels of Dpp signalling. dad is expressed in the posterior wing compartment in the same region where pMad is active. Other Dpp-target genes, like optomotor blind and spalt, are also expressed in this region (Foronda, 2009).

The brinker (brk) gene is a special case as it is negatively regulated by Dpp activity. Therefore, it is expressed in the lateral regions of the wing disc where the levels of Dpp activity are lower. The domain of Dpp activity in the proximal posterior compartment should therefore repress brk activity in that region. The comparison of pMad and brk activities in this region clearly shows they occupy mutually exclusive domains, supporting the idea that brk is repressed by the posterior pMad activity. The staining of adult wings of brk-lacZ genotype also argues in the same direction because the region of brk expression does not coincide but abut with that containing Dpp activity. Although the haltere disc was not analyzed with the same detail, there is also a posterior region of pMad activity in the zone where dpp is expressed (Foronda, 2009).

pMad and brk expression were examined in wing discs from second instar larvae. These exhibit the central domain of pMad activity but there is no detectable staining in the posterior compartment. This result is consistent with the late appearance of the posterior dpp transcription domain and indicates that this domain functions only during the second half of the proliferation phase of the disc (Foronda, 2009).

Having shown that the posterior dpp expression domain acts as a source of morphogen the question was addressed whether it has a functional role. Previous experiments analysing dpp mutant clones did not report significant alterations in the posterior wing. However, given the diffusible nature of the Dpp product it is possible that the lack of Dpp in the mutant clones could be rescued by Dpp emanating from neighbour wildtype cells (Foronda, 2009).

An experiment was designed in which all cells in the posterior compartment would be homozygous for the dppd12 mutation, which eliminates dpp activity in the wing disc without affecting embryonic or pupal expression. In discs of genotype dppd12 ck FRT40A/M (2)24F arm-Z FRT40A; hh-gal4/UAS-Flp the high levels of flipase generated by the hh-gal4 driver would induce FRT-mediated mitotic recombination in virtually all the cells in the posterior compartment. The dpp M+ clones produced will have proliferation advantage and are expected to fill the posterior compartment. They can be identified in the disc because they lose the arm-lacZ transgene, and in the adult wing because they are homozygous for the cuticular marker crinkled (ck) (Foronda, 2009).

These clones entirely fill the posterior compartment. Staining for pMad activity shows the normal pattern in the centre of the disc, but the posterior pMad domain is completely absent, in clear contrast with wildtype discs (Foronda, 2009).

Since the genotype used allows good viability of the flies containing dpp M+ clones, a large number of adult wings was examined. In nearly every case the entire posterior compartment is marked with ck, indicating that it lacks dpp expression. The pattern and size of these wings is normal except in the proximal posterior region: the alula does not form and the pattern that appears in its place resembles that of a more distal region, which is specified by Dpp of anterior origin. The axillary cord is much reduced in size and has lost all its characteristic long hairs; some sclerites are also missing. This loss of structures does not appear to be due to death of proximal cells; no indication of caspase activity was found in this region. Moreover, the addition of the apoptosis inhibitor P35 in the posterior compartment does not modify the phenotype of the dpp M+ compartments (Foronda, 2009).

The favored interpretation of the wing phenotype is that in absence of the organising activity of the posterior Dpp, the proximal region is only patterned by Dpp of anterior provenance. It is interesting to note that even though dpp is not expressed in the alula cells, this structure is affected. This result illustrates the role of the posterior Dpp as an organizer, since it affects patterning in a non-autonomous manner (Foronda, 2009).

One intriguing question was if this posterior Dpp could form these proximal structures without Dpp of anterior origin. A transgenic strain carrying UAS-shmiR-dpp2 construct was used which has been shown to degrade the mRNA of Dpp. The smhiR-dpp over-expression in the anterior Dpp domain (by using ptc-gal4 or dppblnk-gal4) is able to silence efficiently Dpp gene activity, as shown by absence of pMad staining and the extended brk expression domain. Neither the posterior dpp expression nor the formation of alula and axillary cord are affected in both size and morphology. It is concluded that Dpp of posterior origin is necessary and sufficient to pattern these structures (Foronda, 2009).

dpp transcription in the P compartment is an intriguing finding and suggests a novel mode of dpp activation. The normal activation of dpp in the A compartment requires Hh signalling, which is blocked in the P compartment. Alternatively, it was possible that a local diminution of en activity in the proximal region of the P compartment would allow Hh and dpp activation by the standard mechanism. This question was addressed by examining in this region the expression of Cubitus interruptus (ci), a transcription factor that is essential for Hh signalling, and of patched (ptc), a Hh target gene. It was found that neither ci nor ptc are expressed in the posterior. However, there was the possibility that these two genes were expressed at low, undetectable levels. To test this possibility in full, Dpp activity was examined in clones of cells mutant for the smoothened (smo) gene, which would be unable to transduce the Hh signal. The result demonstrates that the posterior expression of dpp does not require Hh signalling and must therefore be activated by a different mechanism (Foronda, 2009).

The approach taken to identify the factor/s behind this posterior Dpp expression was to look for candidate genes or signalling pathways which are expressed in the corresponding place in the posterior compartment. The first one was vein, a ligand of EGFR signalling pathway, which coexpresses with Dpp in late 3rd instar wing disc. vein is the only EGFR ligand required for a proper wing development, so it was a good candidate. The elimination of all posterior vein function has effect neither on posterior Dpp function nor on hinge morphology. Other genes were tested based on expression and/or mutant phenotype in the alula, i.e., homothorax, Zfh2 and empty spiracles, among others. None of them affected Dpp expression (Foronda, 2009).

Another likely candidate was mirror, a member of the Iroquois complex (iro-C), for which a role in alula and axillary cord formation has been described. mirr expression was examined using the mirr-lacZ line and it was found that mirr is expressed in the presumptive alula and axillary cord region (Foronda, 2009).

M+ mirr clones were made to generate posterior compartments that were wholly mirr. They show an adult phenotype more extreme than that of dpp compartments: the alula and the axillary cord are entirely missing. In the discs dpp expression (shown by pMad staining) in the posterior compartment is lost, and consequently brk expression is up-regulated in the presumptive alula region. This result indicates that mirr is necessary for posterior dpp expression. In contrast, Dpp is not required for mirr expression, since the lack of posterior Dpp does not have an effect on mirr-LacZ transcription (Foronda, 2009).

Since the preceding results might suggest a mirr-mediated dpp activation gain of function clones of mirr were generated and whether they gave rise to ectopic Dpp activity was checked. NI significant up-regulation of dpp associated with those clones was detected. These experiments demonstrate that mirr activity is necessary for posterior dpp expression, but it is not sufficient to induce it. Therefore there must be other factors involved in posterior dpp activation (Foronda, 2009).

These results report a novel organizer role of Dpp that occurs during the third instar and is necessary and sufficient to pattern the proximal posterior region of the wing. This is achieved by an hh-independent, mirr-dependent activation of dpp in the posterior compartment (Foronda, 2009).

These findings provide a more complete picture of the development of the wing disc. There is evidence that the three signalling Dpp, Wg and Hh pathways are necessary for normal wing pattern and originate at compartment borders. The results indicate an unforeseen complexity of the function of the Dpp pathway. dpp becomes active in two different body domains, which have independent temporal and spatial regulation. The anterior domain is required for distal wing growth and patterning, whereas the P compartment domain is responsible for the formation of the posterior-proximal structures of the wing. Moreover, the source of Dpp in the posterior domain does not appear to be a compartment border, since no lineage restriction has been reported in the region. A comparable situation has been described for leg development, in which the Dpp and Wg signals originate at the A/P border, but the source of the EFGR signal is not a lineage border (Foronda, 2009).

The fact that this new dpp expression domain is common to wings and halteres may have some evolutionary significance, since this may be an attribute of dorsal thoracic appendages. These results suggest that the posterior tier of dpp expression may have appeared before the mesothoracic and metathoracic appendages diverged. It is therefore, possible that the new model of dorsal appendage development proposed may occur in other species of insects (Foronda, 2009).

Dpp signaling activity requires Pentagone to scale with tissue size in the growing Drosophila wing imaginal disc

The wing of the fruit fly, Drosophila melanogaster, with its simple, two-dimensional structure, is a model organ well suited for a systems biology approach. The wing arises from an epithelial sac referred to as the wing imaginal disc, which undergoes a phase of massive growth and concomitant patterning during larval stages. The Decapentaplegic (Dpp) morphogen plays a central role in wing formation with its ability to co-coordinately regulate patterning and growth. This study asked whether the Dpp signaling activity scales, i.e. expands proportionally, with the growing wing imaginal disc. Using new methods for spatial and temporal quantification of Dpp activity and its scaling properties, it was found that the Dpp response scales with the size of the growing tissue. Notably, scaling is not perfect at all positions in the field and the scaling of target gene domains is ensured specifically where they define vein positions. It was also found that the target gene domains are not defined at constant concentration thresholds of the downstream Dpp activity gradients P-Mad and Brinker. Most interestingly, Pentagone (Pent: see Flybase Magu), an important secreted feedback regulator of the pathway, plays a central role in scaling and acts as an expander of the Dpp gradient during disc growth (Hararatoglu, 2011).

This study measured Dpp pathway activity using an antibody specific to the phosphorylated form of Mad, and compared the P-Mad levels in space and time with the activity levels of direct target genes, such as brk, which plays key roles in both growth and patterning. Transcription of brk is directly repressed via P-Mad binding at defined silencer elements (SEs), resulting in inversely graded brk expression. Brk is the only known regulator affecting the positioning of the expression boundary of omb, while sal and dad translate input from both P-Mad and Brk into their expression boundaries. The dynamics of all of these readouts were analysed using antibodies where possible, to avoid potential misinterpretations due to reporter stability (Hararatoglu, 2011).

P-Mad levels scale very well posterior to 0.4 Lp (Lp stands for the length of the posterior compartment) with the exception of TC5 profiles near the D/V boundary. Previous studies that examined P-Mad scaling reached contradictory conclusions: the P-Mad gradients in late stage discs were reported to correlate with tissue size in a previous study and to have no correlation in another. Similarly, examination of P-Mad gradients across discs of different sizes led to the conclusion that the gradient did not expand, but in another study the P-Mad gradient was shown to scale with tissue size. It is believed that most of the confusion can be attributed to different profile extraction protocols as well as to the use of various definitions of scaling, as discussed below (Hararatoglu, 2011).

Since P-Mad is an early signature of the activation of the Dpp signaling pathway, it was of interest to find out how its scaling properties translate to its immediate key target, the brk gene. In addition to brk being directly repressed by P-Mad, the Brk protein itself is necessary for graded brk transcription. The range of Brk expression was found to strictly follow P-Mad in both wild-type and pent mutant discs. Similar to what was observe for P-Mad, Brk also shows very good scaling for positions posterior to 0.4 Lp. By contrast, levels of Brk increase steadily as the discs grow and cannot be explained by P-Mad dynamics alone. This increase in Brk levels could be due to the build-up of the unknown activator of brk transcription or, alternatively, the SEs in brk could become desensitized to the repressive input of P-Mad. Regardless of the cause of the increase, cells at a given relative position experience increasing levels of the Brk repressor over time (Hararatoglu, 2011).

Traditionally, Dpp and P-Mad gradients have been described by a decaying exponential with characteristic decay length λ. This decay length is different for each profile and corresponds to the position at which the protein levels have decreased by a factor e. The correlation between the decay length and the tissue size has been used as a proxy for scaling, e.g. one study found no significant scaling for the Dpp and P-Mad profiles at the end of 3rd instar stage. Similarly, the width of the P-Mad profile has been used to characterize the spread of the gradient and it was concluded that the width of the P-Mad profile is constant during growth. In contrast, the current study detected that the P-Mad profile expands as the tissue grows. This discrepancy may be due to the fact the previous study lacked time classes TC3 and TC4 in their sample collection, the period where the P-Mad gradient expands before contracting again when measured close to the D/V boundary. Thus, possibly the measurements of the P-Mad profiles were done in the vicinity of the D/V boundary, where at TC5 P-Mad has a sharp profile reminiscent of 30 h younger discs. Hence, the choice of position can significantly alter the final interpretations of the data (Hararatoglu, 2011).

Consistent with these results, another study (Wartlick, 2011) recently showed that the decay lengths of Dpp-GFP, P-Mad, brk-GFP, and dad-RFP do correlate with the length and the area of the posterior compartment during tissue growth. Importantly, the Wartlick study also assessed scaling qualitatively in the whole field by looking at the collapse of the profiles in relative positions and normalized intensities. This method has two advantages: it does not require any fitting of the profiles, and it shows scaling at all positions and not just at the characteristic decay length position (i.e. x = λ) (Hararatoglu, 2011).

A recent mathematical model termed 'expansion-repression integral feedback control' suggests that scaling can emerge as a natural consequence of a feedback loop (BenZvi, 2010). The hypothetical 'expander' molecule facilitates the spread of the morphogen and in turn is repressed by it; scaling is achieved given that the expander is stable and diffusible. The known properties of Pent fit the requirements of this hypothetical agent: Pent is secreted, required for Dpp spreading, and pent transcription is directly inhibited by Dpp signaling. However, it is not known how stable Pent is, and pent transcription is never abolished in the entire field in which the gradient acts during larval stages. To test whether Pent could be a key player involved in scaling of Dpp activity during disc growth, the analyses were repeated at all time points in the absence of Pent. It was found that the P-Mad and Brk gradients indeed fail to scale with the tissue size in this mutant background. Scaling of dad-GFP and Omb are also strongly affected, while Sal still exhibits some degree of Pent-independent scaling in the anterior compartment. Importantly, while the function of Pent is essential for proper scaling of the Dpp activity gradient, it is noted that Pent alone cannot account for the observed selective scaling of Omb and Sal domain boundaries. Scaling of these target genes specifically in those regions in which they have a patterning function points to the involvement of additional players, which will be the subject of future research. Hence, the current findings strongly suggest that Pent is a very good candidate to be the expander in the 'expansion-repression integral feedback control' model and therefore provide the first mechanistic insights into the question of scaling in wing patterning. The exact biochemical functions of Pent have to be determined in order to get a more mechanical view of gradient scaling in the developing wing imaginal disc (Hararatoglu, 2011).

More than 40 years ago, Lewis Wolpert proposed the French flag model to explain pattern formation by morphogens. This study tested whether the activity gradients downstream of Dpp, namely P-Mad and Brk, are read out by their target genes at constant concentration thresholds. Thus, average P-Mad and Brk concentrations were measured at Dad, Omb, and Sal expression boundaries across development. It was found that the amount of P-Mad at these boundaries slightly increased (Dad), slightly decreased (Sal), or was constant throughout development (Omb). Among these three targets, the Omb domain is the widest and it corresponds to a region where the P-Mad gradients scale perfectly; as a result, P-Mad levels fluctuate very little at the Omb domain boundary. Interestingly, the domain boundary of Omb is thought to solely depend on Brk and hence constant P-Mad levels might be a mere coincidence. Remarkably, all the target genes that were considered respond to significantly increasing levels of Brk, suggesting that the target genes desensitize to Brk over time, so that more and more Brk can be tolerated at the domain boundary. Alternatively, if it is considered that the domain boundaries of dad-GFP and Sal do not respond to constant P-Mad levels either, another explanation could be that Brk and P-Mad signals are combined in a non-additive fashion in order to define the boundary position of the target genes. Following this assumption, a simple combination of these signals was sought that is constant at the target gene domain boundary for all TCs. It is proposed that Brk and the unknown activator of Omb could be similarly combined in order to determine the Omb domain boundary (Hararatoglu, 2011).

This paper's data was used to further test a model that was recently proposed to explain the uniform growth in the wing imaginal disc (Wartlick, 2011). The model poses that the temporal changes in Dpp signaling levels drive tissue growth; cells divide when they experience a relative increase of 50% in the levels of Dpp signaling. Since it is the relative differences and not the absolute amount of Dpp signal that regulate cell divisions, the model can account for the uniform growth of the wing disc. Since the relative increase in Dpp activity slows down, the cell cycles lengthen as the disc grows. Growth stops when the cell division time exceeds 30 h. The model of Wartlick is based on the finding that Dpp activity scales with tissue size and that cells at a given relative position experience increasing levels of Dpp signaling over time. In contrast, a general temporal increase was not observed in the level of Dpp signaling at a given relative position in this study. P-Mad is the most upstream and the most dynamic readout available for the activity of the Dpp pathway and it was found that the relative increase in P-Mad levels throughout development is not significantly different from zero at most relative positions. Why is the increase in Dpp-GFP levels not reflected in P-Mad levels? A potential explanation for this might be that the observed accumulation of Dpp-GFP was due to the stability and accumulation of Gal4 since Dpp-GFP was under UAS control. The Wartlick study showed that the half-life of the Dpp-GFP fusion protein is only 20 min, but the Gal4 stability was not considered. Alternatively, the system could get desensitized over time and more and more Dpp would be required to lead to similar P-Mad levels. Finally, increases in Dad levels could counteract the increase in Dpp levels, since Dad is an inhibitory Smad (Hararatoglu, 2011).

Wartlick (2011) monitored Dpp signaling levels using a dad-RFP reporter and found a 5-fold increase in the course of 36 h. In the current analysis, a similar tool, dad-GFP, was used and the Wartlick results could not be fully reproduced. Though it was also found that dad-GFP scales with tissue size, its levels increase merely 2-fold over 40 h, and this increase takes place only in the medial 25% region of the disc, while cells in the lateral part experience a decrease in dad-GFP levels. This disparity in the fold increases is likely due to the higher stability of RFP, since the enhancer used was identical in both studies. Additionally, it was found that the levels of Brk, another direct target of the pathway with a very well-established role in suppressing growth in lateral regions, increase in average 4-fold in the interval studied, an observation not reported by Wartlick. In the lateral areas, increase in Dpp activity (if present) is below detection levels and would be opposed by increasing Brk levels. Importantly, increasing Brk levels, if they were to depend solely on Dpp, would suggest decreasing Dpp activity in lateral areas as Brk expression is directly suppressed by Dpp activity. Hence, the data raise serious questions about the validity of this uniform growth model, especially in the lateral regions of the pouch. An alternative model is favored that does not rely on Dpp activity alone to explain uniform growth in the wing disc (Hararatoglu, 2011).

Ecdysteroids affect Drosophila ovarian stem cell niche formation and early germline differentiation

Steroid hormones are required in Drosophila for progression of oogenesis during late stages of egg maturation. This study shows that ecdysteroids regulate progression through the early steps of germ cell lineage. Upon ecdysone signalling deficit germline stem cell progeny delay switching on a differentiation programme. This differentiation impediment is associated with reduced TGF-β signalling in the germline and increased levels of cell adhesion complexes and cytoskeletal proteins in somatic escort cells. A co-activator of the ecdysone receptor, Taiman is the spatially restricted regulator of the ecdysone signalling pathway in soma. Additionally, when ecdysone signalling is perturbed during the process of somatic stem cell niche establishment enlarged functional niches able to host additional stem cells are formed (König, 2011).

This study shows that in Drosophila ecdysone signalling regulates differentiation of a GSC daughter and modulates ovarian stem cell niche size. The delay in GSC progeny differentiation correlates with reduced expression levels of TGF-β pathway components. Based on expression patterns it appears that germarial somatic cells, niche and ECs are the critical sites of ecdysteroid action and a co-activator of ecdysone receptor, Taiman is the spatially restricted regulator of ecdysone signalling in soma. During adulthood the ecdysone pathway has a specific role in EC differentiation and soma-germline cell contact establishment. In addition, during development the ecdysone signalling pathway has a role in somatic niche formation (König, 2011).

Ecdysteroids in general control major developmental transformations such as metamorphosis and morphogenesis in Drosophila. Different tissues and even different cell types within the same tissue respond to this broad signalling in a specific fashion and in a timely manner. In the developing Drosophila ovary steroid hormone receptors are expressed in a well-timed mode, high levels coinciding with proliferative and immature stages and low levels preceding reduced DNA replication and differentiation. Mutations in ecdysone pathway components affect ovarian morphogenesis, including heterochronic delay or acceleration in the onset of terminal filament differentiation. During the niche establishment the levels of both ecdysone receptors, EcR and USP are greatly downregulated in anterior somatic cells that will contribute to the niche per se. This study shows that perturbation of ecdysone signalling in pre-adult ovarian soma leads to the formation of enlarged niches. The specific response to systemic hormonal signalling in niche precursors is achieved by a specific function of the ecdysone receptor co-activator Taiman. When timely regulation of ecdysone signalling does not occur, more cells are recruited to become niche cells resulting in enlarged niches that are capable to host more stem cells. These data first show that ecdysone steroid hormonal signalling regulates the formation of the adult stem cell niche and suggest that a developmental tuning of ecdysone signalling controls the number of anterior somatic cells that will differentiate into cap cells (König, 2011).

It is logical that stem cell division and germline differentiation are regulated by some systemic signalling depending on the general state of the organism, which depends on age, nutrition, environmental conditions and so on. Hormones are great candidates for this type of regulation as they act in a paracrine fashion and their levels are changing in response to ever-changing external and internal conditions. Steroid binding to nuclear receptors in vertebrates triggers a conformational switch accompanied by increased histone acetylation that permits transcriptional co-activators binding and the transcription initiation complex assembly. In Drosophila, the trithorax-related protein, a histone H3 methyltransferase that like Taiman belongs to the p160 class of co-activators, and an ISWI-containing ATP-dependent chromatin remodelling complex (NURF), that regulates transcription by catalysing nucleosome sliding, both bind EcR in an ecdysone-dependent manner, showing that chromatin modifications can mediate response to this general signalling. Transcriptional regulation has a key role in GSC maintenance and differentiation, for example, the TGF-β ligand Dpp secreted by niche cells induces phosphorylation of the transcription factor Mad in GSCs that in turn suppresses transcription of the differentiation factor Bam. In addition, it has been shown recently that in Drosophila adult GSC ecdysone modulates the strength of TGF-β signalling through a functional interaction with the chromatin remodelling factors ISWI and Nurf301, a subunit of the ISWI-containing NURF chromatin remodelling complex (Ables, 2010). Therefore, it is plausible that ecdysone regulates Mad expression cell autonomously via chromatin modifications. Since pMad directly suppresses a differentiation factor Bam, it is expected that Bam would be expressed in pMad-negative cells. Interestingly, the findings show that ecdysone deficit decreases amounts of phosphorylated Mad in GSCs and also cell non-autonomously suppresses Bam in SSCs. As SSCs that express neither pMad nor Bam are accumulated when the ecdysone pathway is perturbed it suggests that there should be an alternative mechanism of Bam regulation. Even though eventually this still can be done on the level of chromatin modification, the data suggest that the origin of this soma-generated signal may be associated with cell adhesion protein levels. Further understanding of the nature of this signalling is of a great interest (König, 2011).

The progression of oogenesis within the germarium requires cooperation between two stem cell types, germline and somatic (escort) stem cells. In Drosophila, reciprocal signals between germline and escort (in female) or somatic cyst (in male) cells can inhibit reversion to the stem cell state and restrict germ cell proliferation and cyst growth. Therefore, the non-autonomous ecdysone effect can be explained by the necessity of two stem cell types that share the same niche (GSC and ESC) to coordinate their division and progeny differentiation. This coordination is most likely achieved via adhesive cues, as disruption of ecdysone signalling affects turnover of adhesion complexes and cytoskeletal proteins in somatic ECs: mutant cells exhibited abnormal accumulation of DE-Cadherin, β-catenin/Armadillo and Adducin (König, 2011).

Cell adhesion has a crucial role in Drosophila stem cells; GSCs are recruited to and maintained in their niches via cell adhesion. Two major components of this adhesion process, DE-Cadherin and Armadillo/β-catenin, accumulate at high levels in the junctions between GSCs and niche cells, while in the developing cystoblasts and escort cells levels of these proteins are strongly reduced. Levels of DE-Cadherin in GSCs are regulated by various signals, for example, nutrition activation of insulin signalling or chemokine activation of STAT, and this study shows that in ESCs it is regulated by steroid hormone signalling. Possibly, these two stem cell types respond to different signals but then differentiation of their progeny is synchronised via cell contacts. While hormones, growth factors and cytokines certainly manage stem cell maintenance and differentiation, the evidence also reveals that the responses to hormonal stimuli are strongly modified by adhesive cues (König, 2011).

Specificity to endocrine signalling can be achieved via availability of co-factors in the targeted tissue. Tai is a spatially restricted co-factor that cooperates with the EcR/USP nuclear receptor complex to define appropriate responses to globally available hormonal signals. Tai-positive regulation of ecdysone signalling can be alleviated by Abrupt via direct binding of these two proteins that prevents Tai association with EcR/USP (Jang, 2009). Abrupt has been shown to be downregulated by JAK/STAT signalling (Jang, 2009). Interestingly, JAK/STAT signalling also has a critical role in ovarian niche function and controls the morphology and proliferation of ESCs as well as GSCs. JAK/STAT signalling may interact with ecdysone pathway components in ECs to further modulate cell type-specific responses to global endocrine signalling. A combination of regulated by different signalling pathway factors that are also spatially and timely restricted builds a network that ensures the specificity of systemic signalling (König, 2011).

Knowledge of how steroids regulate stem cells and their niche has a great potential for stem cell and regenerative medicine. The current findings open the way for a detailed analysis of a role for steroid hormones in niche development and regulation of germline differentiation via adjacent soma (König, 2011).

BMP signaling dynamics in the follicle cells of multiple Drosophila species

The dorsal anterior region of the follicle cells (FCs) in the developing Drosophila egg gives rise to the respiratory eggshell appendages. These tubular structures display a wide range of qualitative and quantitative variations across Drosophila species, providing a remarkable example of a rapidly evolving morphology. In D. melanogaster, the bone morphogenetic protein (BMP) signaling pathway is an important regulator of FCs patterning and dorsal appendages morphology. To explore the mechanisms underlying the diversification of eggshell patterning, BMP signaling was analyzed in the FCs of 16 Drosophila species that span 45 million years of evolution. The spatial patterns of BMP signaling in the FCs were found to be dynamic and exhibit a range of interspecies variation. In most of the species examined, the dynamics of BMP signaling correlate with the expression of the type I BMP receptor thickveins (tkv). This correlation suggests that interspecies variations of tkv expression are responsible for the diversification of BMP signaling during oogenesis. This model was supported by genetic manipulations of tkv expression in the FCs of D. melanogaster that successfully recapitulated the signaling diversities found in the other species. These results suggest that regulation of receptor expression mediates spatial diversification of BMP signaling in Drosophila oogenesis, and they provide insight into a mechanism underlying the evolution of eggshell patterning (Niepielko, 2011).

In FCs of D. melanogaster, dynamics of BMP signaling are regulated by the dynamics of tkv. In most Drosophila species, a correlation between tkv expression and BMP signaling dynamics was found. Remarkably, ectopic expression/depletion of tkv was sufficient to diversify BMP signaling in D. melanogaster. These perturbations successfully transformed the pattern of BMP signaling found in D. melanogaster into the diverse patterns of BMP signaling naturally found across Drosophila species, supporting the claim that tkv plays a major role in specifying the distribution of BMP signaling in the FCs. In D. melanogaster, a similar mechanism restricts the distribution of BMP signaling in wing and haltere imaginal discs (Niepielko, 2011).

In D. melanogaster, the dynamics of tkv are regulated jointly by BR and BMP signaling. Thus, the late patterns of BR, TKV, and P-MAD are observed in a similar group of cells. Surprisingly, in some species, the pattern of P-MAD correlated with the pattern of tkv; however, these patterns did not fully overlap the BR domain. Specifically, in addition to overlapping the BR domain, tkv was expressed in the adjacent cells that lacked BR expression. It is proposed that, in addition to being regulated by BR and BMP signaling, tkv is also regulated by transcription factors that are expressed in the adjacent domain such as Jra and Fos/Kayak (Niepielko, 2011 and references therein).

Of particular interest are species with three DAs' eggshell, due to the absence of tkv from the BR domain. It is suggested that this signaling pattern provides an example for decoupling of the regulation of tkv by BR from its regulation by BMP signaling. A model is proposed by which tkv is regulated by BMP signaling (anterior); however, it lost its regulation by BR. Of note, the mechanism governing tkv patterning is still unknown, and the established tkv enhancer trap in the Drosophila wing failed to recapitulate the patterns of tkv in the FCs. Thus, the proposed modifications in tkv regulation remain to be experimentally validated (Niepielko, 2011).

In D. melanogaster, early BMP signaling appears as an anterior stripe, reflecting the anterior secretion of the ligand DPP that signals through a uniformly expressed tkv receptor. Thus, in species from the virilis repleta groups, the uniform expression of tkv could account for the anterior stripe domain of late BMP signaling. Indeed, ectopic expression of tkv in all FCs prevented the dorsal anterior repression of BMP signaling in D. melanogaster, which was consistent with the pattern found in flies from the virilis repleta groups (Niepielko, 2011).

In the first three patterning classes, spatial modifications in the late patterns of tkv provided a reasonable explanation for the diversity in late patterns of BMP signaling. In the virilis repleta groups, late expression of tkv was uniform in all FCs; at the same time, BMP signaling was patterned. While the possibility cannot be excluded that a second copy of tkv is present in the non-sequenced species, it is proposed that in these species other BMP components have evolved to gain control of BMP signaling dynamics. In D. melanogaster, the disruption of saxophone (sax), a type I BMP receptor, deformed operculum size and DAs' morphologies. Thus, SAX is a potential regulator of the BMP signaling dynamics in the virilis repleta groups. Also, additional mechanisms were shown to regulate BMP signaling across animals including intracellular and extracellular inhibitors, co-receptors, levels of ligand expression, combinations of ligands, and interactions with other signaling pathways. These mechanisms should be studied systematically in order to determine which BMP component controls the DV phase of signaling in these species (Niepielko, 2011).

In D. melanogaster, the early phase of BMP signaling prevents the anterior domain of the follicle cells from acquiring DA cell fate. This mechanism is based on the inhibition of br expression by the anterior BMP signaling. Thus, it is not surprising that disruption of early BMP signaling is associated with eggshell deformations including modifications in the numbers and shapes of DAs'. Due to the high sensitivity of the eggshell's structures to changes in BMP signaling, it is speculated that small differences in early BMP signaling could guide the natural variations in numbers and shapes of DAs found across Drosophila species (Niepielko, 2011).

In D. melanogaster, the late phase of BMP signaling is associated with the repression of br mRNA in the dorsolateral patches; however, its role in eggshell morphology was not explicitly explored. Depletion of BMP signaling from the BR domain did not affect early BR patterning and operculum size; however, this perturbation deformed DAs' morphology possibly due to late migration of BR cells. Interestingly, similar morphologies were found by disrupting Cad74A, a cadherin gene regulated by BMP signaling that was found to be important for proper eggshell morphology. BMP signaling regulates cadherins in the pupal retina of D. melanogaster and in the human renal epithelial cells. It is proposed that late BMP signaling is involved in the morphological processes of DAs' formation by affecting cell adhesion molecules. Other cadherin genes are expressed or repressed in the DAs' forming cells, and it will be interesting to study how their regulation by BMP signaling affects DAs' morphogenesis (Niepielko, 2011).

Neurophysiological defects and neuronal gene deregulation in Drosophila mir-124 mutants

miR-124 is conserved in sequence and neuronal expression across the animal kingdom and is predicted to have hundreds of mRNA targets. Diverse defects in neural development and function were reported from miR-124 antisense studies in vertebrates, but a nematode knockout of mir-124 surprisingly lacked detectable phenotypes. To provide genetic insight from Drosophila, its single mir-124 locus was deleted, and it was found to be dispensable for gross aspects of neural specification and differentiation. In contrast, a variety of mutant phenotypes were detected that were rescuable by a mir-124 genomic transgene, including short lifespan, increased dendrite variation, impaired larval locomotion, and aberrant synaptic release at the NMJ. These phenotypes reflect extensive requirements of miR-124 even under optimal culture conditions. Comparison of the transcriptomes of cells from wild-type and mir-124 mutant animals, purified on the basis of mir-124 promoter activity, revealed broad upregulation of direct miR-124 targets. However, in contrast to the proposed mutual exclusion model for miR-124 function, its functional targets were relatively highly expressed in miR-124-expressing cells and were not enriched in genes annotated with epidermal expression. A notable aspect of the direct miR-124 network was coordinate targeting of five positive components in the retrograde BMP signaling pathway, whose activation in neurons increases synaptic release at the NMJ, similar to mir-124 mutants. Derepression of the direct miR-124 target network also had many secondary effects, including over-activity of other post-transcriptional repressors and a net incomplete transition from a neuroblast to a neuronal gene expression signature. Altogether, these studies demonstrate complex consequences of miR-124 loss on neural gene expression and neurophysiology (Sun, 2012).

microRNAs (miRNAs) are ~22 nucleotide (nt) regulatory RNAs that function primarily as post-transcriptional repressors. In animals, miRNAs have propensity to target mRNAs via 6-7 nt motifs complementary to their 5' ends, termed 'seed' regions. This limited pairing requirement has allowed most miRNAs to capture large target networks. Analysis of multigenome alignments indicates that typical human miRNAs have hundreds of conserved targets, and that a majority of protein-coding genes are under miRNA control. The extraordinary breadth of animal miRNA:target networks has been extensively validated by transcriptome and proteome studies (Sun, 2012).

miR-124 is strictly conserved in both primary sequence and spatial expression pattern, being restricted to the nervous system of diverse metazoans, including flies, nematodes Aplysia, and all vertebrates studied. Such conservation implies substantial functions of miR-124 in controlling neural gene expression. miR-124 has been a popular model for genomewide investigations of miRNA targeting principles. For example, studies of miR-124 yielded the first demonstration of the downregulation of hundreds of direct targets detected by transcriptome analysis, and that this activity was driven by the miRNA seed region. In addition, miR-124 provided one of the first illustrations of spatially anticorrelated expression of a miRNA and its targets and direct identification of Ago-bound target sites (Sun, 2012).

Functional studies have connected vertebrate miR-124 to various aspects of neural specification or differentiation. Studies in chick ascribed miR-124 as a proneural factor that inhibits the anti-neural phosphatase SCP1. However, no substantial effect of miR-124 on chick neurogenesis was found in a parallel study, although miR-124 was observed to repress neural progenitor genes such as laminin gamma1 and integrin beta1. In the embryonic mammalian brain, miR-124 was reported to direct neural differentiation by targeting polypyrimidine tract binding protein 1 (PTBP1), a global repressor of alternative splicing in non-neural cells. In the adult mammalian brain, miR-124 promoted neural differentiation of the immediate progenitors, the transit-amplifying cells (TAs). Here, miR-124 was shown to directly target the transcription factor Sox9, which maintains TAs and is downregulated during neural differentiation. Other mammalian studies bolster the concept that miR-124 promotes neurogenesis or neural differentiation. One mechanism involves direct repression by miR-124 of Baf53a, a neural progenitor-specific chromatin regulator that must be exchanged for a neural-specific homolog to consolidate neural fate. However, complicating the picture is the recent report that Xenopus miR-124 represses neurogenesis by directly targeting the proneural bHLH factor NeuroD1 (Sun, 2012 and references therein).

All vertebrate miR-124 loss-of-function studies have relied on antisense strategies and have yet to be validated by bona fide mutant alleles. However, as the three vertebrate mir-124 loci are co-expressed in the nervous system, analysis of the null situation will require a triple knockout. So far, a mir-124 knockout has only been described in C. elegans, which harbors a single copy of this gene. Like most other miRNA mutants in this species, the loss of miR-124 did not cause obvious developmental, physiological or behavioral phenotypes. Nevertheless, comparison of gene expression in mir-124-expressing cells from wildtype and mir-124 mutant animals revealed strong enrichment in miR-124 target sites amongst upregulated transcripts, revealing the impact of miR-124 on neuronal gene expression (Clark, 2010). The broad, but phenotypically-tolerated, misregulation of miR-124 targets in this species is potentially consistent with the 'fine-tuning' model for miRNA regulation (Sun, 2012).

This study analyzed a knockout of the sole mir-124 gene in Drosophila. Although this mutant is viable and exhibits grossly normal patterning, numerous phenotypes were documented, including short lifespan, increased variation in the number of dendritic branches of sensory neurons, decreased locomotion and aberrant synaptic release at CNS motoneuron synapses. All of these phenotypes were rescued by a single copy of a 19 kilobase (kb) genomic transgene encompassing the mir-124 locus. A transcriptional reporter of mir-124 was generated that recapitulated the CNS expression of endogenous pri-mir-124, and this was used to purify mir-124-expressing cells from stage-matched wild-type and mir-124-mutant embryos. Transcriptome analysis revealed strong enrichment of direct miR-124 targets amongst genes upregulated in mir-124-mutant cells. The miR-124 target network included coordinate repression of multiple components in the retrograde BMP signaling pathway, whose activity controls synaptic release. Loss of miR-124 further correlated with increased activity of other neural miRNAs and the neural translational regulator Pumilio, and had the net effect of impairing transition from the neuroblast to neuronal gene expression signature. Altogether, it was demonstrated that endogenous miR-124 has substantial impact on CNS gene expression, which underlie its requirement for organismal behavior and physiology (Sun, 2012).

These studies of Drosophila mir-124 demonstrate that its loss is compatible with grossly normal neural development and differentiation, despite broad changes in gene expression and global upregulation of direct miR-124 targets. Nevertheless, many clear defects are detected in these mutants, including short lifespan of adult males, defective larval locomotion, and aberrant synaptic transmission. The latter phenotype is perhaps reminiscent of reports that inhibition of Aplysia miR-124 similarly results in an increase in evoked EPSP amplitude. These phenotypes were confirmed phenotypes to be due to miR-124 loss, as shown by their rescue by a mir-124 genomic transgene. Importantly, these phenotypes were obvious even under optimal culture conditions, demonstrating palpable requirements for this miRNA in the intact animal. It remains to be seen if synaptic overactivity in the mir-124 mutant can be directly linked to the behavioral defects observed at the organismal level. The electrophysiological defects in mir-124 mutants phenocopy activation of BMP signaling at the synapse, and miR-124 directly targets multiple components of this pathway. Still, it remains possible that the many other gene expression changes in mir-124 mutant neurons contribute to its loss of function phenotype. The detailed in vivo transcriptome-wide analysis of endogenous miR-124 targets sets the stage for future studies of how individual targets might affect different settings of miR-124 function (Sun, 2012).

Only a handful of other miRNA mutants are lethal or exhibit overt morphological defects, suggesting that many miRNAs serve as robustness factors. For example, a Drosophila mir-7 mutant exhibits minor cell specification defects, but these are enhanced by heat shock. In addition, the introduction of many C. elegans 'benign' miRNA mutants into genetically sensitized backgrounds uncovers a high frequency of phenotypes. Interestingly, miR-124 is not required for normal dendrite formation per se, but its absence caused a broader distribution of dendrite numbers on ddaD and ddaE neurons, i.e. a 'robustness' defect. It is speculated that environmental or genetic stress may reveal additional requirements for miR-124 in development and differentiation of the nervous system (Sun, 2012).

In light of the broad roles ascribed to endogenous miR-124 in neurogenesis, neural differentiation, and neural physiology (Gao, 2010), all from antisense strategies, the extensive negative data from the current Drosophila mir-124 knockout are equally compelling. While the relevant neural subpopulation may not have been examined, these studies indicate that miR-124 is not required for gross aspects of neurogenesis and differentiation in the embryonic and larval nervous system. Similarly, C. elegans deleted for mir-124, which is expressed mostly in ciliated sensory neurons, do not reveal obvious defects in neural development (Clark, 2010). Given that these invertebrate orthologs of miR-124 are identical in sequence to their vertebrate counterparts, and are highly and specifically expressed in their respective nervous systems, there is not strong reason a priori to suspect that miR-124 should not have comparable requirements amongst different animals. The analysis of vertebrate mir-124 knockouts is therefore highly anticipated (Sun, 2012).

The Drosophila system has been critical for elucidating fundamental features of miRNA target recognition in animals, and for studying specific miRNA-target interactions that mediate phenotype. However, it has been little-used to analyze the effects of miRNA-mediated gene regulation in the animal at the transcriptome-wide level. Perhaps the clearest example is the broad upregulation of maternal transcripts in early embryos lacking the mir-309 cluster. However, most miRNAs are tissue or cell-specific, and while it is much simpler to profile transcripts from whole flies, the inclusion of irrelevant cells can mask the action of the miRNA. For example, only 4/200 transcripts upregulated in mir-8 mutant pupae appeared to be direct conserved targets (Sun, 2012).

By purifying cognate miRNA-expressing cells from wild-type and miRNA-mutant backgrounds, this study succeeded in assessing transcriptome-wide effects of genetic removal of miR-124 with precision. The data provide a new perspective on the utilization of 'anti-targeting' in Drosophila. Previously, miR-124 was selected as a particularly compelling case in which its Drosophila targets were depleted for in situ terms related to nervous system development, and enriched for terms related to epidermal development. Since these tissues derive from a common developmental progenitor, the neuroectoderm, this led to a model in which miR-124 may solidify the neural fate by widespread suppression of epidermal genes that should be absent from neurons. This bioinformatic correlation has not been confirmed using an independently-derived set of miRNA targets (Sun, 2012).

Nevertheless, two observations suggest that the feature of mutual exclusion in the Drosophila miR-124 network is of subtle consequence. First, derepressed target genes were not enriched for epidermally-expressed genes. This is consistent with the view that on the transcriptome-wide level, the exclusion of epidermal genes from miR-124-expressing cells is primarily enforced by transcriptional mechanisms. Second, miR-124 targets were preferentially amongst the higher-expressed transcripts in miR-124+ cells, even in wild-type. Moreover, as well-conserved targets were expressed at overall higher absolute levels than poorly-conserved targets in miR-124+ cells, it is concluded that a dominant feature of the miR-124 target network has selected for substantial co-expression of the miRNA and its targets, perhaps to fine-tune their levels. This viewpoint is consistent with analyses of miR-124 targets in human, indicating a unifying theme for this particular miRNA across animals (Sun, 2012).

Early manifestations of the miRNA world emerged from pervasive control of the C. elegans heterochronic pathway and the D. melanogaster Notch pathway by miRNAs, and a few similar situations have been documented, i.e. direct targeting throughout the branched amino acid catabolism pathway by miR-277 or repression of multiple components of fatty acid metabolism by miR-33. Nevertheless, it is rare for such dedicated target networks to be seen amongst the miRNA oeuvre. Amongst the broad network of miR-124 targets, coordinate targeting of multiple components of the retrograde BMP signaling pathway is striking, including all three receptors (Sax/Tkv/Wit), the downstream transcription factor (Mad) and its cofactor (Medea). It was recently shown that misexpression of activated Sax and Tkv receptors in motoneurons increases evoked excitatory junctional potentials without affecting spontaneous activity, very similar to that of mir-124 mutants. This study extends this finding by analysis of activated Tkv alone. Therefore, deregulation of BMP signaling may contribute to the electrophysiological defects observed in mir-124 mutants (Sun, 2012).

Still, a 'one size fits all' description of miR-124 activity is not appropriate, since a number of functional miR-124 targets were observed whose predominant activities are in epidermal or other non-neural derivatives. Thus, the large miR-124 network accommodates a range of target properties. Derepression of a sufficient number of such non-neural transcripts may contribute collectively to the incomplete capacity of mir-124 mutant cells to transition from a neuroblast to neuronal gene expression signature (Sun, 2012).

One may speculate that dysfunction of miRNAs, which have large networks of targets, may trigger global changes in other modes of gene regulation. For example, overexpression of individual miRNAs or siRNAs can de-repress endogenous regulation via non-cognate miRNAs, possibly reflecting a titration mechanism. In addition to a global effect on neuroblast-to-neural transition, it was observed that genes downregulated upon in vivo loss of miR-124 were enriched for seeds of K box miRNAs and miR-10-5p. This is potentially consistent with a model in which absence of this abundant miRNA frees up AGO1 complexes to accept other neural miRNAs, yielding their overactivity. Another plausible mechanism might be that miR-124 represses a transcriptional repressor of these other miRNAs (Sun, 2012).

Pumilio binding sites were strongly associated with downregulated transcripts in mir-124 mutants. Pumilio is well-characterized as a neural RNA binding protein and translational regulator, and affects synaptic function and dendrite morphogenesis, which was also observed to be miR-124-regulated settings. Predictions of conserved miRNA binding sites (e.g., TargetScan or mirSVR) did not identify miR-124 target sites in the annotated pumilio 3' UTR or CDS; however modENCODE data revealed that pumilio transcription extends ~2 kb downstream of its annotated 3' end. The regulatory potential of such long pumilio 3' UTR isoforms remains to be studied. Other possibilities are that miR-124 regulates a transcriptional regulator of pumilio, or that Pumilio activity is altered in mir-124 mutants. Future studies should address the cross-talk of post-transcriptional regulation in neurons mediated by miR-124, neuronal miRNAs and Pumilio (Sun, 2012).

Postsynaptic glutamate receptors regulate local BMP signaling at the Drosophila neuromuscular junction

Effective communication between pre- and post-synaptic compartments is required for proper synapse development and function. At the Drosophila neuromuscular junction (NMJ), a retrograde BMP signal functions to promote synapse growth, stability and homeostasis and coordinates the growth of synaptic structures. Retrograde BMP signaling triggers accumulation of the pathway effector pMad in motoneuron nuclei and at synaptic termini. Nuclear pMad, in conjunction with transcription factors, modulates the expression of target genes and instructs synaptic growth; a role for synaptic pMad remains to be determined. This study reports that pMad signals are selectively lost at NMJ synapses with reduced postsynaptic sensitivities. Despite this loss of synaptic pMad, nuclear pMad persisted in motoneuron nuclei, and expression of BMP target genes was unaffected, indicating a specific impairment in pMad production/maintenance at synaptic termini. During development, synaptic pMad accumulation followed the arrival and clustering of ionotropic glutamate receptors (iGluRs) at NMJ synapses. Synaptic pMad was lost at NMJ synapses developing at suboptimal levels of iGluRs and Neto, an auxiliary subunit required for functional iGluRs. Genetic manipulations of non-essential iGluR subunits revealed that synaptic pMad signals specifically correlate with the postsynaptic type-A glutamate receptors. Altering type-A receptor activities via protein kinase A (PKA) revealed that synaptic pMad depends on the activity and not the net levels of postsynaptic type-A receptors. Thus, synaptic pMad functions as a local sensor for NMJ synapse activity and has the potential to coordinate synaptic activity with a BMP retrograde signal required for synapse growth and homeostasis (Sulkowski, 2013).

Previous work has described Neto as the first nonchannel subunit required for the clustering of iGluRs and formation of functional synapses at the Drosophila NMJ. Neto and iGluR complexes associate in the striated muscle and depend on each other for targeting and clustering at postsynaptic specializations. This study shows that Neto/iGluR synaptic complexes induce accumulation of pMad at synaptic termini in an activity-dependent manner. The effect of Neto/iGluR clusters on BMP signaling is selective, and limited to synaptic pMad; nuclear accumulation of pMad appears largely independent of postsynaptic glutamate receptors. This study demonstrates that synaptic pMad mirrors the activity of postsynaptic type-A receptors. As such, synaptic pMad may function as an acute sensor for postsynaptic sensitivity. Local fluctuations in synaptic pMad may provide a versatile means to relay changes in synapse activity to presynaptic neurons and coordinate synapse activity status with synapse growth and homeostasis (Sulkowski, 2013).

Drosophila NMJs maintain their evoked potentials remarkably constant during development, from late embryo to the third instar larval stages. This coordination between motoneuron and muscle properties requires active trans-synaptic signaling, including a retrograde BMP signal, which promotes synaptic growth and confers synaptic homeostasis. Nuclear pMad accumulates in motoneurons during late embryogenesis. However, embryos mutant for BMP pathway components hatch into the larval stages, indicating that BMP signaling is not required for the initial assembly of NMJ synapses and instead modulates NMJ growth and development. This study demonstrates that synaptic accumulation of pMad follows GluRIIA arrival at nascent NMJs and depends on optimal levels of synaptic Neto and iGluRs. As type-A receptors have been associated with nascent synapses, and type-B receptors mark mature NMJs, accumulation of synaptic pMad appears to correlate with a growing phase at NMJ synapses. Furthermore, synaptic pMad correlates with the activity and not the net levels of postsynaptic type-A receptors. In fact, expression of a GluRIIA variant with a mutation in the putative ion conduction pore triggered reduction of synaptic pMad levels. Thus, synaptic pMad functions as a molecular sensor for synapse activity and may constitute an important element in synapse plasticity (Sulkowski, 2013).

The synaptic pMad pool has been localized primarily to the presynaptic compartment. However, a contribution for postsynaptic pMad to the pool of synaptic pMad is also possible. Postsynaptic pMad accumulates in response to glia-secreted Mav, which regulates gbb expression and indirectly modulates the Gbb-mediated retrograde signaling (Fuentes-Medel, 2012). RNAi experiments revealed that knockdown of mad in muscle induces a decrease in synaptic pMad, albeit much reduced in amplitude compared with knockdown of mad in motoneurons (Fuentes-Medel, 2012). Also, knockdown of wit in motoneurons, but not in muscle, and knockdown of put in muscle, but not in motoneurons, triggers reduction of synaptic pMad (Fuentes-Medel, 2012). Intriguingly, the synaptic pMad is practically abolished in GluRIIA and neto109 mutants and cannot be further reduced by additional decrease in Mad levels. Whereas loss of postsynaptic pMad could be due to a Mav-dependent feedback mechanism that controls Gbb secretion from the muscle, the absence of presynaptic pMad demonstrates a role for GluRIIA and Neto in modulation of BMP retrograde signaling (Sulkowski, 2013).

As BMP signals are generally short lived, synaptic pMad probably reflects accumulation of active BMP/receptor complexes at synaptic termini. Recent evidence suggests that BMP receptors traffic along the motoneuron axons, with Gbb/receptors complexes moving preferentially in a retrograde direction. By contrast, Mad does not appear to traffic. Thus, Mad is likely to be phosphorylated and maintained locally by a pool of active Gbb/BMP receptor complexes that remain at synaptic termini for the time postsynaptic type-A receptors are active (Sulkowski, 2013).

The activity of type-A glutamate receptors may control synaptic pMad accumulation (1) indirectly via activity-dependent changes that are relayed to both pre- and postsynaptic cells, or (2) directly by influencing the production and signaling of varied Gbb ligand forms or by localizing Gbb activities. For example, inhibition of postsynaptic receptor activity induces trans-synaptic modulation of presynaptic Ca2+ influx. Such Ca2+ influx changes may trigger events that induce a local change in synaptic pMad accumulation. One possibility is that changes in Ca2+ influx may recruit Importin-β11 at presynaptic termini, which in turn mediate synaptic pMad accumulation (Sulkowski, 2013).

At the Drosophila NMJ, Gbb is secreted in the synaptic cleft from both pre- and postsynaptic compartments. The secretion of Gbb is regulated at multiple levels, transcriptionally and post-translationally. Furthermore, the Gbb prodomain could be processed at several cleavage sites to generate Gbb ligands with varying activities. The longer, more active Gbb ligand retains a portion of the prodomain that could influence the formation of Gbb/BMP receptor complexes. Synaptic pMad may result from signaling by selective forms of Gbb. Or type-A receptors could modulate secretion and processing of Gbb in an activity-dependent manner. Understanding the function of different pools and active forms of Gbb within the synaptic cleft will help explain the multiple roles for Gbb at Drosophila NMJs (Sulkowski, 2013).

Alternatively, active postsynaptic type-A receptor complexes may directly engage and stabilize presynaptic Gbb/BMP receptor signaling complexes via trans-synaptic interactions. CUB domains can directly bind BMPs; thus Neto may utilize its extracellular CUB domains to engage Gbb and/or presynaptic BMP receptors. As synaptic pMad mirrors active type-A receptors, such trans-synaptic complexes will depend on Neto in complexes with active type-A receptors. No capture has yet been shown of a direct interaction between Gbb and Neto CUB domains in co-immunoprecipitation experiments. Nonetheless, a trans-synaptic complex that depends on the activity of type-A receptors could offer a versatile means for relaying synapse activity status to the presynaptic neuron via fast assembly and disassembly (Sulkowski, 2013).

Irrespective of the strategy that correlates synaptic pMad pool with the active type-A receptor/Neto complexes, further mechanisms must act to maintain the Gbb/BMP receptor complexes at synapses and protect them from endocytosis and retrograde transport. Such mechanisms must be specific, as general modulators of BMP receptors endocytosis impact both synaptic and nuclear pMad. A candidate for differential control of BMP/receptor complexes is Importin-β11. Loss of synaptic pMad in importin-β11 is rescued by neuronal expression of activated BMP receptors, by blocking retrograde transport, but not by neuronal expression of Mad. As Mad does not appear to traffic, presynaptic Importin-β11 must act upstream of the BMP receptors, perhaps to stabilize active Gbb/BMP receptor complexes at the neuron membrane. By contrast, local pMad cannot be restored at Neto-deprived NMJs by overactivation of presynaptic BMP receptors or by blocking retrograde transport. As neto and gbb interact genetically, it is tempting to speculate that postsynaptic Neto/type-A complexes localize Gbb activities and stabilize Gbb/BMP receptor complexes from the extracellular side. Additional extracellular factors, for example heparan proteoglycans, or intracellular modulators, such as Nemo kinase, may control the distribution of sticky Gbb molecules within the synaptic cleft and their binding to BMP receptors, or may stabilize Gbb/BMP receptor complexes at synaptic termini (Sulkowski, 2013).

Synaptic pMad may act locally and/or in coordination with the transcriptional control of BMP target genes to ensure proper growth and development of the synaptic structures. A presynaptic pool of pMad maintained by Importin-β11 neuronal activities ensures normal NMJ structure and function. Like importin-β11, GluRIIA and Neto-deprived synapses show a significantly reduced number of boutons. Intriguingly, the absence of GluRIIA induces up to 20% reduction in bouton numbers, whereas knockdown of GluRIIB does not appear to affect NMJ growth. Although the amplitude of the growth phenotypes observed in normal culturing conditions (25°C) was modest, this phenomenon may explain the requirement for GluRIIA reported for activity-dependent NMJ development (at 29°C). Furthermore, knockdown of Neto or any iGluR essential subunit affect synaptic pMad and NMJ growth in a dose-dependent manner. Not significant changes were found in nuclear pMad or expression of BMP target genes in GluRIIA or Neto-deprived animals, but the restoration of synaptic pMad by presynaptic constitutively active BMP receptors rescues the morphology and physiology of importin-β11 mutant NMJs. The smaller NMJs observed in the absence of local pMad may reflect a direct contribution of synaptic pMad to retrograde BMP signaling, a pathway that provides an instructive signal for NMJ growth. Thus, BMP signaling may integrate synapse activity status with the control of synapse growth (Sulkowski, 2013).

Synaptic pMad may also contribute to synapse stability. Mutants in BMP signaling pathway have an increased number of 'synaptic footprints': regions of the NMJ where the terminal nerve once resided and has retracted. It has been proposed that Gbb binding to its receptors activates the Williams Syndrome-associated Kinase LIMK1 to stabilize the NMJ. Synaptic pMad may further contribute to the stabilization of synapse contacts by engaging in interactions that anchor the Gbb/BMP receptor complexes at synaptic termini. During neural tube closure, local pSmad1/5/8 mediates stabilization of BMP signaling complexes at tight junction via binding to apical polarity complexes. Flies may utilize a similar anchor mechanism that relies on pMad-mediated interactions for stabilizing BMP signaling complexes and other components at synaptic junctions. Local active BMP signaling complexes are thought to function in this manner in the maintenance of stemness and in epithelial-to-mesenchymal transition (Sulkowski, 2013).

Separate from its role in synapse growth and stability, BMP signaling is required presynaptically to maintain the competence of motoneurons to express homeostatic plasticity. The requirements for BMP signaling components for the rapid induction of presynaptic response may include a role for synaptic pMad in relaying acute perturbations of postsynaptic receptor function to the presynaptic compartment. At the very least, attenuation of local pMad signals, when postsynaptic type-A receptors are lost or inactive, may release local Gbb/BMP receptor complexes and allow them to traffic to neuron soma and increase the BMP transcriptional response, promoting expression of presynaptic components and neurotransmitter release. In addition, synaptic pMad-dependent complexes may influence the composition and/or activity of postsynaptic glutamate receptors. Although future experiments will be needed to address the nature and function of local pMad-containing complexes, the current findings clearly demonstrate that synaptic pMad constitutes an exquisite monitor of synapse activity status, which has the potential to relay information about synapse activity to both pre- and postsynaptic compartments and contribute to synaptic plasticity. As BMP signaling plays a crucial role in synaptic growth and homeostasis at the Drosophila NMJ, the use of synaptic pMad as a sensor for synapse activity may enable the BMP signaling pathway to monitor synapse activity then function to adjust synaptic growth and stability during development and homeostasis (Sulkowski, 2013).

Disruption of axonal transport perturbs Bone Morphogenetic Protein (BMP) - signaling and contributes to synaptic abnormalities in rwo neurodegenerative diseases

Formation of new synapses or maintenance of existing synapses requires the delivery of synaptic components from the soma to the nerve termini via axonal transport. One pathway that is important in synapse formation, maintenance and function of the Drosophila neuromuscular junction (NMJ) is the bone morphogenetic protein (BMP)-signaling pathway. This study shows that perturbations in axonal transport directly disrupt BMP signaling, as measured by its downstream signal, phospho Mad (p-Mad). Components of the BMP pathway genetically interact with both kinesin-1 and dynein motor proteins. Thick vein (TKV) vesicle motility is also perturbed by reductions in kinesin-1 or dynein motors. Interestingly, dynein mutations severely disrupted p-Mad signaling while kinesin-1 mutants showed a mild reduction in p-Mad signal intensity. Similar to mutants in components of the BMP pathway, both kinesin-1 and dynein motor protein mutants also show synaptic morphological defects. Strikingly TKV motility and p-Mad signaling are disrupted in larvae expressing two human disease proteins; expansions of glutamine repeats (polyQ77) and human amyloid precursor protein (APP; see Drosophila Appl) with a familial Alzheimer's disease (AD) mutation (APPswe). Consistent with axonal transport defects, larvae expressing these disease proteins show accumulations of synaptic proteins along axons and synaptic abnormalities. Taken together these results suggest that similar to the NGF-TrkA signaling endosome, a BMP signaling endosome that directly interacts with molecular motors likely exists. Thus problems in axonal transport occurs early, perturbs BMP signaling, and likely contributes to the synaptic abnormalities observed in these two diseases (Kang, 2014 - Open access: 25127478).

Effects of Mutation or Deletion

Mothers against dpp is required for dpp function. Mad was identified in screens to identify genes that interact with dpp. Mad loss-of-function mutations interact with dpp alleles to enhance embryonic dorsal-ventral patterning defects, as well as adult appendage defects, suggesting a role for MAD in mediating some aspect of DPP function. In support of this, homozygous Mad mutant animals exhibit defects in midgut morphogenesis, imaginal disk development and embryonic dorsal-ventral patterning reminiscent of dpp mutant phenotypes (Sekelsky, 1995).

Salivary gland formation in the Drosophila embryo is linked to the expression of the homeotic gene Sex combs reduced (Scr). When Scr function is missing, salivary glands do not form, and when Scr is expressed everywhere, salivary glands form in new places. However, not every cell that expresses Scr is recruited to a salivary gland fate. Along the anterior-posterior axis, the posteriorly expressed proteins encoded by the teashirt (tsh) and Abdominal-B (Abd-B) genes block Scr activation of salivary gland genes. Along the dorsal-ventral axis, the secreted signaling molecule encoded by decapentaplegic prevents activation of salivary gland genes by Scr in dorsal regions of parasegment 2. Five downstream components in the Dpp signaling cascade required to block salivary gland gene activation have been identified: two known receptors (the type I receptor encoded by the thick veins gene and the type II receptor encoded by the punt gene); two of the four known Drosophila members of the Smad family of proteins which transduce signals from the receptors to the nucleus [Mothers against dpp (Mad) and Medea (Med)], and a large zinc-finger transcription factor encoded by the schnurri (shn) gene. The expression patterns of d-CrebA and Trachealess were examined in embryos missing zygotic function of schnurri. In embryos homozygous for shn, a dorsal expansion of salivary gland protein expression is observed. The presence of amnioserosa, an extreme dorsal cell type, suggests that embryos lacking zygotic shn function are not ventralized, as are embryos missing maternal and zygotic function of tkv, pt, Mad, or Med or missing zygotic function of dpp. These results reveal how anterior-posterior and dorsal-ventral patterning information is integrated at the level of organ-specific gene expression (Henderson, 1999).

To determine roles for Medea during larval development, clones mutant for Medea have been examined in the eye. Dpp has an important role in the initiation and progression of the morphogenetic furrow. Mad clones in the posterior of the eye result in the loss of eye structures, which are instead replaced by head cuticular structures. These clones showed the ectopic expression of wingless (wg), a gene that is normally repressed by dpp signaling and is required at the lateral margins of the eye disk to regulate the proper timing of furrow initiation and progression. Hence clones of Mad mutant cells were unable to transduce the Dpp signal and are unable to initiate the morphogenetic furrow. Clones of the strong Medea alleles, Med 1 and Med 26 were induced, and these clones gave very similar phenotypes to Mad clones, such as loss of eye tissue. Such clones are observed only at the margins of the eye, most commonly the posterior margin, where the furrow initiates. This indicates that Medea has overlapping functions with Mad in dpp signaling during furrow initiation. Clonal analysis with Medea has also revealed abnormalities in other tissues, in keeping with its involvement in dpp signaling. For example, partial duplications of the leg are observed, a phenotype reported in the dorsal regions of the leg for clones of the Dpp receptor, punt. These analyses strongly suggest a closely related function for Medea and dpp during imaginal disc development (Das, 1998).

Since Mad and Medea are separately mutable, it is expected that they function non-redundantly and cannot substitute for each other. Consistent with this model, ubiquitous expression of Mad (Ubi-Mad) cannot rescue Medea lethality. To further examine the relationship between these two Smads, a sensitized assay system was used. This assay utilizes the dominant maternal effect lethality of Mad and Medea with dpp. The extent of this lethality depends on the strength of the Mad or Medea allele and the dpp allele with which it is crossed. Crossing a strong, hypomorphic dpp allele, dpp hr27, to the strongest available alleles of Mad, (Mad 12) or Medea (Med 1), results in 100% lethality of both dpp classes among the progeny. As expected, Ubi-Mad can rescue the maternal effect lethality of Mad 12, and Ubi-Medea rescues that of Med 1. The effects of introducing Ubi-Medea or Ubi-Mad from Mad/+ or Medea/+ females, respectively, were examined. Interestingly, a Ubi-Medea transgene can reduce the maternal effect lethality of Mad 12 /+ females with dpp from 100% to 12%, while Ubi-Mad reduces that of Med 1/+ females to 68%. To assay whether this rescue is simply due to increased levels of dpp pathway components, a Ubi-tkv line was usedin the same assay system. While this line is able to rescue a tkv mutant, it cannot rescue the maternal effect lethality associated with Mad or Medea. The lower extent of Ubi-Mad rescue of Medea maternal effect lethality, may be due to the fact that Med 1 may be an antimorphic allele. Alternatively, this may be indicative of an important aspect of Smad function. While it is clear that Mad and Medea cannot substitute for each other, the genetic data argue that a reduction in one class of Smads can be at least partially compensated by augmenting the dosage of the other Smad class. This compensation may be a Smad-specific feature, as elevated levels of tkv do not yield the same results. The simplest explanation for these genetic observations is that increased levels of one class of Smads may enhance the ability of the other class to signal (Das, 1998).

Stem cells are thought to occupy special local environments, or niches, established by neighboring cells that give them the capability for self-renewal. Each ovariole in the Drosophila ovary contains two germline stem cells surrounded by a group of differentiated somatic cells that express hedgehog and wingless. The BMP2/4 homolog decapentaplegic (dpp) is specifically required to maintain female germline stem cells and promote their division. Overexpression of dpp blocks germline stem cell differentiation. Overexpressing dpp for 3 days after eclosion produces tumorous germaria. Large germline cells filling germarial regions 1 and 2a contain spectrosomes but showed no evidence of cyst formation. In regions 2b and 3, 16-cell cysts are observed that probably derive from differentiated cystoblasts, or cysts that had formed before the first heat shock. This phenotype is very similar to that of bag of marbles (bam) and benign gonial cell neoplasm (bgcn) mutants. These results suggest that ectopic Dpp inhibits cystoblast differentiation but does not block cyst formation and maturation (Xie, 1998).

Mutations in dpp or its receptor (saxophone) accelerate stem cell loss and retard stem cell division. Mutant germline stem cell clones were constructed to show that the dpp signal is directly received by germline stem cells. punt, thickveins, mad, Medea and Dad are all shown to be required cell-autonomously for germline stem cell maintenance; punt, tkv, mad, and Med are shown to be required cell-autonomously to stimulate germline stem cell division. During aging, the number and activity of stem cells is thought to be reduced. The level of dpp signaling is shown to control the life span and division rate of germline stem cells. Reduced dpp signaling causes premature stem cell loss. Perhaps more surprising is the observation that putative increases in signaling, caused by removal of Dad activity from stem cells, causes them to be maintained longer. This finding suggests that dpp signaling not only is necessary, but may sometimes be rate limiting for stem cell maintenance. This is the first example where stem cell life span has been extended in an intact organism. Thus, dpp signaling helps define a niche that controls germline stem cell proliferation (Xie, 1998).

Mad acts as a dominant enhancer of vrille phenotypes in wing. About 10% of Mad6+/+ vri2 flies show a wing phenotype. The L5 vein is shortened and sometimes the posterior cross vein is also shortened and extra vein material is observed along the L2 vein. The same phenotype is observed with vri1 whereas with the other alleles the effect is weaker. Since this phenotype in not observed in the Mad/+ and vri/+ controls, it is concluded that it is due to the association of both genes. In order to investigate a possible interaction between vri and dpp in wing, a dominant effect of vri was sought in a dpp- context. The dpphr4/dppd6 phenotype consists of a reduction of wing to about one half of the wild type size and no defects in eyes or legs. When one dose of vri is associated with this genotype in dpphr4 vri2/dppd6 + flies, a further reduction in wing size is observed with reduction of veins. Furthermore, eyes are smaller with a rough aspect and legs are truncated. The enhancement of dpp phenotypes by vri2 is always observed, although the strength of the enhancement is variable. The same phenotypes are observed with vri1 (George, 1997).

TGF-beta comprise a superfamily of secreted proteins with diverse functions in patterning and cell division control. TGF-beta signaling has been implicated in synapse assembly and plasticity in both vertebrate and invertebrate systems. wishful thinking, a Drosophila gene that encodes a protein related to BMP type II receptors, has been shown to be required for the normal function and development of the neuromuscular junction (NMJ). These findings suggest that a TGF-beta-related ligand activates a signaling cascade involving type I and II receptors and the Smad family of transcription factors to orchestrate the assembly of the NMJ. This study demonstrates that the TGF-beta type I receptor Saxophone and the downstream transcription factor Mothers against dpp (Mad) are essential for the normal structural and functional development of the Drosophila NMJ, a synapse that displays activity-dependent plasticity (Rawson, 2003).

A positive role for Short gastrulation in modulating BMP signaling during dorsoventral patterning in the Drosophila embryo

Positional information in the dorsoventral axis of the Drosophila embryo is encoded by a BMP activity gradient formed by synergistic signaling between the BMP family members Decapentaplegic and Screw. short gastrulation, which is functionally homologous to Xenopus Chordin, is expressed in the ventrolateral regions of the embryo and has been shown to act as a local antagonist of BMP signaling. Sog has a second function, which is to promote BMP signaling on the dorsal side of the embryo. A weak, homozygous-viable sog mutant is enhanced to lethality by reduction in the activities of the Smad family members Mad or Medea, and this lethality is caused by defects in the molecular specification and subsequent cellular differentiation of the dorsal-most cell type, the amnioserosa. While previous data had suggested that the negative function of Sog is directed against Scw, data are presented that suggest that the positive activity of Sog is directed towards Dpp. Chordin shares the same apparent ligand specificity as does Sog, preferentially inhibiting Scw but not Dpp activity. However, in Drosophila assays, Chordin does not have the same capacity to elevate BMP signaling as does Sog, identifying a functional difference in the otherwise well conserved process of dorsoventral pattern formation in arthropods and chordates (Decotto, 2001).

Morphogen gradients, once a purely theoretical concept, are now viewed as central players in the establishment of cell identity in a broad range of developmental processes. However, the exact biological mechanisms used to establish and maintain a morphogen gradient vary, depending on the biological context. In the Drosophila embryo, while Dpp can act in a dose-dependent fashion to specify different cell fates along the DV axis, in vivo its activity is modulated spatially by other components of the patterning system. In particular, Sog, a diffusible BMP-binding protein, has been shown to inhibit BMP signaling ventrally by preventing ligand access to the BMP receptors. A novel aspect of Sog’s function has been characterized in this study. Specifically, Sog functions cell non-autonomously to elevate BMP signaling on the dorsal side of the embryo. Thus, the interpretation of any experiment to elucidate the role of Sog in the control of dorsoventral patterning must take into account the two apparently opposing functions of the protein (Decotto, 2001).

Loss-of-function mutations in Mad or Medea have been identified as dominant enhancers of a weak homozygous-viable sog mutation, and the enhanced embryos have been shown to have defects in amnioserosa specification. Furthermore, synthetic lethality between weak homozygous-viable alleles of sog and zen has been demonstrated, indicating that both are required for maximal production of amnioserosa. Lastly, there was a dramatic decrease in the level of zen transcription in sogP129D embryos that were derived from Mad/+ females, compared to the level of zen transcription in either genotype alone. Taken together, these results unambiguously demonstrate that the positive action of Sog is exerted before gastrulation to attain the maximal expression of a direct BMP target gene (Decotto, 2001).

Deciphering synergistic and redundant roles of Hedgehog, Decapentaplegic and Delta that drive the wave of differentiation in Drosophila eye development

In Drosophila, a wave of differentiation progresses across the retinal field in response to signals from posterior cells. Hedgehog (Hh), Decapentaplegic (Dpp) and Notch (N) signaling all contribute. Clones of cells mutated for receptors and nuclear effectors of one, two or all three pathways were studied to define systematically the necessary and sufficient roles of each signal. Hh signaling alone is sufficient for progressive differentiation, acting through both the transcriptional activator Ci155 and the Ci75 repressor. In the absence of Ci, Dpp and Notch signaling together provide normal differentiation. Dpp alone suffices for some differentiation, but Notch is not sufficient alone and acts only to enhance the effect of Dpp. Notch acts in part through downregulation of Hairy; Hh signaling downregulates Hairy independently of Notch. One feature of this signaling network is to limit Dpp signaling spatially to a range coincident with Hh (Fu, 2003).

The development of cells mutant for all three transcription factors, Mad, Su(H), and ci is a helpful starting point, since they may reflect a 'ground state' of eye development that requires extracellular signals to differentiate. Mad Su(H) ci cells fail to express the atonal or senseless genes that initiate R8 differentiation, and, consequently, fail to support retinal differentiation. This shows that the absence of Ci75 is not sufficient for differentiation. Dpp alone can induce Ato [e.g., in Su(H) ci clones], but N and Dpp signaling together are required to activate Atonal with normal kinetics, as occurs in ci-mutant cells. N signaling alone (in tkv ci clones) is insufficient. In the presence of Ci, prompt differentiation requires Hh to downregulate Ci75, and differentiation is delayed in Smo clones that lack this input. The normal role of Hh is not just to remove Ci75 thus permitting Dpp and N to work, because Atonal is turned on normally in Mad Su(H) clones that do not respond to Dpp or N signals. Such differentiation depends exclusively on Hh yet progresses normally, except that a neurogenic phenotype reflects dependence of lateral inhibition on Su(H). Hh depends positively on ci to drive differentiation in Mad Su(H) cells and, therefore, requires Ci155. The positive role of ci can also be inferred from the delayed differentiation of Su(H) ci clones in comparison with Su(H) clones (Fu, 2003).

Hairy is downregulated redundantly by Hh and N signaling. Prolonged Hairy expression is not sufficient to block differentiation completely but it does antagonize it (e.g., in Su(H) ci clones). Downregulation of Hairy in response to Hh as well as N explains why both ci and Su(H) mutant clones can differentiate promptly, and why N enhances differentiation in response to Dpp but is not required for differentiation in response to Hh (Fu, 2003).

Comparison between Mad Su(H) ci cells and Su(H) ci cells shows that Dpp signaling is sufficient to initiate eye differentiation in its normal location in the absence of Hh or N signals, but such differentiation is delayed. The normal timing of differentiation is restored by combined Dpp and N signals (in ci clones). This is the basis for the ectopic differentiation on co-expression of Dpp and Dl ahead of the furrow (Fu, 2003).

Superficially, these results differ from previous ectopic expression studies that concluded that Dpp signaling alone was not sufficient to induce ectopic differentiation in all locations. This discrepancy is probably explained by the baseline repressor activity of Su(H) protein. Previous work shows that without N signaling, repressor activity of Su(H) protein retards differentiation. Dpp signaling is sufficient for differentiation in the experiments where the Su(H) gene has been deleted. In the presence of the Su(H) gene, Dpp may be most effective at locations where there is little Su(H) repressor activity, such as close to the morphogenetic furrow where N signaling is active (Fu, 2003).

Comparison between Mad Su(H) ci cells, which do not differentiate, and Mad ci or tkv ci cells, which differentiate slowly or not at all, shows that Notch signaling alone is insufficient for differentiation. Premature differentiation reported when N is activated ectopically ahead of the furrow must reflect endogenous Dpp signaling at such locations (Fu, 2003).

These experiments reveal an outline of the mechanisms of Hh, Dpp and N redundancy. First, the results show that Mad and Ci independently reinforce differentiation, presumably through the transcription of target genes because Mad is sufficient for differentiation in the absence of Ci, and vice versa. The results show unequivocally that the transcriptional activator Ci155 activates differentiation in addition to Ci75 antagonizing differentiation (Fu, 2003).

It was surprising to find that Dpp stabilizes Ci155 in the absence of Smo, which suggests Dpp input into Hh signal transduction. Although the requirement for smo-dependent input through fused makes it unlikely that Ci155 is functional in smo clones, Ci155 accumulation might be associated with reduced Ci75 levels. Ci75 is shown to repress differentiation in smo clones because smo ci clones differentiation normally. Ci155 stabilization cannot be due to an indirect effect of Dpp signaling on Hh, Ptc or Smo expression levels because the effect is detected in the absence of smo, and, therefore, reflects an effect on Hh signal transduction components downstream of Smo. One idea is that Dpp signaling (or Dpp-induced differentiation) may replace SCFSlimb processing of Ci (which cleaves Ci155 to Ci75) with Cullin3-mediated Ci degradation, just as normally occurs posterior to the morphogenetic furrow. In a smo clone, Ci155 would accumulate because Smo is required for Cullin3 to degrade Ci. However, the SCFSlimb-to-Cullin3 switch may not be the only effect of Dpp on Ci processing, because Tkv slightly enhances Ci155 accumulation even when smo is present (Fu, 2003).

Finally, downregulation of Hairy by N requires the Su(H) gene. N also overcomes baseline repressor activity of Su(H) protein to promote progression of differentiation. This role of N must be independent of Hairy (Fu, 2003).

Dl, Hh and Dpp are generally thought to signal over very different distances. How can signals of such different range substitute for one another to permit normal eye development? Dpp is transcribed in response to Hh signaling and is produced where Ci155 levels are highest. Dl is regulated by Hh indirectly through Ato and Ato-dependent Egfr activity in differentiating cells. Hh is expressed most posteriorly of the three, in differentiating photoreceptors (Fu, 2003).

Eye differentiation uses Hh to progress through cells unable to respond to Dpp (tkv, Mad) or N (Su(H)). The range of Hh diffusion depends in part on the shape of the morphogenetic furrow cells. The Dpp that drives differentiation through ci-mutant cells unable to respond to Hh must diffuse from outside the ci clones because Dpp synthesis is Hh dependent. Large ci clones develop normally so Dpp diffusion cannot be limiting (dpp-mutant clones offer no information about the range of Dpp because they express and differentiate in response to Hh). Instead, the rate of progression in response to Dpp is controlled by Dl. Dl signals over (at most) one or two cell diameters at the morphogenetic furrow (Fu, 2003).

The previous view of eye patterning was influenced by the morphogen function of Hh and Dpp in other discs. It was thought that domains of Ato and Hairy expression reflected increasing concentrations of Hh and Dpp. The data shows that, in the eye, the combination of signals is important. Differentiation is triggered where Dl and/or Hh synergize with Dpp, regardless of where the source of Dpp is. The additional requirements limit Dpp to initiating differentiation at the same locations that Hh does (Fu, 2003).

dpp acting through mad is probably a signal for stimulating germline stem cell clonal expansion

Stem cell niches are specific regulatory microenvironments formed by neighboring stromal cells. Owing to difficulties in identifying stem cells and their niches in many systems, mechanisms that control niche formation and stem cell recruitment remain elusive. In the Drosophila ovary, two or three germline stem cells (GSCs) have recently been shown to reside in a niche, in which terminal filaments (TFs) and cap cells are two major components. Signals from newly formed niches promote clonal expansion of GSCs during niche formation in the Drosophila ovary. After the formation of TFs and cap cells, anterior primordial germ cells (PGCs) adjacent to TFs/cap cells can develop into GSCs at the early pupal stage while the rest directly differentiate. The anterior PGCs are very mitotically active and exhibit two division patterns with respect to cap cells. One of these patterns generates two daughters that both contact cap cells and potentially become GSCs. Lineage tracing study confirms that one PGC can generate two or three GSCs to occupy a whole niche ('clonal expansion'). decapentaplegic is expressed in anterior somatic cells of the gonad, including TFs/cap cells. dpp overexpression promotes PGC proliferation and causes the accumulation of more PGCs in the gonad. A single PGC mutant for thick veins, encoding an essential Dpp receptor, loses the ability to clonally populate a niche. Therefore, Dpp is probably one of the mitotic signals that promote the clonal expansion of GSCs in a niche. This study also suggests that signals from newly formed niche cells are important for expanding stem cells and populating niches (Zhu, 2003).

This demonstrates how an adult GSC niche is populated with stem cells in the Drosophila ovary. Before niche formation, all PGCs proliferate as pre-stem cells and are undifferentiated. As niche formation starts, PGCs divide into two distinct subpopulations: anterior PGCs adjacent to cap cells start to acquire stem cell identity, and the remaining PGCs directly proceed to differentiation. GSCs in one niche can come from one PGC. Dpp is likely involved in stimulating clonal expansion of GSCs during niche formation. This study suggests that signals from newly formed niches are important for expanding GSCs and most likely for populating nascent adult GSC niches (Zhu, 2003).

How stem cell identity is established initially remains elusive even in the well-studied stem cell systems -- Drosophila ovary and testis. In the primitive female gonads before the pupal stage, PGCs appear to undergo symmetric division to generate germ cells with the identical pre-stem cell fate. Several studies suggest that GSCs are established at the early pupal stage. At the early pupal stage, there are 136 germ cells on average in each gonad. The adult ovary, which is composed of 12-16 ovarioles with two or three GSCs per ovariole (average of 2.5), contains about 30 to 40 GSCs. Therefore, at the most, 20%-30% of PGCs in the early pupal gonad are recruited to niches and turn into GSCs (Zhu, 2003).

How is a particular germ cell selected and recruited to niches, and how does it become a GSC? Positional information is known to be very important for cell-fate determination in various developmental processes. In this study, a developmental approach was taken to investigate when key niche components form, and how PGCs are subdivided into GSCs and differentiated germ cells. The expression of bam is associated with germ cell differentiation in the adult ovary. Using bam expression as an indicator for germ cell differentiation, it has been shown that no PGCs in late third instar larval gonads have differentiated. In early pupal gonads (about 0-4 hours after pupation), all the PGCs that are not in contact with TFs/cap cells are differentiated; therefore, the PGCs that contact newly formed cap cells remain undifferentiated and become GSCs. Possibly, newly formed TFs/cap cells directly prevent the most anterior PGCs from differentiation when an unknown developmental signal triggers PGC differentiation around the larval-pupal transition stage. This study demonstrates that the stem cell fate of PGCs is determined by their position, i.e. juxtaposition to TFs/cap cells (Zhu, 2003).

The next important question is how these anterior PGCs populate niches. In this study, it has been show that the PGCs in contact with newly formed cap cells at the early pupal stage divide more frequently than the rest of the PGCs. The division patterns are very interesting: one division pattern generates two daughters that are both in contact with cap cells; the other pattern generates only one daughter that is in contact with cap cells. As in the adult ovary, two daughters that are in contact with cap cells can both become GSCs. This is verified by the observation that one marked PGC in the gonad at the late third-instar larval stage can generate two or three GSCs in a niche. The results also indicate that the stem cells in a niche can come from multiple PGCs. Whether GSCs in a niche come from one or multiple PGCs probably depends on whether one or multiple PGCs directly contact cap cells within the developing niche. If only one PGC contacts cap cells, it probably has an opportunity to generate two or three germ cells that contact cap cells and become GSCs. This study shows that newly formed niches do not simply recruit existing PGCs and turn them into GSCs, but also stimulate PGCs to proliferate and produce more GSCs (Zhu, 2003).

The clonal expansion of GSCs in a niche clearly requires the newly established stem cell to divide rapidly and generate a daughter that occupies the same niche, which further prevents other neighboring precursor cells from entering it. Consistent with this prediction, it was observed that the anterior row of germ cells at the early pupal stage is more mitotically active than the rest of the germ cells based on the BrdU incorporation assay. dpp is known to be important for maintaining GSCs and stimulating their division in the adult ovary. dpp is expressed in TFs/cap cells and other anterior somatic cells, and PGCs close to cap cells are capable of responding to dpp. Furthermore, overexpressing dpp promotes PGC proliferation. To demonstrate the necessity of dpp signaling in stimulating GSC clonal expansion, it has been shown that a PGC mutant for tkv, an essential dpp receptor, fails to clonally populate a niche. All these results demonstrate that dpp is probably a signal for stimulating GSC clonal expansion (Zhu, 2003).

As in the adult ovary, hh is also expressed in terminal filaments and cap cells in developing female gonads. Hh has been shown to play a minor role in modulating GSC division. Wingless (Wg) protein is expressed in terminal filaments and cap cells. Its expression in developing female gonads has not been examined. Because wg, dpp and hh often work together to regulate many developmental processes in Drosophila, it is possible that hh and wg could also cooperate with dpp to regulate PGC proliferation and modulate GSC clonal expansion in niches (Zhu, 2003).

PGCs in the gonad do not show any signs of differentiation until the larval-to-pupal transition. At the early pupal stage, only the PGCs in the anterior row remain undifferentiated, but the rest have already differentiated. It seems that a developmental signal(s) starts to appear and then induces the differentiation of PGCs during the transition from larva to pupa. Such a developmental signal could be mediated by a steroid-like hormone ecdysone. Interestingly, during most of the third instar larval stage, the ecdysteroid levels are very low but begin to rise and peak just before pupation. The ecdysteroid peak could be potentially responsible for the initial differentiation of germ cells in the gonad of the larva ready for pupation. It is also possible that the hormone is not a direct signal but controls the production of the signal(s). Somehow, the signals from the anterior somatic cells antagonize the differentiating signals and thus prevent the anterior row of the PGCs from differentiation. One of the signals that prevent PGCs from differentiation could be encoded by dpp. Dpp is known to prevent GSCs from differentiation in the adult ovary. In this study, 2.5% of the marked tkv mutant PGCs and none of the marked mad mutant PGCs before the third instar larval stage were recruited to niches or were maintained as GSCs before adulthood. The failure of tkv and mad mutant GSCs to be maintained in niches could be explained by the role of dpp in preventing PGCs from differentiation. It could also be explained by other possibilities, such as defects in the formation of adherens junctions between cap cells and GSCs. Whether dpp is a signal for maintaining the undifferentiated state of PGCs during early ovarian development remains undetermined. Therefore, the signals that maintain the undifferentiated state of PGCs from TFs/cap cells remain to be identified (Zhu, 2003).

p38 mitogen-activated protein kinase can be involved in transforming growth factor beta superfamily signal transduction in Drosophila wing morphogenesis

Thick veins is likely to target p38b, a MAP kinase implicated in Dpp signal transduction. Two Drosophila homologs of p38, Mpk2 (also known as p38a or simply p38) and p38b, have been identified on the basis of their homology to mammalian p38 and to one another. p38b is maternally expressed and is present ubiquitously during embryonic development (Han, 1998). The chromosomal region around the p38b locus has been well characterized genetically. However, a p38b transgene was unable to rescue any of the known mutations mapping to this region. Likewise, attempts to isolate a mutant of p38b were unsuccessful. These failures are possibly due to the functional redundancy of the two p38 homologs. Various alternative methods were therefore use to interfere with endogenous p38(s) in order to investigate its function. A dominant-negative allele of p38b, designated D-p38bDN, was generated by replacing the Thr-183 of the MAPKK target site with Ala, analogous to the change in ERK2 that produces a dominant-negative allele (Adachi-Yamada, 1999).

Two lines were prepared which express D-p38bDN at different levels: D-p38bDN-S (Strong), which expresses high levels, and D-p38bDN-W (Weak), which expresses low levels. When two copies of the D-p38bDN-S transgene are expressed in the wing, a certain fraction of adult flies that escape death exhibit ectopic vein fragments around the end of the longitudinal vein L2 and a reduction in the distance between L4 and L5. Both of these features have also been observed with some mutant alleles of decapentaplegic and thick veins. This wing phenotype is rescued by coexpression of the wild type p38b+ transgene. When two copies of the D-p38bDN-S transgene are weakly expressed in the wing of a dpp mutant, the vein phenotype of dpp is strongly enhanced. These phenotypes suggest the involvement of Drosophila p38(s) in Dpp function in the early and late stages of wing pattern development. Dpp is known to play a dual role during wing development, acting as a morphogen and mitogen at early stages, while activating vein differentiation at later stages (Adachi-Yamada, 1999).

To examine whether p38(s) functions in the Dpp signaling pathway, the genetic interaction was examined between p38(s) and a constitutively active mutant of Tkv (TkvCA). Two classes of tkvCA insertions, tkvCA-S (Strong) and tkvCA-W (Weak), were used. When tkvCA-S is expressed, normal wing venation is severely distorted and extensive production of fragments of vein material is observed. The abdominal-cuticle pattern also appears irregular. This wing phenotype suggests that TkvCA may influence Dpp action during vein formation. Ectopic coexpression of dpp+ and tkv+ causes similar phenotypes, indicating that these TkvCA-induced aberrations are indeed the result of an increase in Dpp signaling. It was thus expected that reducing the levels of downstream components would suppress tkvCA. In fact, reducing by one-half the gene dosage of Mothers against dpp (Mad), a well-documented Dpp-signaling factor, significantly suppresses the tkvCA wing phenotype (Adachi-Yamada, 1999).

To investigate whether p38b is activated by Tkv signaling, a preliminary biochemical characterization of p38b was carried out. Immediately after heat treatment of flies, the amount of p38b immunoprecipitated by anti-p-Tyr antibody was found to increase considerably, demonstrating that p38b is tyrosine phosphorylated following heat shock, like mammalian p38. The site of tyrosine phosphorylation is expected to be in the 'activation loop' region recognized by MAPKK, as is the case in mammalian p38. Thus, a test was performed to see whether an anti-phospho-p38 (anti-p-p38) antibody raised against a phosphorylated peptide from the activation loop of mammalian p38 could cross-react with p38b. This anti-p-p38 antibody detects a protein with a calculated size of 42 kDa whose amount increases immediately after heat shock. This protein is also more abundant in the flies overproducing p38b regardless of heat treatment. Therefore, it has been concluded that anti-phospho-p38 can cross-react with the phosphorylated from of p38b and can be used to assay recombinant p38b phosphorylation in vitro. Treatment of p38b with recombinant human MKK6, a MAPKK that activates p38, causes a marked increase in the level of p38b, as detected with anti-phospho-Tyr and anti-p-p38 antibodies, and a drastic increase in the level of Drosophila p38-dependent phosphorylation of recombinant human activating transcription factor 2 (ATF2), a physiological substrate for mammalian p38. The correlation between the phosphorylation state and kinase activity of p38b indicates that the anti-p-p38 antibody recognizes the active form of p38b. This allowed activation of p38b by TkvCA to be examined in vivo. The amount of active p38b was found to be slightly but significantly higher in larvae carrying ectopically expressed tkvCA relative to that in wild-type Canton-S larvae. However, it has been reported that p38a protein expressed in yeast, which was presumed to have the same molecular mass as p38b, is also recognized by anti-p-p38 antibody. It is therefore possible that p38b, or both D-p38's, may be activated by Tkv signaling in vivo (Adachi-Yamada, 1999).

Mad and spermatogenesis

The continuous and steady supply of transient cell types such as skin, blood and gut depends crucially on the controlled proliferation of stem cells and their transit amplifying progeny. Although it is thought that signaling to and from support cells might play a key role in these processes, few signals that might mediate this interaction have been identified. During spermatogenesis in Drosophila, the asymmetric division of each germ line stem cell results in its self-renewal and the production of a committed progenitor that undergoes four mitotic divisions before differentiating while remaining in intimate contact with somatic support cells. TGF-ß signaling pathway components punt and schnurri have been shown to be required in the somatic support cells to restrict germ cell proliferation. This study showns, by contrast, that the maintenance and proliferation of germ line stem cells and their progeny depends upon their ability to transduce the activity of a somatically expressed TGF-ß ligand, the BMP5/8 ortholog Glass Bottom Boat. TGF-ß signaling represses the expression of the Bam protein, which is both necessary and sufficient for germ cell differentiation, thereby maintaining germ line stem cells and spermatogonia in their proliferative state (Shivdasani, 2003).

In order to test the requirement for TGF-β signaling in the germ line, the behavior of marked germ line clones lacking the activity of various TGF-β signaling pathway components was investigated. Germ line stem cells mutant for tkv or put (a type II TGF-β receptor) and spermatocytes lacking the activity of tkv, put, or mad (a transcription factor required for the regulation of TGF-β target genes) were generated but did not persist to the same extent as wild-type clones, as evidenced by assessing the ratio of the number of testes containing at least one germ line clone to the number of testes containing wild-type control clones. Sporadically (approximately 4% of cases), cysts containing eight, rather than 16, spermatocytes were observed, implying that the fourth spermatogonial division had not been complete. Such a scenario might have arisen due to the transient persistence of Tkv, Mad, or Put protein after the respective wild-type allele was lost. Together, these clonal analysis data suggest that TGF-β signaling is required for both germ line stem cell maintenance and spermatogonial proliferation. No requirement was found for schnurri (shn), the product of which is frequently required in Dpp signaling, in the germ line for these processes (Shivdasani, 2003).

DPP-mediated TGFβ signaling regulates juvenile hormone biosynthesis by activating the expression of juvenile hormone acid methyltransferase

Juvenile hormone (JH) biosynthesis in the corpus allatum (CA) is regulated by neuropeptides and neurotransmitters produced in the brain. However, little is known about how these neural signals induce changes in JH biosynthesis. This study reports a novel function of TGFβ signaling in transferring brain signals into transcriptional changes of JH acid methyltransferase (jhamt), a key regulatory enzyme of JH biosynthesis. A Drosophila genetic screen identified that Tkv and Mad are required for JH-mediated suppression of broad (br) expression in young larvae. Further investigation demonstrated that TGFβ signaling stimulates JH biosynthesis by upregulating jhamt expression. Moreover, dpp hypomorphic mutants also induces precocious br expression. The pupal lethality of these dpp mutants is partially rescued by an exogenous JH agonist. Finally, dpp is specifically expressed in the CA cells of ring glands, and its expression profile in the CA correlates with that of jhamt and matched JH levels in the hemolymph. Reduced dpp expression was detected in larvae mutant for Nmdar1, a CA-expressed glutamate receptor. Taken together, it is concluded that the neurotransmitter glutamate promotes dpp expression in the CA, which stimulates JH biosynthesis through Tkv and Mad by upregulating jhamt transcription at the early larval stages to prevent premature metamorphosis (Huang, 2011).

The functions of the TGFβ superfamily and other morphogens in regulating insect metamorphosis are rarely reported. In two independent genetic screens, it was discovered that Drosophila TGFβ signaling controls two different aspects of insect metamorphosis. In a previous study, it was found that Baboon (Babo) and dSmad2-mediated TGFβ signaling regulates larval neuron remodeling, which is part of the insect central nervous system metamorphosis induced by 20E during the pupal stage. Further investigation revealed that Babo/dSmad2-mediated TGFβ signaling controls larval neuron remodeling through regulating the expression of EcR-B1, a specific isoform of the 20E receptor (Huang, 2011).

This paper reports several findings. First, br is precociously expressed in 2nd instar tkv and Mad mutant larvae. Second, the precocious br expression phenotype in tkv and Mad mutant larvae can be suppressed by exogenous JH agonist (JHA). Third, Tkv and Mad repressed br expression in a non-cell-autonomous manner. Fourth, the presence of Mad in the CA is sufficient to repress br expression in the fat body (FB). Fifth, jhamt mRNA levels and JHAMT activity were significantly reduced in the Mad-deficient larvae. These results demonstrate that Tkv- and Mad-mediated signaling is required in the CA to activate jhamt expression and thus JH biosynthesis, which in turn controls insect metamorphosis (Huang, 2011).

The Drosophila genome encodes two TGFβ type II receptors, Punt (Put) and Wishful thinking (Wit). The genetic screen failed to identify a role for either of these receptors in the regulation of JH biosynthesis. Put and Wit are most probably functionally redundant in this biological event, as in the case of TGFβ-mediated mushroom body neuron remodeling (Huang, 2011).

Dpp is a key morphogen that controls dorsal/ventral polarity, segmental compartment determination and imaginal disc patterning. Dpp function usually depends on its gradient distribution. In an attempt to identify the ligand for Tkv/Mad-mediated TGFβ signaling in the CA, a novel, gradient-independent role for Dpp was discovered that controls JH biosynthesis. Dpp is the ligand of Tkv, which regulates jhamt transcription. Loss of Dpp, even RNAi reduction of Dpp in the CA specifically, causes precocious br expression at the early larval stages, which phenocopies tkv and Mad mutants. Phenotypes of dpp, including precocious br expression and lethality, are at least partially rescued by JHA treatment or ectopic jhamt expression in the CA. Notably, dpp-lacZ is strictly expressed in the CA cells, but not in the other two types of endocrine cells in the ring gland: the prothoracic gland and corpus cardiacum cells. The developmental expression profile of dpp in the CA is always consistent with that of jhamt. Finally, dpp expression in the CA may be directly controlled by neurotransmitter signals in the brain, which is supported by reduced dpp and jhamt transcription levels in the Nmdar1 mutant wandering larvae (Huang, 2011).

Several lines of evidence suggest that Met is a crucial regulator at or near the top of a JH signaling hierarchy, possibly acting as a JH receptor. However, null Met mutants of Drosophila are completely viable, which is unexpected if Met is a JH receptor. A recent investigation indicated that another Drosophila bHLH-PAS protein, Germ cell-expressed (Gce), which has more than 50% homology to Met, may function redundantly to Met in transducing JH signaling (Baumann, 2010). Because Met is on the X chromosome in the fly genome, it was not covered by the genetic screen. The br protein in the FBs of a Met null allele, Met27, was tested at the 2nd instar larval stage, and precocious br expression was observed. Importantly, this precocious br expression phenotype could not be suppressed by exogenous JHA. This result not only supports the previous reports regarding the function of Met in transducing JH signaling but also suggests that the precocious br expression is a more sensitive indicator for the reduced JH activity in Drosophila compared with precocious metamorphosis, lethality and other phenotypes (Huang, 2011).

Kr-h1 was reported to act downstream of Met in mediating JH action. Studies in both Drosophila and Tribolium reveal that, at the pupal stages, exogenous JHA induces Kr-h1 expression, which in turn upregulates br expression. The genetic screen successfully identified that Kr-h1 is cell-autonomously required for the suppression of br expression at young larval stages. Precocious br expression occurred in the FBs of Kr-h1 mutants and was not suppressed by JHA treatment. Therefore, these studies further suggest that Kr-h1 functions as a JH signaling component in mediating insect metamorphosis. However, the finding shows that, at the larval stages of Drosophila, the JH-induced Kr-h1 suppresses, rather than stimulates, br expression. This result is consistent with the facts that Kr-h1 functions to prevent Tribolium metamorphosis and Br is a crucial factor in promoting pupa formation (Huang, 2011).

In summary, this study has found a novel function of Dpp, Tkv and Mad-mediated TGFβ signaling in controlling insect metamorphosis. As summarized in a model, the brain sends neurotransmitters, such as glutamate, to the CA through neuronal axons. Glutamate interacts with its receptor (NMDAR) on the surface of CA cells to induce dpp expression. Dpp protein produced and secreted by CA cells forms a complex with TGFβ type I receptor (Tkv) and type II receptor on the membrane of CA cells, followed by phosphorylation and activation of Tkv. Activated Tkv in turn phosphorylates Mad, which is imported into the nucleus together with co-Smad and stimulates jhamt expression. JHAMT in CA cells transforms JH acid into JH, which is released into hemolymph. The presence of JH in young larvae prevents premature metamorphosis through Met/Gce and Kr-h1 by suppressing the expression of br, a crucial gene in initiating insect metamorphosis (Huang, 2011).

Mothers against dpp: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | References

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