Gene name - glass bottom boat
Synonyms - Transforming growth factor beta at 60A
Cytological map position - 60A1--60A3
Function - ligand
Symbol - gbb
FlyBase ID: FBgn0024234
Genetic map position - 2-
Classification - TGF-beta superfamily
Cellular location - secreted
Transforming growth factor beta at 60A, known in the literature as 60A, is a member of the TGFbeta superfamily that bears a closer resemblence to vertebrate osteogenic protein (OP-1 or BMP-7) than to BMP-4 or to BMP-4's homolog in Drosophila, Decapentaplegic. Although the gene will be referred to here as Tgfbeta-60A, it is more correctly termed glass bottom boat (gbb). Other close relatives of Tgfbeta-60A in vertebrates are BMP-5 and BMP-6. It is likely that the two evolutionary lineages, one giving rise to Dpp and its homologs, and a second giving rise to Tgfbeta-60A and its homologs, diverged well before the divergence of insects and mammals (Wharton, 1991).
Little about Tgf-beta-60A was known beyond its expression pattern because mutants were unavailable, but a 1998 paper by Chen changed all that. Tgfbeta-60A expression had been previously found to be pronounced in mesodermal cells and in cells of the stomadeal and posterior midgut invaginations, foregut and hindgut cells and in the endodermal cells of the anterior and posterior midgut (Doctor, 1992). Chen sought out dominant enhancer mutations magnifying the effects of a hypomorphic allele of thick veins (tkv), a type I receptor for dpp. Hypomorphic alleles result in partial loss of function of a gene, and dominant enhancers worsten the phenotypic effects of these hypomorphic alleles. Enhancing mutations were found in Mad, Medea, punt and thick veins, all of which are known components of the dpp signaling pathway and in Tgfbeta-60A. Phenotypic analysis of Tgfbeta-60A single mutants and tkv 6;Tgfbeta-60A double mutants revealed both dpp-independent and dpp-dependent functions for Tgfbeta-60A. Tgfbeta-60A mutants lack the first constriction of the embryonic midgut and Antennapedia expression in parasegment 6, indicating that 60A is required for the formation of the first constriction, possibly through regulating Antp expression. This function is independent of dpp signaling, since mutations in dpp or its receptors only disrupt the formation of the second but not the first constriction. This also suggests that there is either redundancy or that a different receptor system is responsible for mediating Tgfbeta-60A signaling to pattern the first constriction. It would be interesting to see if AtrI (Childs, 1993), a type I receptor resembling the type I activin receptor, and STK-D (Ruberte, 1995), a type II receptor in Drosophila, both of unknown function, are mediators of 60A signaling at the site of the first constriction (Chen, 1998).
The fact that Tgfbeta-60A mutations are dominant enhancers of a sensitized dpp pathway implicates Tgfbeta-60A in potentiating dpp signaling (for review, see Raftery, 1999). This is most obvious in the visceral mesoderm of the midgut where dpp signaling is required to regulate homeotic gene expression and to maintain its own expression through a positive feedback mechanism. Although dpp signaling in the visceral mesoderm appears intact in Tgfbeta-60A mutants, a requirement for Tgfbeta-60A is revealed in tkv 6 Tgfbeta-60A double mutants. When dpp signaling is attenuated through a mutant tkv receptor, eliminating Tgfbeta-60A function reduces the signaling to below threshold level. The derepression of Sex combs reduced in the anterior midgut and the loss of expression of dpp target genes (wingless, Ultrabithorax and dpp) in the visceral mesoderm and labial in the endoderm are consistent with inadequate dpp signaling. A similar requirement for Tgfbeta-60A is observed during dorsal closure of the embryonic ectoderm. The enhanced phenotypes of the adult appendages closely resemble those of the Tgfbeta-60A hypomorphic mutants, suggesting that Tgfbeta-60A activity is also required for imaginal disc patterning. It is interesting that the imaginal discs are more sensitive than are mesoderm or endoderm to the reduction of Tgfbeta-60A function, as a 50% reduction in Tgfbeta-60A function is sufficient to produce a phenotype in a tkv 6 genetic background. This may reflect a differential threshold requirement for dpp signaling in different tissues (Chen, 1998).
Tgfbeta-60A may form functional heterodimers with Decapentaplegic. In a signaling system with multiple interacting dimeric ligands, the interpretation of any single mutant phenotypes must consider the effect of losing both homomeric and possible heteromeric ligands. Therefore, the functions of the dpp pathway may be a composite input from Dpp homodimers, and Dpp/Scw and Dpp/Tgfbeta-60A heterodimers. Alternatively, Tgfbeta-60A homodimers may function in an additive fashion with Dpp homodimers at sites of overlapping expression. However, the loss-of-function phenotypes of dpp are as severe as the loss-of- function phenotypes of its downstream components, such as tkv or Mad, suggesting that there is very little signaling, if any at all, from Tgfbeta-60A homodimers in dpp-dependent events. Therefore, it is unlikely that Tgfbeta-60A homodimers play a significant role in dpp-dependent processes. Rather, it is thought that Dpp/Tgfbeta-60A heterodimers form at sites of overlapping expression and participate with Dpp homodimers in multiple signaling events. The broad distribution of Tgfbeta-60A proteins provides an opportunity for forming Dpp/Tgfbeta-60A heterodimers. Unlike scw null mutations, no obvious disruption of dpp signaling is observed in Tgfbeta-60A null mutants, suggesting that Dpp/Tgfbeta-60A heterodimers are not as limiting as Dpp/Scw heterodimers, but function in partially redundant manner with Dpp homodimers (Chen, 1998).
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 [(Wishful thinking (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).
Bases in 5' UTR - 171
Exons - 1
Bases in 3' UTR - 130
The Tgfbeta-60A gene, a member of the transforming growth factor beta superfamily of signaling proteins, has been identified in Drosophila. From its inferred protein sequence it is predicted that the precursor is secreted and processed to release a growth factor-like molecule. The putative precursor contains an amino-terminal signal sequence and four N-linked glycosylation sites, consistent with the proposed secreted nature of this protein. The TGF-beta homologous sequences are located in 100 amino acids of the C-terminal region. The multibasic cleavage sites, RSKR and RKRKK (positions 322 and 331, respectively), found upstream of the TGF-beta homologous region are candidate sites for the proteolytic cleavage of the precursor molecule. As would be expected based on the primers used to isolate this gene, the C-terminal sequence of the Tgfbeta-60A gene exhibits expecially high levels of sequence identity in amino acid residues that are conserved within the Vg-1/dpp subgroup. The putative Tgfbeta-60A protein shows greater sequence similarity to three vertebrate family members (human bone morphogenetic proteins 5, 6, and 7) than to its only Drosophila relative, the protein product of the dpp gene. This observation suggests that the duplication event that gave rise to the two transforming growth factor beta-like proteins in Drosophila predates the divergence of chordates and arthropods (Wharton, 1991).
Using PCR methods, a second Drosophila gene in the TGF-beta family has been identified. It encodes a protein product that is more similar to the TGF-beta-related human bone morphogenetic proteins (BMPs) 5, 6, and 7 than it is to the Drosophila dpp gene product. All seven cysteines are precisely conserved between Tgfbeta-60A and other family members Because of its localization on the polytene chromosome map, this gene is termed Tgfbeta-60A. Expression of a Tgfbeta-60A cDNA in Drosophila S2 cells was used to determine that Tgfbeta-60A encodes a preproprotein that is processed to yield secreted amino- and carboxy-terminal polypeptides (Doctor, 1992).
date revised: 5 June 98
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