The gastrulation defective gene of Drosophila melanogaster is a member of the serine protease superfamily

Abstract

The establishment of dorsal-ventral polarity in the oocyte involves two sets of genes. One set belongs to the gurken-torpedo signaling pathway and affects the development of the egg chorion as well as the polarity of the embryo. The second set of genes affects only the dorsal-ventral polarity of the embryo but not the eggshell. gastrulation defective is one of the earliest acting of this second set of maternally required genes. We have cloned and characterized the gastrulation defective gene and determined that it encodes a protein structurally related to the serine protease superfamily, which also includes the Snake, Easter, and Nudel proteins. These data provide additional support for the involvement of a protease cascade in generating an asymmetric signal (i.e., asymmetric Spätzle activity) during establishment of dorsal-ventral polarity in the Drosophila embryo.

Figure 1

Correlation of the genetic and molecular maps and the transcripts of gd and tsg. The top line displays the recombination distances in centimorgans between the loci in this region (cf. Materials and Methods). They are depicted according to their orientation along the X chromosome with proximal to the right. The approximate locations of breakpoints for several inversions were determined by Southern blotting as follows: In A97 (51.9/49.3kb), In A101 (44.5/42.5), In N66 (38.1/36.1) and In A78 (32/27.6). The bars represent DNA that is present in the deficiency chromosomes Df(1)HF368, Df(1)m13, and Df(1)RC29. The lightly shaded areas represent the uncertainty of the breakpoints, and the dashes indicate the DNA which is missing. The portion of the chromosomal walk used to identify the gd locus is shown immediately below the breakpoints with the extent of overlapping λ phage clones 320, 348, K3, and C8 indicated in kilobases. The positions of the 9.3-kb EcoRI and 10.2-kb HindIII transformation subclones are shown expanded below the chromosome walk as is the region that was sequenced. The sequenced region abuts other sequenced regions from the tsg gene and beyond. A partial restriction map of the 9.3-kb EcoRI subclone is displayed with E = EcoRI, H = HindIII, Bg = BglII, and B = BamHI. The extent and orientation of the 2.1-kb gd and 1.0-kb tsg transcripts are shown. The smaller lightly shaded blocks indicate the primary transcripts, and the protein coding regions are shown as larger darker blocks.

Figure 2

Transcript map of the gd region. mRNA from stages throughout the entire life cycle were probed. The results obtained with three probes are shown here (i.e., a 1.3-kb HindIII and 3.1-kb BamHI fragments and the entire K3 phage DNA). The 2.1-kb transcript seen in early embryos (0–3.5 h) and faintly in females is the only transcript in the region with a developmental pattern consistent with the genetics of gd. It is the only transcript detected by the 1.3-kb HindIII fragment. The band in females and embryos is of similar size although this result is distorted here by a curve in the migration front. The 3.1-kb BamHI probe hybridizes to three bands whereas the entire K3 phage hybridizes to the 2.8-, the 1.9-, and a 4.7-kb transcript. Additional probing with other subclones permitted ordering the transcripts as shown (21). The tsg transcript has been identified by transformation (32). The 4.7-kb transcript likely corresponds to the furrowed gene (Corces, V.G., Johns Hopkins Univ., personal communication). The 2.8-kb transcript appears specific to males, and the 1.9-kb transcript is specific to females. From the hybridization pattern, we infer that these are likely alternative splicing variants of a single gene. The 1.3-kb transcript may have a large splicing variant as well. We have not yet identified any genetic lesions that may correspond to these transcription units.

Figure 3

Alignment of GD with related proteins. (A) Diagram of the gene structure. Region shown in the alignment below is the Ser protease domain with diagonal lines. A hydrophobic secretion signal appears at the N terminus and a hydrophobic tail at the C terminus. Putative N-linked glycosylation sites (NXT/S) are noted by inverted Ys. (B) Each protein is shown beginning with the activating cleavage site (|) and showing the catalytic portion of the protein. Amino acid numbers of the Drosophila protein are shown above the alignment. The putative cleavage site listed for GD is atypical. The H D S residues of the catalytic triad (marked with ∗) are conserved in GD as are most of the Cys bridges with the exception of the penultimate C indicated by (?). A number of other residues are conserved as well. Residues shown in bold correspond to some of the key residues that define distinct structural domains of serine proteases (40). Molecular modeling suggests that the sequence differences seen here could be accommodated into a folded protein that preserved the catalytic site and the substrate groove. The sequences used in this alignment are chicken urokinase P15120, human factor IX clotting enzyme P00740, horseshoe crab proclotting enzyme precursor P21902, crab coagulation factor B A48050, and Bos taurus chymotrypsin National Center for Biotechnology Information sequence ID: 442949.

Figure 4

In situ hybridization to gd mRNA in ovaries. (A) gd mRNA first appears in the germarium. Levels continue to build in the nurse cells and oocytes of the vitellarium. Expression also begins to appear in the follicle cells (fc) as the oocytes move into stage 10. (B) Extensive transcription in both nurse cells and in follicle cells is seen in stage 10 oocytes. In oocytes where the D/V axis can be inferred from the position of the oocyte nucleus, a rough ventral to dorsal gradient of transcript often can be detected. (D) By stage 13, residual gd transcripts remain in the nurse cells as well as the surrounding follicle cells. (C) Sense strand control probe shows no detectable background staining.

Figure 5

Physical characterization of the GD protein. (A) Western blot of protein extracts from OreR females and staged embryos probed with antisera raised against the GD peptide. The peptide produced by in vitro translation of synthetic gd mRNA is 60 kDa (not shown), and the peptides detected in vivo are smaller. (B) Western blot of protein extracts from ovaries of various gd mutant females suggests that some gd alleles affect the protein detected by this antiserum.