Interallelic complementation at the Drosophila melanogaster gastrulation defective locus defines discrete functional domains of the protein.

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, we examined the complementation behavior of additional mutant alleles and sequenced alleles in all complementation groups. 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 we performed SP6 RNA microinjection 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. We propose a model in which Gd is expressed during late oogenesis and bound within the perivitelline space but only becomes catalytically active during embryogenesis.

Multiple isoforms of the Drosophila Spätzle protein are encoded by alternatively spliced maternal mRNAs in the precellular blastoderm embryo.

The spätzle gene is required for proper specification of positional information along the dorsal-ventral axis of the Drosophila embryo and for induction of the innate immune response to fungal infection. It has been shown to encode a precursor of a Nerve Growth Factor-like ligand which is also a member of the cysknot protein superfamily. In dorsal-ventral patterning, the most widely accepted model of the pathway places Spätzle at the end of a ventrally restricted protease cascade that results in the proteolytic processing of the precursor form of Spätzle to an active ligand which is thought to bind to the Toll receptor. Here we show that the spätzle gene encodes at least ten different protein isoforms as a result of complex alternative splicing in precellular blastoderm embryos. Multiple transcripts are clearly present up until the time of cellularization, at which point most transcripts can no longer be detected. Nine isoforms were expressed and at least five are efficiently secreted in a heterologous protein expression system. RNA microinjection experiments demonstrate that three isoforms completely rescue embryos from spätzle null mothers, while most of the others rescue to a lesser extent. The phenotypic rescue activities of several isoforms and the relevance of these isoforms to the generation of the ventralizing signal are discussed.

Proteolytic processing of the Drosophila Spätzle protein by easter generates a dimeric NGF-like molecule with ventralising activity.

Biochemical interactions underlying the generation of the ventralising signal during Drosophila embryogenesis were investigated by the expression of recombinant Easter and Spätzle proteins. An active form of Easter protease cleaves the Spätzle protein, generating a carboxyterminal polypeptide fragment which, when microinjected into the perivitelline space of a spätzle deficient embryo, directs production of ventrolateral pattern elements. This Spätzle carboxyterminal fragment is a disulfide-linked dimer and modelling suggests that the core disulfide bonds and dimer arrangement of this fragment are highly similar to vertebrate nerve growth factor. Thus Spätzle is a member of a new family of neurotrophin-like signalling molecules in invertebrate development.

Structure and regulation of a complex locus: the cut gene of Drosophila.

The cut locus encodes a homeobox protein that is localized in the nuclei of a variety of tissues and is required for proper morphogenesis of those tissues. Cut protein is required in embryonic and adult external sensory organs, where its absence results in conversion of the organs to chordotonal organs. Expression of cut also occurs in the Malpighian tubules, spiracles, central nervous system, and a number of other tissues. Gypsy transposon insertions upstream of the cut promoter block expression in subsets of these tissues. The effect of the gypsy insertions is polar, with those farthest from the promoter affecting the fewest tissues. The hypothesis that gypsy insertions block a series of tissue-specific enhancer elements that are distributed over a region of 80 kb upstream of the promoter predicts the location of the enhancers for cut expression in each of the tissues in which it is active in embryos. DNA fragments from this region drive expression of a reporter gene in each of the embryonic tissues in which endogenous cut gene is expressed. Each tissue has its own enhancer, and none of the enhancers require the activity of the endogenous cut gene to function.

Effect of wing scalloping mutations on cut expression and sense organ differentiation in the Drosophila wing margin.

A number of wing scalloping mutations have been examined to determine their effects on the mutant phenotype of cut mutations and on the expression of the Cut protein. The mutations fall into two broad classes, those which interact synergistically with weak cut wing mutations to produce a more extreme wing phenotype than either mutation alone and those that have a simple additive effect with weak cut wing mutations. The synergistically interacting mutations are alleles of the Notch, Serrate and scalloped genes. These mutations affect development of the wing margin in a manner similar to the cut wing mutations. The mutations inactivate the cut transcriptional enhancer for the wing margin mechanoreceptors and noninnervated bristles and prevent differentiation of the organs. Surprisingly, reduction of Notch activity in the wing margin does not have the effect of converting epidermal cells to a neural fate as it does in other tissues of ectodermal origin. Rather, it prevents the differentiation of the wing margin mechanoreceptors and noninnervated bristles.

Expression of the cut locus in the Drosophila wing margin is required for cell type specification and is regulated by a distant enhancer.

The cut locus is a complex gene whose function is necessary for specification of a number of cell types, including the external sensory organs. The cut wing class of mutations of the cut locus are homozygous viable and lack tissue from the wing margin, which is normally composed of external sensory organs and noninnervated bristles. Expression of cut was examined in the developing wings of wild-type and mutant pupae using an antiserum against Cut protein. Cut is expressed in all of the external sensory organs of the wing and the noninnervated bristles of the posterior margin. The cut wing class of mutations prevents Cut expression specifically in the wing margin mechanoreceptors and noninnervated bristles, apparently preventing neural differentiation. The transformed cells die soon after differentiation would have occurred. We identify an enhancer, located about 80 kb upstream of the cut gene promoter, that confers expression in the cells of the mechanoreceptors and noninnervated bristles from a heterologous promoter. The 27 gypsy retrotransposon insertions that prevent expression in these margin cells, all occur between this enhancer and the promoter. These gypsy insertions probably interfere with the interaction between the enhancer and the cut gene promoter.