Transforming growth factor beta at 60A

DEVELOPMENTAL BIOLOGY

Embryonic

The Tgfbeta-60A gene is expressed throughout development with peaks of transcription during early embryogenesis, in pupae, and in adult males, but is expressed in adult females only at low levels, if at all (Wharton, 1991).

The Tgfbeta-60A transcripts and protein are first detected at the onset of gastrulation, in ectodermal and mesodermal cells and are observed throughout the extending germ band. Staining is particularly pronounced in mesodermal cells and in cells of the stomadeal and posterior midgut invaginations. The Tgfbeta-60a polypeptide is first detected after stage 7, somewhat later than the stage at which the mRNA is first detected. By germ band extension, the protein is readily detected in the extended germ band and, similar to mRNA localization, is most readly detected in cells of the mesoderm and of the stomodeal invagination and the posterior midgut. Neither mRNA nor polypeptide are observed prior to gastrulation, indicating that there is little maternal contribution. As the germ band retracts, and throughout later stages of embryonic development, the Tgfbeta-60A transcript and protein are most readily detected in cells of the developing foregut and hindgut. In particular, enhanced expression is observed in the endodermal cells of the anterior and posterior midgut of stage 12 embryos. At the same stage, mRNA and protein are also detected in cells of the visceral mesoderm and the gastric caecae. Following dorsal closure, mRNA becomes more difficult to detect, consistent with a decrease in transcript levels during late embryogenesis. During later stages of embryogenesis, the protein is readily detected in cells of the foregut and hindgut and in the anterior and posterior midgut (Doctor, 1992).

Gbb expression in the neurohemal organ regulates CNS expression of FMRFa expression by retrograde signaling through a Bmp receptor

Amidated neuropeptides of the FMRFamide class regulate numerous physiological processes including synaptic efficacy at the Drosophila neuromuscular junction (NMJ). Mutations in wishful thinking (wit), a gene encoding a Drosophila Bmp type 2 receptor that is required for proper neurotransmitter release at the neuromuscular junction, also eliminates expression of FMRFa in that subset of neuroendocrine cells (Tv neurons) which provide the systemic supply of FMRFa peptides. Gbb, a Bmp ligand expressed in the segmentally repeated neurohemal organ associated with the ventral cord, provides a retrograde signal that helps specify the peptidergic phenotype of the Tv neurons. Supplying FMRFa in neurosecretory cells partially rescues the wit lethal phenotype without rescuing the primary morphological or electrophysiological defects of wit mutants. It is proposed that Wit and Gbb globally regulate NMJ function by controlling both the growth and transmitter release properties of the synapse as well as the expression of systemic modulators of NMJ synaptic activity (Marqués, 2003).

wishful thinking is primarily expressed in, and required for, proper nervous system function. Mutations in wit result in pharate lethality caused, in part, by defects in the growth and physiology of motoneuron synapses. Mutations in wit also affect the peptidergic phenotype of certain FMRFa-expressing cells found in the ventral cord. In particular, FMRFa expression is eliminated in the Tv neurons that contribute to the systemic supply of FMRFa peptides through release at the neurohemal organ. The regulation of FMRFa expression in Tv neurons is mediated by the Bmp ligand Gbb, since gbb null mutations also eliminate FMRFa expression in Tv neurons. Furthermore, supplying Gbb to the dorsal neurohemal cells restores FMRFa expression in Tv neurons. Since Tv neuron axons arborize onto the neurohemal cells, this strongly suggests that Gbb signals in a retrograde manner to specify the peptidergic phenotype of Tv neurons. Consistent with this view, overexpression in neuroendocrine cells of Dynamitin or a dominant-negative form of p150/Glued, both components of the Dynactin/Dynein motor complex was found to eliminate FMRFa expression in the Tv neurons. Finally, it is shown that providing FMRFa in neuroendocrine cells using the Gal4/UAS system partially rescues the lethal phenotype of wit mutants, even though they still exhibit structural and physiological synaptic defects. It is suggested that Bmp signaling provides a global cue that not only regulates the growth of the NMJ synapses locally but also controls their systemic modulation by the neuroendocrine system (Marqués, 2003).

The Drosophila genome contains seven TGFß type ligands. Three of these, Dpp, Screw and Gbb, have been shown to transduce Bmp-type signals (Mad) and to use the type I receptors Tkv and Sax. Two others, Activin and Activin-like protein, transduce signals through Smad2. The signaling pathways used by Maverick and Myoglianin remain untested. Among the three Bmp-type ligands, Gbb seemed a likely candidate for controlling expression of FMRFa, since it is broadly expressed, at least in embryos, and can signal through Wit to regulate P-Mad accumulation in motoneurons and tissue culture cells. gbb is strongly expressed in the larval brain lobes and much more weakly in the ventral ganglia. Interestingly, gbb shows enriched expression in the NHO relative to other ventral ganglia neurons. Thus, Gbb is expressed in the correct place to be a FMRFa regulating ligand (Marqués, 2003).

In Drosophila, FMRFamide peptides have been shown to enhance synaptic transmission and muscle twitch tension when perfused onto standard larval nerve-muscle preparations; however, their in vivo role(s) are not known as no mutations in the FMRFa gene have been identified. As with most neuropeptides, FMRFamide related peptides are thought to act as neuromodulators and neurohormones. The Tv-produced FMRFamide related peptides are released into the hemolymph through the neurohemal organ and hence are able to act on every tissue in the animal that is not blocked to hemalymph contact. It has been hypothesized that the lethality of wit mutants is due to the lack of proper synaptic transmission at the NMJ, resulting in the animals not being able to eclose from the pupal case. The lack of systemic FMRFamide described in this study would be expected to further decrease synaptic efficiency and the ability of wit mutants to eclose. The fact that loss of FMRFa does contribute to the lethal phenotype is supported by the partial rescue of wit mutants by overexpression of FMRFa. These results are consistent with the view that in vivo, FMRFa peptides probably enhance NMJ synaptic activity similar to their in vitro documented effects on standard larval electrophysiological preparations (Marqués, 2003).

It is important to note that although the lethal phenotype is partially reversed, the morphological and physiological synaptic defects reported for wit mutants are not rescued by overexpression of FMRFa. The simplest interpretation is that the excess of FMRFamide related peptides enhances the efficiency of wit mutant synapses in vivo without correcting the underlying developmental defects. Although one might expect a significant improvement of the electrophysiological phenotype, this is not detected, probably because the excess FMRFamide related peptides are either washed off the preparation during standard dissection prior to recording or act for only short periods (Marqués, 2003).

How Wit signaling regulates FMRFa expression is not clear. Since Smads are well known to act as transcriptional co-activators or co-repressors, the simplest explanation is that Mad directly regulates activation of FMRFa transcription, perhaps by forming a complex with Ap. However, other indirect mechanisms are also possible and this issue will only be resolved once the FMRFa promoter is fully characterized. It is also not clear whether Gbb is the only ligand that regulates FMRFa expression through Wit. In some developmental contexts, such as wing imaginal disc patterning, Gbb acts in combination with Dpp, another Bmp-type ligand. No expression of dpp has been detected in the NHO. However, it could be that one of the as yet uncharacterized ligands, Maverick or Myoglianin, could be a partner with Gbb in regulating FMRFa expression. Conversely, it seems clear that regulating the peptidergic phenotype of the six Tv neurons is not the only role of Gbb signaling. There are hundreds of neurons that receive Bmp signaling as indicated by P-Mad nuclear localization Most of them appear to be motoneurons, which require Wit/Gbb signaling to achieve proper synaptic growth but not to specify their neurotransmitter phenotype. Given that Smads act as co-transcriptional regulators, the fact that the same signal (nuclear translocation of P-Mad) results in different phenotypic outcomes in different neurons can probably be ascribed to the presence of a different set of transcription factors available in each cell type. The Tv neurons receiving the Bmp signal express apterous, a transcription factor required in those cells for FMRFa transcription, and maybe other factors that are required, in addition to the Wit signal, to activate FMRFa (Marqués, 2003).

Another important issue to resolve is whether Gbb is constitutively released from the NHO, or is synthesized and released as part of a feedback mechanism to modulate muscle contractions. It might be that efficient muscle contraction under normal conditions requires a constant level of FMRFamide related peptides that are produced in response to a constitutive Gbb signal. Alternatively, Gbb production or release might be regulated by a sensing mechanism that would activate the pathway in response to an increased demand for FMRFamide related peptides, owing to increased locomotor activity or other stimuli, such as compensating for a synaptic developmental defect. Muscle-derived Gbb acts through neuronal Wit to convey a retrograde signal essential for NMJ synapse growth and maturation. In that context, it appears that the role of Bmp signaling is to coordinate muscle growth with synapse maturation to ensure proper synaptic efficiency. Thus, the Wit/Gbb pathway acts as a two-step regulator of NMJ function. First, there is a developmental role in which Wit signaling is required for proper synaptic growth during larval development. Second, Wit signaling is required to achieve the neuromodulatory effect of circulating FMRFamide related peptides that are required for optimal synaptic transmission. Lack of either one of these inputs probably results in a substantial decrease of the EJCs. These two examples suggest that the Gbb/Wit pathway is of general importance in neural retrograde signaling and it is speculated that it may be used in the nervous system for other as yet uncharacterized developmental and physiological purposes (Marqués, 2003).

Tv neurosecretory cells form part of a cluster of four apterous-expressing neurons on each side of the three thoracic ganglia. The axons of the Tv neurons extend proximally and dorsally to join the contralateral axon, and form a median nerve that swells and arborizes onto a group of neurons and glial cells that constitute the neurohemal organ. In wit mutants, these structures develop normally, but the Tv neuron fail to activate FMRFa transcription. Using the Gal4/UAS system Wit's requirement for FMRFa expression was narrowed down to the Tv neurons. Since these neurons accumulate nuclear P-Mad, the results strongly suggest that Wit is required in the Tv neurons themselves, as opposed to forming part of an indirect signal relay mechanism. It appears likely that the source of Gbb in this signaling system is the NHO, since gbb is expressed in the NHO and replenishing Gbb in the NHO of gbb mutants rescues FMRFa expression in the Tv neurons. These experiments do not exclude the possibility that signaling might occur at the cell soma of the Tv neurons in vivo or that the source of the diffusible ligand could be a different tissue under physiological conditions. However, the dependence of nuclear P-Mad accumulation and FMRFa expression in Tv neurons on Dynein-mediated retrograde transport strongly suggests that signaling is taking place at the Tv axon terminal. This dependency on Dynein motors is not a general requirement for FMRFa expression in all neurons because subesophageal ganglion neurons are not affected by overexpression of dominant-negative Glued or Dynamitin. Nor is the consequence of disrupting this motor likely to exert its effect at the level of P-Mad translocation to the nucleus, since nuclear accumulation of P-Mad in epithelial and mesodermal cells is not effected by retrograde transport disruption. Only in the nervous system is P-Mad accumulation specifically affected, consistent with a role for a retrograde transport mechanism in moving some component of this signaling pathway from the synapse to the nucleus (Marqués, 2003).

Regulation of stem cell maintenance and transit amplifying cell proliferation by Gbb signaling in Drosophila 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).

Spermatogenesis in adult Drosophila commences in the germinal proliferation center at the apical tip of the testis. To identify factors that can influence the regulation of cell proliferation in spermatogenesis, a number of signal transduction pathway components were screened using the GAL4/UAS targeted overexpression system. Various lines carrying transgenes under the transcriptional control of the UAS enhancer were crossed to lines carrying nanos-GAL4:VP16 (nos-GAL4), in which the GAL4 transcriptional activator is expressed only in the germ line. The testes of the progeny of such crosses were screened for morphological abnormalities (Shivdasani, 2003).

Overexpression of decapentaplegic results in testes containing large, opaque, spherical structures and large numbers of small cells resembling germ line stem cells (GSCs) and spermatogonia, but no spermatocytes or mature spermatids. Wild-type GSCs and gonialblasts contain a spectrin-rich organelle known as the spectrosome, which is spherical in shape, while spermatogonia and spermatocytes contain spectrin-rich structures known as fusomes, which are linear and branched in appearance. UAS-dpp/+; nos-GAL4/+ (UAS-dpp) testes contain similar numbers of spectrosome-containing cells to wild-type testes but many more fusome-containing cells, as shown by immunostaining using antibodies to α-spectrin, suggesting that the ectopic cells resemble spermatogonia rather than GSCs or gonialblasts (Shivdasani, 2003).

To investigate the behavior of germ cells in UAS-dpp testes, cell proliferation and cell death were examined. Immunostaining with anti-phosphorylated histone 3 (PH3) antibodies revealed cysts of germ cells undergoing synchronous mitotic division, another characteristic of spermatogonia, but exceeding the usual four rounds of mitosis. Staining with acridine orange revealed significantly more cell death in all the UAS-dpp testes examined. Taken together, these data suggest that in UAS-dpp testes, spermatogonia fail to cease mitotic division after four rounds but continue to divide synchronously and ultimately die (Shivdasani, 2003).

Similar phenotypes were observed in testes in which an activated form of the type I TGF-β receptor Thickveins was overexpressed in the germ line (UAS-tkv*), suggesting that the observed germ cell overproliferation phenotype in UAS-dpp testes seems to be due to a direct effect of dpp on germ cells rather than an indirect effect via the soma. Since there is no increase in the number of GSCs in either UAS-dpp or UAS-tkv* testes, it follows that high-level TGF-β signaling in the germ line is sufficient to induce spermatogonial overproliferation but is not sufficient to specify GSC identity (Shivdasani, 2003).

To investigate whether TGF-β signaling is required for germ cell proliferation, the inhibitory SMAD daughters against dpp (dad) (which has been shown to antagonize TGF-β signaling) was overexpressed in the germ line using nos-GAL4. Testes of such animals raised at 25°C exhibit a range of phenotypes, perhaps reflecting the strength of the UAS-dad transgenic line. In 27% of cases (43/161), testes resembled those of wild-type animals. In 21% of cases (34/161), testes appeared smaller and thinner than those of wild-type animals, with a visible reduction in the number of germ cells. In the remaining 52% of cases (84/161), testes had degenerated and completely lacked GSCs, spermatogonia, and spermatocytes, as indicated by the absence of Vasa protein, which is normally expressed in all germ cells. By contrast, overexpression of brinker (brk), a transcriptional repressor of many Dpp target genes, had no effect on testes (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).

To establish whether Dpp function is required for male germ cell proliferation, the testes was analyzed of animals transheterozygous for a temperature-sensitive combination of dpp alleles -- dpp^hr27/hr56 that had been raised at 18°C and shifted to the restrictive temperature of 29°C upon eclosion. After 7 days, the testes of such animals did not exhibit any overt morphological abnormalities and contained germ cells in all stages and in quantities indistinguishable from controls. Similar experiments were undertaken with a transheterozygous combination of temperature-sensitive alleles of punt. Adult males of the genotype put^135/10460 were moved to the restrictive temperature of 29°C after eclosion. After 7 days, their testes were smaller and thinner than controls and exhibited an apparent reduction in the number of early germ cells, particularly spermatogonia and GSCs. These data indicate that while TGF-β signaling appears essential for germ cell proliferation, dpp is unlikely to play a major role in this process. Consistent with this notion, no dpp mRNA expression was detected in the testis by in situ hybridization. In situ hybridization was conducted to investigate the expression of two closely related homologs of dpp: screw (scw) and glass bottom boat (gbb). Whereas no expression of scw mRNA was detected in testes, gbb transcript was detected in the area corresponding to the germinal proliferation center, specifically in the somatic cyst cells (Shivdasani, 2003).

Given that gbb is expressed in the region where GSC and spermatogonial proliferation take place, whether loss of function of gbb has any effect on germ cell proliferation was tested. Examination of the testes of gbb^1/4 adult males raised at 18°C revealed them to be significantly smaller than wild-type, with a dramatic reduction in the number of germ cells of all stages, particularly GSCs, spermatogonia, and spermatocytes. Immunostaining with antibodies to Vasa protein revealed that in the most extreme cases, testes from gbb mutant animals lacked GSCs, spermatogonia, and spermatocytes altogether. Similar phenotypes were observed in gbb^4/4 males. These phenotypes could not be rescued by ectopically expressing gbb in a gbb^4/4 mutant background using nos GAL4, thus confirming that these phenotypes are indeed due to a reduction in wild-type gbb function (Shivdasani, 2003).

Cyst cells outside the germinal proliferation center do not express gbb, implying that the cessation of proliferation of spermatogonia may be directly linked to the loss of gbb activity. The effects of overexpressing gbb were investigated using nos-GAL4 or the hub and early cyst cell-specific driver patched-GAL4. Surprisingly, none of these drivers yielded a phenotype resembling that produced when dpp or tkv* is overexpressed (Shivdasani, 2003).

This could be because Gbb is a less potent ligand than Dpp or that Gbb is posttranscriptionally regulated. Consistent with these notions are previous observations regarding the relative potency of the two ligands in orchestrating growth and patterning in imaginal discs and the RNA and protein expression patterns of Gbb in wing discs. Alternatively, it is possible that Gbb acts in tandem with another ligand as a heterodimer. In this case, loss of function of Gbb would be sufficient to produce a loss-of-function phenotype, whereas overexpression of Gbb alone may not be sufficient to achieve a gain of function effect (Shivdasani, 2003).

In an interesting parallel, a mouse ortholog of Gbb, BMP8b, has been shown to be required for the survival and proliferation of primordial germ cells in males. However, these studies reported that BMP8b is expressed in the germ line rather than the soma, and it was not established whether BMP8b acts on the germ line in an autocrine manner or indirectly via the soma (Shivdasani, 2003).

The mechanisms by which Gbb might regulate germ cell proliferation were explored next. One candidate that might interact with the pathway is the bags of marbles (bam) gene since, as with the activation of high-level TGF-β signaling in the male germ line, the loss of bam function is sufficient to induce the overproliferation of spermatogonia-like cells, but not GSCs (Shivdasani, 2003).

It has been reported that cytoplasmic Bam (Bam-C) is expressed in 2- to 16-cell spermatogonia, but not in GSCs, gonialblasts, or spermatocytes. Bam-C levels appear to be highest in late-stage spermatogonia, those farthest away from the apical hub, which are about to cease mitosis and differentiate into spermatocytes. Overexpression of bam using a heat-shock-bam transgene is sufficient to eliminate germ line stem cells in the ovary, but not in the testis. However, since it is not possible to achieve sustained, high-level, targeted overexpression with a heat-shock transgene, this analysis does not exclude a similar activity for Bam in the germ line of both sexes. By driving sustained, high-level overexpression of bam in GSCs and spermatogonia using nos-GAL4, it was found that testes of such animals resemble UAS-dad testes, being dramatically reduced in size, lacking early germ cells, and containing only mature spermatids. These expression and phenotypic data suggest that bam might be required for the differentiation of spermatogonia into spermatocytes. Loss of bam function might forbid differentiation, thereby maintaining spermatogonia in a proliferative state (Shivdasani, 2003).

The similarity between the TGF-β gain of function and bam loss of function phenotypes suggests that TGF-β signaling might act to repress bam activity. Testes were examined in which clones of cells expressing tkv* had been generated; such clones did not express Bam-C even though they overproliferated. It is therefore possible that TGF-β signaling might promote germ cell proliferation by repressing the activity of Bam, thus preventing premature differentiation of GSCs and amplifying spermatogonia, thereby maintaining them in a proliferative state. This possibility was tested by generating germ line clones that lacked both Bam activity and the ability to transduce the TGF-β signal. Such germ cells, doubly mutant for bam and put, behave as bam mutant clones and overproliferate as small cells resembling spermatogonia (Shivdasani, 2003).

It is proposed that Gbb acts as a short-range signal, emanating from cyst cells, signaling only to the GSCs and spermatogonia they enclose, thereby repressing Bam activity and maintaining germ cells in a proliferative state. Such short-range signaling by Gbb is consistent with the independent proliferation and differentiation of individual cysts. As each cyst ages, diminishing Gbb levels result in less TGF-β signal transduction in the spermatogonia, which in turn results in increasing Bam levels. Bam levels might constitute a counting mechanism such that once Bam levels reach a certain threshold, spermatogonia exit the cell cycle and commence differentiation into spermatocytes. This would be consistent with spermatogonia undergoing exactly four mitotic divisions. Bam activity thus forges an intimate link between proliferation and differentiation such that the former can only proceed if the latter is suppressed (Shivdasani, 2003).

By contrast with the ovary, in which TGF-β signaling appears to be both necessary and sufficient for GSC maintenance, the pathway appears necessary in the testis for GSC maintenance but is not sufficient to specify GSC fate. This is presumably because male GSCs are required to transduce the JAK/STAT signaling pathway in order to self-renew. Since only GSCs adjacent to the hub are thought to transduce this pathway, it is only these cells that are capable of retaining stem cell identity (Shivdasani, 2003).

It has been reported that loss of function of put and shn in somatic cyst cells results in spermatogonial overproliferation. Taken together with the data presented in this study, these observations suggest the intriguing possibility of two opposing roles for TGF-β signaling in the regulation of germ cell proliferation: a requirement in the cyst cells to restrict spermatogonial proliferation and a requirement in the germ line to maintain GSCs and promote the proliferation of spermatogonia. It is noted, however, that the previous study did not find a requirement for either the type I TGF-β receptors Tkv or Sax or the transcription factor Mad in the cyst cells, nor was the requisite TGF-β ligand for this process identified (Shivdasani, 2003 and references therein).

The results of this and other studies highlight the significance of bilateral communication between the germ line and soma, and the importance of somatic support and guidance of the germ line during gametogenesis. The emergence of specific roles for well-known developmental signaling pathways in gametogenesis is enabling the attainment of a comprehensive understanding of the inductive interactions required to guide germ cells through the stereotypical sequence of events from germ line stem cell to gamete and is affording insights into how fundamental biological processes such as asymmetric cell division and controlled mitotic division are regulated (Shivdasani, 2003).

Thus spermatogenesis in Drosophila might constitute a relatively simple model system in which to investigate such processes and may unveil general paradigms that may be applicable to more complex systems in which the relationship between stem cells, proliferating progeny, and their neighboring support cells is less well understood (Shivdasani, 2003).

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).

During Drosophila larval growth, muscle surface area increases dramatically, on the order of 100-fold. To maintain constant synaptic output, nerve terminals adjust both bouton number and active zones per bouton to compensate for the increase in muscle size during development. Evidence is provided that Gbb, a BMP-type ligand, is produced in muscles and signals in part by a retrograde mechanism through BMP receptors located in presynaptic nerve terminals to regulate synaptic growth and function at the Drosophila neuromuscular junction (McCabe, 2003).

The partial rescue of P-Mad accumulation, bouton number, and neurotransmission when Gbb is expressed in muscles is consistent with the notion that Gbb can provide a retrograde signal from the synapse to the neuron cell body. Although the data indicate that a component of the BMP signal is provided by a retrograde mechanism, an additional signal also seems to be required in either the motoneuron cell body or the CNS. Gbb is expressed in the CNS in addition to muscle. Resupplying Gbb in the muscles of gbb mutants does significantly restore P-Mad accumulation in motoneurons; however, rescue either ubiquitously or in both the CNS and muscle is significantly better than muscle rescue alone. Furthermore, complete rescue of the defects of neurotransmitter release in gbb mutants can be achieved by restoration of Gbb in all neurons, but only partially by restoration in muscles or in motoneurons alone. This contrasts with the requirement of Gbb for neuromuscular junction growth, which appears to primarily require muscle expression. It is possible that this is a quantitative issue; however, staining with a Gbb antibody suggests that both the muscle and elav drivers provide much higher levels of Gbb protein than is present under endogenous conditions. Thus, full rescue may require BMP signaling in both the CNS and at the synapse (McCabe, 2003).

The retrograde requirement for Gbb in synapse structural growth is further supported by experiments inhibiting dynein motor function by overexpression of dominant-negative Glued protein (ΔGl). RNAi-mediated depletion of Arp-1/centractin as well as overexpression of dominant P150/Glued reveal a requirement for dynactin to stabilize NMJ synapses (Eaton, 2002). The degree of net synapse growth appears to be determined by a balance of synapse expansion and retraction (Eaton, 2002). Expression of a dominant-negative Glued protein in the presynaptic cell reduces synaptic bouton number and produces synaptic ultrastructure defects, which are remarkably similar to those described for mutations in the BMP signaling pathway. These ultrastructure defects include membrane detachments along active zones and increased numbers of large vesicles in the presynaptic nerve (Eaton, 2002). Two possible models have been put forth to explain the requirement for dynactin at nerve termini: either it affects local properties, perhaps by altering microtubule stability and dynamics, or it interferes with a retrograde signal. These models are not mutually exclusive and, as described in this study, presynaptic expression of ΔGlu interferes with accumulation of P-Mad in motoneurons. This led to the conclusion that at least some portion of the ΔGl overexpression phenotype is attributable to interference with the retrograde BMP signal. It is suggested that perhaps ΔGl-induced retraction defects result from local disruptions in microtubule stability as suggested by (Eaton, 2002, while other phenotypes, such as reduced bouton number and active zone defects, are the result of disruption in BMP signaling. When synaptic function was examined in animals overexpressing dominant P150/Glued, quantal content was found to be reduced by 40%; however, mEJP amplitude and frequency was unaffected (Eaton, 2002). This contrasts with the findings for mutants of gbb where an 85% reduction in neurotransmitter release is found but also a 3-fold decrease in mEJP frequency. This is consistent with a disconnect between the retrograde requirement for Gbb in synapse structural growth and the requirement for Gbb in neurotransmitter release (McCabe, 2003).

It is interesting to note that bilateral NMJ signaling is not without precedent. Recently, Wingless (Wg) has been shown to be essential for both pre- and postsynaptic differentiation. In this case, Wg is made in the presynaptic cell, but its dual pre- and post-synaptic requirement indicates that either it signals in both an autocrine and juxtacrine manner or that the postsynaptic cell sends back a second signal that is responsible for presynaptic differentiation. Likewise, Gbb may have both a pre- and postsynaptic role. It is unlikely that Wg is regulating the Gbb signal, since accumulation of P-Mad is still seen in motoneurons of wg mutants. It remains to be determined if Gbb might influence the Wg signal (McCabe, 2003).

Gbb may also have a general role as a retrograde signaling ligand in the CNS, since Gbb is also required for specifying the FMRFa peptidergic phenotype of Tv neurons. Tv neurons innervate a specialized secretory structure known as the neurohemal organ (NHO) that is responsible for systemic release of FMRFa peptides. In this case, the Gbb signal originates in the NHO and controls the FMRFa peptidergic phenotype of Tv neurons. As is found in this study, expression of dominant-negative Glued also blocks the Gbb signal, providing evidence that this signal may also be retrograde. Also, an unknown retrograde signal that controls the homeostasis of neurotransmitter release at the NMJ is modulated by postsynaptic CaMKII (Haghighi, 2003). This homeostatic retrograde signal requires presynaptic Wit, implicating BMP signaling in this form of plasticity, though it remains to be determined if Gbb is involved (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).

In support of a possible endosome model, recent studies of TGF-β signaling in Mv1Lu and Cos-7 cells indicate that Smad phosphorylation and subsequent release from receptors does not occur efficiently until dynamin has excised a budded vesicle, presumably containing the activated receptor complex, from the plasma membrane. Efficient signaling also requires Smad Anchor for Receptor Activation (SARA), an endosomally localized protein. In Drosophila, there is conflicting data concerning the requirement for receptor internalization for signal propagation. Clones of wing disc cells mutant for the Drosophila α adaptin gene are still able to express the Dpp target gene spalt, suggesting that endocytosis prior to vesicle formation is not required for signal propagation. However, expression of a dominant-negative version of Rab5 to block formation of endocytic vesicles in wing discs has recently been shown to at least partially interfere with Dpp signaling. Since clonal analysis requires that preexisting protein be depleted before phenotypic consequences are manifested, while the dominant-negative methodology does not, it may be that perdurance of α adaptin protein in clones obscures the involvement of endocytosis for Dpp signaling. Thus, it is possible that like TGF-β, BMP-type signals may also emanate from a signaling endosome. Consistent with this view is the observation that overexpression of the SARA FYVE endosomal localization domain has been shown to disrupt BMP-induced transcriptional responses in HeLa cells (McCabe, 2003 and references therein).

Of particular relevance to the involvement of endosomes in mediating BMP signaling at synapses is the recent observation that mutations in the spinster (spin) gene greatly enhance synaptic growth. Spin is a putative multipass transmembrane protein localized to the late endosomes/lysosomes. Mutations in spin disrupt the morphology of late endosomes and lead to a 2-fold overgrowth in bouton number at the NMJ. This overgrowth can be suppressed by mutations in wit, suggesting that elimination of Spin leads to enhanced BMP signaling. Although the molecular mechanism responsible for the increased signal is unclear, the combined data support the idea that BMP signaling is likely modulated by intracellular trafficking of various signaling components at the level of the endosome. If these endosomes are capable of binding to dynein motors, then, like the Trk containing endosomes, they may also be transported along microtubules back to the cell body. Other models, such as wave activation of receptors, may also be possible, although if this is the case, then it is not clear why overexpression of ΔGl should interfere with BMP signaling as described above (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).

A final issue to consider is what regulates Gbb delivery at the synapse. Two alternatives seem most probable. Either it constitutively bathes the synapse, or it is released in response to a presynaptic stimulus. A third possibility, assuming that more than one BMP ligand is activating the pathway, is that one of the ligands would be constitutively released, acting as a background synapse growth stimulus, and the second ligand would be released in response to developmental or physiological stimuli, such as muscle growth or increased synaptic activity (McCabe, 2003).

Retrograde signaling plays an important role in synaptic homeostasis, growth, and plasticity. A retrograde signal at the neuromuscular junction (NMJ) of Drosophila controls the homeostasis of neurotransmitter release. This retrograde signal is regulated by the postsynaptic activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII). Reducing CaMKII activity in muscles enhances the signal and increases neurotransmitter release, while constitutive activation of CaMKII in muscles inhibits the signal and decreases neurotransmitter release. Postsynaptic inhibition of CaMKII increases the number of presynaptic, vesicle-associated T bars at the active zones. Consistently, it is shown that glutamate receptor mutants also have a higher number of T bars; this increase is suppressed by postsynaptic activation of CaMKII. Furthermore, presynaptic BMP receptor Wishful thinking is required for the retrograde signal to function. These results indicate that CaMKII plays a key role in the retrograde control of homeostasis of synaptic transmission at the NMJ of Drosophila (Haghighi, 2003).

It has been demonstrated that a BMP type II receptor, wishful thinking (wit), is required for both growth and function of the NMJ in Drosophila. To further explore the mechanism by which motor neurons respond to the retrograde signal, whether the retrograde enhancement of quantal content can occur in wit mutants was examined. The results indicate that the retrograde signal cannot increase neurotransmitter release in the absence of Wit. Activation of the retrograde signal by either postsynaptic expression of GluRIIA^M/R or postsynaptic inhibition of CaMKII did not lead to any increase in quantal content. These results indicate a requirement for wit presynaptically for the functioning of the retrograde mechanism that controls the homeostasis of neurotransmitter release at the NMJ of Drosophila and that postsynaptic inhibition of CaMKII requires the function of presynaptic BMP signaling to enhance quantal release (Haghighi, 2003).

Glass bottom boat (Gbb), a BMP ortholog, functions as a retrograde ligand for Wit at the Drosophila NMJ. Mutations in gbb lead to NMJ defects similar to those observed in wit mutants, and postsynaptic transgenic expression of Gbb can rescue many of these defects. In light of these findings, it is possible that there is a link between postsynaptic activity of CaMKII and the level and function of Gbb at the NMJ of Drosophila (Haghighi, 2003).

Effects of Mutation or Deletion (part 1/2)

A screen was carried out for dominant enhancer mutations magnifying the effects of a hypomorphic allele of thick veins (tkv), a type I receptor for dpp. tkv 6 is a mutation in a splice acceptor site that results in aberrant in-frame splicing, deleting two extracellular amino acids of the receptor. When expressed in COS1 cells, the mutant receptor fails to bind BMP-2 homodimers. However, tkv 6 behaves genetically as a hypomorph. In contrast to the embryonic lethal tkv null alleles, tkv 6 is homozygous viable: the only visible phenotype is the thickened wing veins. All other imaginal-disc-derived structures of tkv 6 homozygotes appear normal. Interestingly, tkv 6/Df(2L)tkv2 flies are phenotypically identical to tkv 6 homozygotes. To test if tkv 6 is a suitable genetic background for a modifier screen, the effects of lowering the activity of other known dpp pathway components were examined. Heterozygous mutations in shn or punt enhance the tkv 6 homozygous phenotype. In the tkv 6 background, shn IB is a dominant enhancer of the venation pattern in the wing and the proximal/distal patterning of the leg. In the wing, longitudinal vein 2 fails to reach the wing margin. In the leg, distal elements such as claws and distal tarsal segments are deleted. Such phenotypes are reminiscent of hypomorphic dpp phenotypes. punt 135 also enhances the tkv 6 phenotypes. Based on these observations, it was reasoned that the dpp signaling output through the mutant receptor tkv 6 is near the threshold for proper patterning of the imaginal discs. The tkv 6 mutation is therefore an appropriate genetic background for identifying new components essential for mediating dpp signaling. Enhancers of tkv 6 are phenotypically similar to dpp mutants. The enhancers are recessive lethal in a wild-type background. tkv 6 homozygotes that are heterozygous for the enhancer mutations have defects in imaginal disc development. During pupal development, the dorsal proximal region of the two wing imaginal discs fuse to form the adult notum. A heterozygous mutation, mapping to Tgfbeta-60A, causes deletions of distal and dorsal structures and occasional duplication of ventrolateral structures such as sex combs on male prothoracic legs. These phenotypes are indistinguishable from those of dpp disk alleles, suggesting that this enhancer acts in the dpp signal transduction pathway (Chen, 1998).

Meiotic mapping and complementation tests have established seven complementation groups for the enhancers. New alleles of tkv, Mad, Medea and punt have been recovered. Of the five Mad alleles, three have point mutations in the coding region. Missense mutations were found in both new punt alleles. The tkv D17 and Med D5 allelism is based on genetic non-complementation. A tkv transgene rescues the lethality of tkv D17 homozygotes, supporting the view that D17 is a tkv allele. Genetic and molecular characterizations of the D4 complementation group reveal that it corresponds to the Tgfbeta-60A gene. Tgfbeta-60A encodes a BMP-7 homolog, isolated on the basis of its sequence homology. Its function has been unknown due to the lack of mutations in Tgfbeta-60A. Three alleles of Tgfbeta-60A were confirmed by sequencing the mutant alleles. 60A D8 and 60A D20 are nonsense mutations in the prodomain due to single nucleotide substitutions. 60A D4 has one nucleotide deletion, causing a frame-shift premature stop in the prodomain (Chen, 1998).

Previous studies have established Dppís role as a morphogen in patterning the embryonic ectoderm. dpp signaling is also required for dorsal closure of the embryonic ectoderm. How, if at all, does the level of Tgfbeta-60A affect the phenotype of the embryonic ectoderm? The cuticle phenotypes of single and double mutants were compared. Since tkv 6 homozygotes are viable and Tgfbeta-60A mutants show no obvious defects until late in development, the cuticular patterns of these mutants are essentially normal. However, tkv 6 Tgfbeta-60A homozygote embryos die and exhibit head defects and an excessive ventral curvature. Although the double mutant cuticles bear some resemblance to hypomorphic dpp mutants, they do not exhibit an obvious expansion of the ventral denticle belts. However, the double mutant phenotype suggests that when dpp signaling is compromised in the embryonic ectoderm, removing Tgfbeta-60A activity further attenuates dpp signaling. The relatively mild phenotype of the double mutant embryo might reflect partial rescue by the maternal contribution of wild-type Tkv receptors. Indeed, a quarter of the embryos produced by mothers homozygous for tkv 6 and heterozygous for Tgfbeta-60A exhibit a dorsal open phenotype similar to that of zygotic tkv null embryos. Therefore, in the absence of maternally provided wild-type Tkv, tkv 6 Tgfbeta-60A double mutant embryos exhibit a phenotype indicative of defective dpp signaling during the process of dorsal closure (Chen, 1998).

These results provide the first in vivo evidence for the involvement of Tgfbeta-60A in the dpp pathway. It is proposed that Tgfbeta-60A activity is required to maintain the optimal signaling capacity of the dpp pathway, possibly by forming biologically active heterodimers with Dpp proteins (Chen, 1998).

Reported assays of the bone morphogenetic proteins (BMPs) have not, in general, revealed specific functions for the different proteins, belying the specificity implied by the evolutionary conservation and distinct expression patterns of the genes encoding BMPs. Assays of developmental function have been used to show that the two Drosophila homologs of the BMPs, Decapentaplegic (Dpp) and Tgfbeta-60A, which both induce ectopic bone formation in mammalian assay systems, have distinct effects in Drosophila development. A binary expression system using the yeast transcriptional activator GAL4 directed identical patterns of tissue and temporally specific dpp and Tgfbeta-60A expression. When dpp enhancer elements drive GAL4 expression, GAL4-responsive dpp transgenes rescue dpp mutant phenotypes, but GAL4-responsive Tgfbeta-60A transgenes do not. Ectopic ectodermal expression of dpp during gastrulation respecifies the dorsal/ventral pattern of the embryo. In contrast, ectopic Tgfbeta-60A expression had no detectable effect on embryonic development but leads to defects in adult structures or lethality during metamorphosis. Expression of Tgfbeta-60A in cells expressing dpp does not interfere with dpp functions, indicating that dysfunctional heterodimers do not form at sufficient levels to inhibit Dpp. These specific developmental responses in Drosophila indicate that in vivo functions of BMP-like factors can be more specific than indicated by the ectopic bone formation assays and that the Drosophila embryo provides an assay system sensitive to the structural differences that contribute to BMP specificity in vivo (Staehling-Hampton, 1994).

TGF-beta/BMP superfamily members, Gbb-60A and Dpp, cooperate to provide pattern information and establish cell identity in the Drosophila wing

Multiple BMPs are required for growth and patterning of the Drosophila wing. The Drosophila BMP gene, Tgfbeta-60A, exhibits a requirement in wing morphogenesis distinct from that shown previously for dpp. Tgfbeta-60A mutants exhibit a loss of pattern elements in the wing, particularly those derived from cells in the posterior compartment, consistent with the Tgfbeta-60A mRNA and protein expression pattern. Individuals homozygous for null alleles of the Tgfbeta-60A gene, exhibit embryonic defects in gut morphogenesis and result in early larval lethality. Allelic combinations between a hypomorphic 60A allele and a null allele or a deficiency deleting the 60A locus (hypomorph/deletion) result in later larval lethality with only rare adult escapers (<1%). Third instar larvae of such a genotype appear transparent and smaller than wild-type larvae. These larvae develop more slowly than wild type and never attain wild-type size. The transparency appears to be due to a defect in both the quantity and quality of the fat body, as well as a dramatic reduction in imaginal tissues. In addition to a reduction in imaginal disc tissue, other tissues that normally proliferate during larval development, such as areas of the brain, are also reduced. The 60A locus has been named glass bottom boat (gbb) in light of the remarkable transparency of the mutant larvae, but here it will be referred to as Tgfbeta-60A. The rare adult escapers of the hypomorph/deletion exhibit small misshapen wings, which lack the posterior cross vein (PCV), much of longitudinal vein L5, distal portions of L4 and the posterior half of the anterior cross vein (ACV). The mutant wings are small, narrow and pointed, with a loss of intervein material, especially between veins L2/L3, L4/L5 and posterior to L5. In addition to the change in wing shape and the loss of veins, a thinning of vein L2 and some ectopic vein material in the intervein region flanking L2 is common. No abnormalities were found along the margin in the position or type of wing margin bristles, however, ectopic margin bristles were often seen along a vein or in intervein tissue in the distal regions of the wing. Defects in these mutant flies are not limited to the wings: a reduction in the size of the eye, with the presence of supernumerary vibrissae, an increase in the number of thoracic bristles, the presence of misshapen leg segments and female sterility have all been observed. The mutant wing phenotype of homozygous hypomorph adult is similar to, but less severe than, that observed for the rarer hypomorph/deletion adults. In homozygote hypomorphs, the overall shape of the wing is broader and less pointed, more like wild type. The ACV is often complete and longitudinal veins L4 and L5 are longer (Khalsa, 1998).

Given that homozygote hypomorphs retain some Tgfbeta-60A function, the affect of complete loss of Tgfbeta-60A function on wing patterning and morphogenesis was studied through the production of null clones. Tgfbeta-60A null clones are never recovered in the adult wing if induced during the early period of cell proliferation (before 36 hours AEL); however, clones in all parts of the wing were recovered from later inductions. Clones located in both the anterior and posterior compartments, as well as clones limited to the posterior compartment, exhibit defects in the same pattern elements affected in hypomorphic wings: loss of the PCV, and portions of L5 and L4. Clones strictly limited to the anterior compartment or along the A/P boundary exhibited no wing defects with the exception of very small clones that were located near the ACV. These clones resulted in ectopic vein material anterior to L3. In several wings vein loss was observed in wild-type tissue at a distance from the Tgfbeta-60A-patch. In each case that this non-autonomous effect was found, multiple clones were present in the wing, one in the posterior compartment and another along the A/P boundary, sites that otherwise did not produce abnormalities. This type of vein loss was never observed in the generation of control clones (Khalsa, 1998).

The expression of Tgfbeta-60A in imaginal discs was examined by both whole-mount RNA in situ hybridizations and antibody staining using a Tgfbeta-60A antibody. Tgfbeta-60A mRNA is expressed in the wing disc mainly in the posterior compartment in the pteropleural and medial regions extending into the progenitors of the scutellum. High levels are found within the wing pouch in cells belonging to both the posterior and anterior compartments, with higher levels in the posterior. A small amount of expression is found within the hinge region. In the eye/antennal disc, Tgfbeta-60A mRNA is highest anterior to the morphogenetic furrow and in the medial regions, with lower levels of expression posterior to the morphogenetic furrow. Tgfbeta-60A is expressed throughout the posterior compartment of the leg imaginal discs and within the ventral anterior compartment. Tgfbeta-60A protein is generally detected at locations coincident with Tgfbeta-60A mRNA with one major exception: the presence of a stripe of markedly lower Tgfbeta-60A protein running through the middle of the wing pouch. This stripe of reduced expression is coincident with the prominent stripe of dpp RNA expression in the cells anterior to the anterior/posterior (A/P) boundary of the wing imaginal disc. A weak but consistent reduction in Tgfbeta-60A expression is observed along the dorsal/ventral boundary from which the cells of the wing margin are derived. High levels of Tgfbeta-60A expression exists in a pattern complementary to the localized expression of dpp in the other imaginal discs as well. For example, Tgfbeta-60A expression is absent or at very low levels in the morphogenetic furrow of the eye disc, the site of dpp expression. In addition, the regions of low or absent Tgfbeta-60A expression in the antennal and leg imaginal discs are the sites of dpp expression (Khalsa, 1998).

The structures in the wing that are affected most dramatically by mutations in Tgfbeta-60A, the PCV and L5, are those that are least sensitive to the reduction or absence of dpp. The ACV and longitudinal veins L2 and L4, lying on either side of the A/P boundary, have been shown to be most sensitive to the loss of Dpp signaling, consistent with the proposal that Dpp organizes wing pattern via a morphogen gradient emanating from the A/P boundary. (It has been proposed that longitudinal vein L3 is fated by a different mechanism and as a result is not sensitive to the level of Dpp signaling). Since mutations in the Tgfbeta-60A locus indicate that Tgfbeta-60A is essential for establishing cell identity in the wing, especially within the posterior compartment, the possibility was investigated that Tgfbeta-60A and Dpp signal together to provide positional information for the entire wing. No dominant genetic interactions are observed between alleles of Tgfbeta-60A and dpp, therefore, recombinant chromosomes were constructed using alleles of dpp that result in an overall lowering of dpp expression in the imaginal discs. Individuals heterozygous for such hypomorphic dpp alleles are phenotypically wild type. As homozygotes or in various heteroallelic combinations, these dpp alleles can be lethal or can generate adults with appendage defects ranging from minor loss of vein material to truncations of the entire appendage. An individual heterozygous for a hypomorphic dpp allele and homozygous or transheterozygous for Tgfbeta-60A alleles exhibits wing phenotypes qualitatively different from those observed in the Tgfbeta-60A mutant alone. Tgfbeta-60A mutants heterozygous for a dpp hypomorphic allele exhibit a significant loss of L4 (>60% lack more than half of L4) and a more frequent loss of the ACV. Tgfbeta-60A mutants heterozygous for a more severe dpp allele, not only show a greater loss of L4, ACV and L2 vein material (>90% lack more than half of L4 and 100% lack the entire ACV including a thinning of L2), but also a loss of intervein tissue, especially between L2/L3 and L4/L5, as exhibited by a reduction in the overall size of the wing. In addition to defects in wing patterning, abnormalities were noted in the proximal/distal organization of the legs, a process in which dpp is known to play a central role. The common defects exhibited by the Tgfbeta-60A mutants combined with dpp hypomorphic alleles are truncations and/or apparent fusions of the distal most tarsal segments of the male prothoracic leg. These data indicate that heteroallelic combinations of Tgfbeta-60A and dpp result in phenotypes more pronounced and distinct from phenotypes observed as a result of mutations in either gene alone. These new phenotypes are not simply additive. Individual pattern elements are affected differently as a result of these heteroallelic combinations, for example, a greater loss of L4 is seen while the loss of L5 is reduced. These results suggest that the requirement for both dpp and Tgfbeta-60A in earlier stages of larval development, presumably at times of high cell proliferation, is not met in such complex genotypes. Yet the suppression of the ACV defect indicates that Tgfbeta-60A does not solely act to modulate levels of Dpp signaling; rather, as the ratio of Tgfbeta-60A to dpp changes across the wing imaginal disc, the specification of a pattern element is affected. Different relative levels of Tgfbeta-60A to Dpp signaling would result in different positional information. The readout may be either synergistic or antagonistic, depending on the particular positional point within the developing wing (Khalsa, 1998).

Tgfbeta-60A alleles have been shown to genetically interact with mutations in BMP type I receptor genes, tkv and sax. The Dpp signal is mediated by two different BMP type I receptors, Tkv and Sax, during wing morphogenesis as well as during other stages of development The possibility of a genetic interaction between alleles of Tgfbeta-60A and alleles of tkv or sax was investigated to address the relative importance of these receptors in mediating the signals resulting from the actions of Tgfbeta-60A and Dpp. Recombinants were constructed between gbb-60A 4 or gbb-60A 1 and several alleles of tkv and sax. The addition into a Tgfbeta-60A mutant background of a chromosomal deficiency that removes the tkv locus, results in a severe mutant wing phenotype with a dramatic loss of both the PCV and ACV and most of L4 and L5. In addition, distal gaps are present in L2 and L3. A less extreme phenotype is seen with tkv6, a hypomorphic allele that retains significant receptor function. The observed interaction between tkv and Tgfbeta-60A cannot be explained solely as a secondary consequence of lowering Dpp signaling readout by the mutation of a receptor that mediates Dpp signaling. These data suggest that Tkv is able to mediate Tgfbeta-60A signaling and that it may do so in different ways at different times during development. The effect of reducing the Tgfbeta-60A copy number was investigated in flies compromised for functional Tkv receptor. Reducing Tgfbeta-60A in a tkv mutant background produces a further thickening of wing veins. This result suggests that Tgfbeta-60A may play a role in vein differentiation itself and/or in the tkv/dpp feedback loop important in defining the boundaries of the vein. Genetic combinations used to investigate the potential interaction between Tgfbeta-60A and sax alleles indicate a reduction in viability for Tgfbeta-60A mutant genotypes containing a single copy of a sax null allele. This reduction in viability suggests that lowering both Tgfbeta-60A and sax compromises development. The wing phenotype of the few viable adults recovered is similar to a very severe Tgfbeta-60A mutant wing phenotype, with a substantial loss of L5, complete loss of the PCV and ACV and loss of half of L4. Clearly the levels of Tgfbeta-60A signaling and Sax function are dependent on one another (Khalsa, 1998).

Based on genetic analysis and expression studies, it has been concluded that Tgfbeta-60A must signal primarily as a homodimer to provide patterning information in the wing imaginal disc. Tgfbeta-60A and dpp genetically interact and specific aspects of this interaction are synergistic while others are antagonistic. It is proposed that the positional information received by a cell at a particular location within the wing imaginal disc depends on the balance of Dpp to Tgfbeta-60A signaling. Furthermore, the critical ratio of Tgfbeta-60A to Dpp signaling appears to be mediated by both Tkv and Sax type I receptors (Khalsa, 1998).

Synergistic signaling by two BMP ligands through the SAX and TKV receptors controls wing growth and patterning in Drosophila

In Drosophila wing discs, a morphogen gradient of Dpp has been proposed to be a determinant of the transcriptional response thresholds of the downstream genes sal and omb. Evidence is presented that the concentration of the type I receptor Tkv must be low to allow long-range Dpp diffusion. However, low Tkv receptor concentrations result in low signaling activity. To enhance signaling at low Dpp concentrations, a second ligand, Tgf-beta-60A, has been found to augment Dpp/Tkv activity. Tgf-beta-60A signals primarily through the type I receptor Sax, which synergistically enhances Tkv signaling and is required for proper Omb expression. Omb expression in wing discs is found to require synergistic signaling by multiple ligands and receptors to overcome the limitations imposed on Dpp morphogen function by receptor concentration levels (Haerry, 1998).

While the reduction of Tkv and Put activity affects the whole disc (Sal, Omb and growth), the expression of dominant negative Sax only affects the peripheral region of the disc (Omb and peripheral growth). If the dominant negative receptors function primarily by titrating Dpp, then it is curious why the overexpression phenotypes of dominant negative Sax are different. One possibility is that these receptors do not simply signal in response to Dpp but also in response to the binding of other ligands as well. Of the other two BMP-type ligands that have been described in Drosophila, scw shows no detectable expression at this stage. However, Tgf-beta-60A is expressed broadly in wing discs, and mutant analyses indicate that Tgf-beta-60A is required for normal wing development. Given its role in wing patterning, the effects of heteroallelic Tgf-beta-60A mutations were examined on Sal and Omb expression. Similar to discs expressing dominant negative Sax, Sal expression in Tgf-beta-60A mutant discs is normal while the Omb domain is reduced, particularly in the dorsal compartment. These observations are consistent with the notion that a second BMP-type ligand, Tgf-beta-60A, is required in addition to Dpp for proper Omb expression. Furthermore, the similarity of the Tgf-beta-60A loss-of-function and the dominant negative Sax phenotypes is consistent with recently described genetic interactions between Tgf-beta-60A and sax mutations and suggests that Tgf-beta-60A could signal in part through Sax (Haerry, 1998).

Ubiquitous overexpression of moderate levels of Tgf-beta-60A does not result in excessive disc overgrowth and does not alter the distribution of Sal and Omb. The resulting wings are slightly larger and exhibit minor venation defects along L2 and L5. However, similar to Dpp or TkvA, higher levels of Tgf-beta-60A overexpression expands both Sal and Omb and results in blistered and pigmented adult wings. Since only activated Tkv but not Sax is able to expand Sal and Omb expression, these findings are consistent with the notion that expression of moderate levels of Tgf-beta-60A leads to signaling preferentially through Sax, producing relative mild phenotypes, while higher concentrations of Tgf-beta-60A may also result in signaling through Tkv, producing phenotypes similar to activated Tkv (Haerry, 1998).

An investigation was carried out to determine if Tgf-beta-60A contributes to wing development primarily in the form of homodimers or Tgf-beta-60A/Dpp heterodimers. Results: (1) the level of Tgf-beta-60A mRNA appears to be significantly less than that of DPP, based on RNA in situ hybridization, indicating that heterodimers are not likely to be very abundant assuming similar translational efficiencies. (2) Localized overexpression of Tgf-beta-60A in the dpp-expressing cells does not result in any mutant phenotypes. (3) Expression of Tgf-beta-60A in the posterior compartment results in overgrowth, an expansion of the Sal and Omb domains, and restriction all adult wing defects exclusively to the posterior compartment. Since Tgf-beta-60A expression in this experiment does not overlap with Dpp-secreting cells, no Dpp/Tgf-beta-60A heterodimers should form, since heterodimer formation requires expression of both proteins in the same cell. Therefore, Tgf-beta-60A functions most likely as a homodimer. This finding is consistent with recent genetic analysis showing that clones of Tgf-beta-60A mutant cells that do not include dpp-expressing cells nevertheless produce patterning defects. It has been shown that dominant negative Tkv is more potent than Sax for inhibiting Dpp signaling, while dominant negative Sax is a stronger suppressor than Tkv of Tgf-beta-60A signaling. High levels of Tkv receptor limit Dpp diffusion and restrict Omb expression (Haerry, 1998).

LIM kinase1 controls synaptic stability downstream of the type II BMP receptor and Gbb

The BMP receptor Wishful thinking (Wit) is required for synapse stabilization. In the absence of BMP signaling, synapse disassembly and retraction ensue. Remarkably, downstream Smad-mediated signaling cannot fully account for the stabilizing activity of the BMP receptor. LIM Kinase1 (DLIMK1)-dependent signaling has been identified as a second, parallel pathway that confers the added synapse-stabilizing activity of the BMP receptor. DLIMK1 binds a region of the Wit receptor that is necessary for synaptic stability but is dispensable for Smad-mediated synaptic growth. A genetic analysis demonstrates that DLIMK1 is necessary, presynaptically, for synapse stabilization, but is not necessary for normal synaptic growth or function. Furthermore, presynaptic expression of DLIMK1 in a wit or mad mutant significantly rescues synaptic stability, growth, and function. DLIMK1 localizes near synaptic microtubules and functions independently of ADF/cofilin (Twinstar), highlighting a novel requirement for DLIMK1 during synapse stabilization rather than actin-dependent axon outgrowth (Eaton, 2005).

The canonical bone morphogenic protein (BMP) signaling system has been implicated in diverse cellular and developmental processes ranging from cell growth to tissue patterning. At the Drosophila NMJ, a BMP signaling system has been identified that controls synaptic growth via canonical Smad-mediated signaling to the cell body. It has been demonstrated that mutations in the BMP ligand glass bottom boat (gbb), the type I and type II BMP receptors thick veins (tkv) and wishful thinking (wit), and the Smad homologs mad and medea all significantly impair synaptic growth and function. These data define a retrograde trophic signaling system that functions through transcriptional mechanisms in the cell soma to control motoneuron synaptic growth. This study demonstrates that BMP signaling at the Drosophila NMJ is not only required for normal synaptic growth, but also for synaptic stabilization. In the absence of BMP signaling, significant increases in synapse retraction and disassembly are observed. Signaling downstream of the BMP receptors can be genetically separated into two pathways: Smad-dependent synaptic growth and LIM Kinase1-dependent synaptic stability (Eaton, 2005).

LIM Kinase1 (LIMK1) is a cytoplasmic serine/threonine kinase that was originally isolated in screens for novel kinases expressed in the nervous system. Findings in LIMK1 knockout mice reveal defects in dendritic spine morphology and activity-dependent plasticity, although neither synaptic growth nor synaptic stability has been specifically analyzed. In the Drosophila central nervous system, DLIMK1 has been implicated in the mechanisms of axonal outgrowth during metamorphosis, acting through ADF/cofilin to modulate the actin cytoskeleton, a mechanism also documented in other tissues (Eaton, 2005 and references therein).

Genetic analyses have defined a new function for DLIMK1 during synaptic stability in comparison with its function in axonal outgrowth. Synaptic DLIMK1 is closely associated with the synaptic microtubule cytoskeleton. In addition, genetic manipulation of ADF/cofilin activity does not affect synaptic stability at the NMJ. These data highlight differences in LIMK1 function during the rapid, dynamic process of axon outgrowth compared to the slower, more prolonged mechanisms that govern synapse stabilization at the NMJ. Together, these data define genetically separable signaling pathways downstream of the BMP receptor that could allow a single trophic signaling event to coordinately control synaptic growth and synaptic stabilization (Eaton, 2005).

An assay to quantify synaptic retraction at the Drosophila NMJ was developed and used to identify molecules involved in synaptic stability. This assay is based on the demonstration that the formation of organized postsynaptic muscle membrane folds, termed the subsynaptic reticulum (SSR), requires the presence of the presynaptic nerve terminal. Therefore, the SSR and proteins that localize to this structure will be present only at sites where the nerve terminal resides, or where it has recently resided. Thus, observed sites of organized postsynaptic SSR that lack opposing presynaptic neuronal markers identify regions of the neuromuscular junction (NMJ) where the nerve terminal once resided and has since retracted. This interpretation has been confirmed in previous studies using light-level, ultrastructural, and electrophysiological analyses. Sites of synapse retraction are referred to as 'retraction events' or 'synaptic footprints' and represent a quantitative assay for synaptic stability. A wide array of pre- and post-synaptic markers have been used to clearly define synaptic retraction events. Synaptic retractions can be identified with equal efficiency using antibodies that recognize diverse presynaptic antigens, including cytoplasmic, membrane-associated, cytoskeleton, or vesicle-associated proteins. Several postsynaptic markers have also been used to quantify synapse retractions, including Discs-large, Shaker-GFP, and the clustered postsynaptic glutamate receptors (Eaton, 2005 and references therein).

The retraction assay was used to test whether BMP signaling in Drosophila motoneurons is required for synapse stabilization. First it was confirmed that mutations in the BMP type II receptor Wit and the BMP ligand Gbb cause a significant decrease in bouton number. These mutations also cause a significant increase in synaptic footprints, demonstrating that synaptic stability is significantly compromised in the absence of BMP signaling. Synaptic footprints were identified in equal numbers using multiple presynaptic markers, including anti-Synapsin and anti-nc82, which recognizes an antigen at the presynaptic active zone. Synaptic footprints can be rescued in the wit mutant background by neuronal expression of the full-length wit transgene, demonstrating that synapse destabilization is caused by the absence of the presynaptic Wit receptor. The number of synapse retractions is slightly, but statistically significantly, less in gbb compared with that in wit. This could be due to the fact that wit is a null mutation, whereas the gbb genotype that was used is not. Null mutations in gbb do not survive through larval development and, therefore, the genetic combination previously used in studies of its effects on synaptic growth was used (Eaton, 2005).

This analysis was extended to mutations that disrupt additional downstream components of the canonical BMP signaling cascade. Mutations in the BMP type I receptor thick-veins (tkv), the Smad homolog mad, and the co-Smad medea, were used. All three mutations decrease bouton numbers to levels that are statistically identical to those observed in the wit and gbb mutations. All three mutations also cause a statistically significant increase in synaptic footprints compared to wild-type, demonstrating that canonical BMP signaling is necessary for synaptic stability as well as for growth (Eaton, 2005).

Continued: Transforming growth factor beta at 60A Effects of mutation part 2/2