In situ hybridization using antisense riboprobes from the gastrulation-defective locus indicates that expression of gd begins in previtellogenic stages. Gd mRNA is seen in the germ line-derived nurse cells of the germarium. Expression continues throughout oogenesis with transcripts from the nurse cells accumulating in the oocytes of the vitellarium. Of interest, at about stage 10, gd expression can be detected in the surrounding follicle cells, although the level of signal is lower than the intense signal seen in the nurse cells at that stage. In some stage 10 oocytes, accumulation of mRNA appears to be somewhat graded along the dorsal-ventral axis with marginally higher levels of mRNA in the ventral follicle cells. By stage 13, residual mRNA remains in the shrinking nurse cells and in the follicle cells surrounding the oocyte (Konrad, 1998).
Within its C-terminal domain homologous to serine proteases, Gd has aspartic acid and histidine residues at the appropriate positions to be part of the catalytic triad necessary for enzymatic activity; however, the best candidate to be the catalytic serine is unusually positioned. Site-directed mutagenesis was used to change this serine to an alanine. Embryos injected with RNA encoding this mutant protein are not rescued and develop the dorsalized phenotype, like uninjected gd mutant embryos, indicating that the mutation destroys the rescuing activity of gd RNA. The putative catalytic aspartic acid was separately mutated, changing this residue to asparagine. Injection of RNA encoding this altered protein fails to rescue ventral and lateral development in gd mutant embryos. The destruction of gd RNA's biological activity by alteration of putative catalytic triad residues is consistent with Gd being an active serine protease (Han, 2000).
The gastrulation defective (gd) locus encodes a novel serine protease that is involved in specifying the dorsal-ventral axis during embryonic development. Mutant alleles of gd have been classified into three complementation groups, two of which exhibit strong interallelic (intragenic) complementation. To understand the molecular basis of this interallelic complementation, the complementation behavior of additional mutant alleles were examined and alleles in all complementation groups were sequenced. The data suggest that there are two discrete functional domains of Gd. A two-domain model of Gd, suggesting that it is structurally similar to mammalian complement factors C2 and B, has been previously proposed. To test this model a SP6 RNA microinjection was performed to assay for activities associated with various domains of Gd. The microinjection data are consistent with the complement factor C2/B-like model. Site-directed mutagenesis suggests that Gd functions as a serine protease. An allele-specific interaction between an autoactivating form of Snake (Snk) and a gd allele altered in the protease domain suggests that Gd directly activates Snk in a protease activation cascade. A model is proposed in which Gd is expressed during late oogenesis and bound within the perivitelline space but only becomes catalytically active during embryogenesis (Ponomareff, 2001).
The C2/B model proposes that Gd, in the process of becoming activated, is cleaved into two separate polypeptide chains. Since all mutations in alleles of the gd2 group map to the presumptive proenzyme polypeptide, these alterations are expected to specifically alter the activity of the propolypeptide chain. In most serine proteases, this polypeptide is involved in regulating the activity of the catalytic chain by modulating interactions with cofactors and other components of activation complexes in protease activation cascades. The alleles comprising the gd10 complementation group have lesions in close proximity to the putative active site serine residue, predicting that they will disrupt the activity of the catalytic chain. Grouping of the alterations to these two regions of Gd would suggest that each part of the Gd protein has an independent biochemical activity (Ponomareff, 2001).
Whereas interallelic complementation is often due to dimerization or multimerization of a protein, this interpretation is not favored for gd for several reasons: (1) dimerization of Gd has been sought using recombinant forms of the protein expressed using the baculovirus system and no evidence of either covalent or noncovalent dimers has been found to date; (2) if proteolytic cleavage at the arginine-lysine pair is part of the normal activation mechanism, then two separate polypeptides would be generated. Complementation would then arise by each allele providing a functional polypeptide consisting of either the catalytic or propolypeptide chain. These two polypeptides appear to have independent biochemical functions and might function sequentially. (3) Alternatively, they may be involved in formation of a larger multiprotein activation complex, something for which there is strong precedent among the coagulation and complement proteases. In this case it might be expected that after activation and cleavage two functional polypeptides may associate in an activation complex that can initiate the protease cascade and direct processing of Ea and Spz (Ponomareff, 2001).
The genetic data indicate that functions mapping to each putative polypeptide chain can in some cases be provided in trans to restore normal function to the system. Microinjection of gdDeltane into embryos from gd10 females restores the dorsal-ventral pattern with correct polarity with respect to the asymmetry of the egg. This indicates that the function of the carboxy-terminal catalytic chain may be provided as late as stage 2 of embryonic development. Microinjection of the propolypeptide into embryos from gd2/gd2 females does not result in rescue. This may be due to the inability of the presumptive proenzyme polypeptide (gdpro) to displace a nonfunctional form of the Gd propolypeptide from an activation complex within the perivitelline space (PVS). Transplantation experiments with perivitelline fluid revealed activities for Snk, Ea, and Spz, although they fail to find Gd activity. This result can be interpreted to indicate that Gd is bound to receptors within the PVS at early times during embryonic development. Perhaps gdpro contains binding sites for such a receptor. gdpro can also dorsalize a wild-type embryo when introduced at stage 2. This dominant negative effect indicates that the Gd propolypeptide can interfere with the biochemical pathway in such a way as to reduce the level of the ventralizing signal. However, this competitive effect might be due to interaction with something other than a membrane receptor for Gd and might exert its effect by complexing with and rendering inaccessible the proenzyme of Snk. Similarly, gdvWF, which comprises the vonWillebrand type A homology region, is also able to produce a dominant negative effect when introduced into wild-type embryos. These data are taken to indicate that other components of the biochemical pathway interact with this region of the presumptive catalytic chain of Gd. The RNA microinjection result is consistent with the idea that this region might be involved in binding of a positive modulator of activation. However, biochemical aspects of the regulation of Gd are clearly complex and further biochemical analysis of the protein will be necessary (Ponomareff, 2001).
The allele-specific interaction between gd10 and XaSnake is consistent with Gd directly activating the Snk zymogen in a protease cascade. When injected into embryos from gd9/gd9 females, the catalytic chain of Gd does not rescue or exhibit a dominant lateralizing or ventralizing effect as do analogous constructs for Snk (snkDeltaNe) and Ea (eaDeltan). Therefore, it would appear that both the propolypeptide as well as the catalytic chain are absolutely required for Gd to efficiently activate the downstream components of the pathway (Ponomareff, 2001).
A role of the propolypeptide in the spatial regulation of Gd activity is supported by the fact that microinjection of gdDeltane into the posterior pole of an embryo from a gd10/gd10 female results in rescue that is uniform along the anterior-posterior axis of the embryo. This contrasts with injection of wild-type gd RNA, which produces phenotypic rescue that is greater near the site of injection and less extreme away from the site of injection. The result of injection of gdDeltane into embryos from gd10/gd10 females suggests that the Gd catalytic chain is capable of freely diffusing within the PVS, while full-length Gd is not. This would argue for a localizing or binding function for the Gd propolypeptide (Ponomareff, 2001).
The following is a model for how Gd functions that fits the temperature sensitive period data and the genetic and molecular data. In this model the two separable functions, that of the propolypeptide chain and that of the catalytic chain, are required at two distinct times. Gd protein would be expressed from maternal mRNA late during oogenesis, secreted, and localized to the plasma membrane surface within the PVS via binding sites within the proenzyme polypeptide. Gd is uniformly distributed relative to the dorsal-ventral axis. It remains bound to the plasma membrane and remains inactive until the syncytial blastoderm stage. At this time, Gd becomes autocatalytically active only on the ventral side and initiates a proteolytic cascade resulting in the ventrally restricted production of a processed form of Spaetzle. This 'localization during oogenesis/activation during embryogenesis' model explains the ability of Gd to restore ventrolateral pattern elements as late as stage 2 of embryogenesis by microinjection of RNA and its failure to normalize the pattern in embryos from gd9/gd9 females. This would be due to the failure to establish the normal distribution of bound Gd within the PVS during embryogenesis because of the nonuniform secretion of Gd into the PVS. Aspects of this model might be tested by generating a heat-shock-inducible form of Gd and a P-element-mediated transformed line (Ponomareff, 2001).
Gd occupies a pivotal role in the process of specifying the dorsal-ventral axis of the embryo. The data are consistent with Gd directly activating Snk and therefore suggest that Gd is the earliest acting of the germ-line-derived proteases in the PVS. Gd appears to be a molecule intimately involved in the interpretation of the ventral prepattern of the egg. Biochemical data from coexpression experiments indicate that Gd activates Snk and triggers a proteolytic cascade comprising Snk, Ea, and Spz and in the process Gd undergoes rather complex proteolytic processing. Some of the sizes of these fragments are consistent with predicted sizes for an active form of Gd from the C2/B model. Taken together, the available data suggest that Gd is responding to spatial prepattern information and to temporal cues for activation and receiving feedback from the cascade that modulates its activity. Since Gd is the focus for these multiple inputs, it may constitute a nexus for regulating the shape of the Dorsal gradient. From this perspective, it may not be surprising that complex genetic interactions might be attributed to the gd locus (Ponomareff, 2001).
Chasan, R., Jin, Y. and Anderson, K. V. (1992). Activation of the easter zymogen is regulated by five other genes to define dorsal-ventral polarity in the Drosophila embryo. Development 115: 607-616. 1425342
Cho, Y. S., Stevens, L. M., Sieverman, K. J., Nguyen, J. and Stein, D. (2012). A ventrally localized protease in the Drosophila egg controls embryo dorsoventral polarity. Curr Biol 22: 1013-1018. PubMed ID: 22578419
DeLotto, L. (2001). Gastrulation defective, a complement factor C2/B-like protease, interprets a ventral prepattern in Drosophila. EMBO Reports 2: 721-726. 11493599
Dissing, M., Giordano, H. and DeLotto, R. (2001). Autoproteolysis and feedback in a protease cascade directing Drosophila dorsal-ventral cell fate. EMBO J. 20: 2387-2393. 11350927
Han, J.-H., et al. (2000). Gastrulation defective is a serine protease involved in activating the receptor toll to polarize the Drosophila embryo. Proc. Natl. Acad. Sci. 97: 9093-9097. 10922064
Konrad, K. D., Goralski, T. J. and Mahowald, A. P. (1988). Developmental genetics of the gastrulation defective locus in Drosophila melanogaster. Dev. Bio. 127: 133-142. 3129326
Konrad, K. D., et al. (1998). The gastrulation defective gene of Drosophila melanogaster is a member of the serine protease superfamily. Proc. Natl. Acad. Sci. 95(12): 6819-6824. PubMed ID: 9618496
LeMosy, E. K., Tan, Y.-Q. and Hashimoto, C. (2001). Activation of a protease cascade involved in patterning the Drosophila embryo. Proc. Natl. Acad. Sci. 98: 5055-5060. 11296245
LeMosy, E. K., et al. (2006). Spatially dependent activation of the patterning protease, Easter. FEBS Letters 580: 2269-2272. 16566925
Misra, S., Hecht, P., Maeda, R. and Anderson, K. V. (1998). Positive and negative regulation of Easter, a member of the serine protease family that controls dorsal-ventral patterning in the Drosophila embryo. Development 125: 1261-1267. 9477324
Ponomareff, G., et al. (2001). Interallelic complementation at the Drosophila melanogaster gastrulation defective locus defines discrete functional domains of the protein. Genetics 159: 635-645. 11606540
Smith, C. L. and DeLotto, R. (1994). Ventralizing signal determined by protease activation in Drosophila embryogenesis. Nature 368: 548-551. 8139688
Stein, D. and Nüsslein-Volhard, C. (1992). Multiple extracellular activities in Drosophila egg perivitelline fluid are required for establishment of embryonic dorsal-ventral polarity. Cell 68: 429-440. 1739964
date revised: 15 April 2013
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