Mutations in the third chromosome gene Serrate are shown to display genetic interactions with specific alleles of the neurogenic locus Notch, which encodes a transmembrane protein with epidermal growth factor (EGF) homology. Embryonic lethal Serrate mutations exhibit epidermal and neuronal defects, which are reminiscent of those produced by mutations in the Drosophila EGF receptor homolog gene. We present the molecular cloning of Serrate and show that it encodes two coordinately expressed transcripts from a genomic interval >30 kb in length. The deduced protein product of 1404 amino acids contains a single transmembrane domain and 14 EGF-like repeats. Thus, Serrate represents another member of the group of EGF-containing loci in Drosophila. Whole-mount in situ hybridization analysis reveals complex temporal and spatial patterns of RNA expression consistent with the epidermal and neuronal defects observed in mutant embryos. Finally, we discuss the implications of Serrate function within the context of other cell-surface molecules known to be involved in the differentiation of ectodermally derived tissues.

The homeotic proboscipedia (pb) locus of the Antennapedia Complex (ANT-C) directs the differentiation of adult labial and maxillary structures. Loss-of-function pb alleles show a transformation of adult mouth parts to legs and affect maxillary palp morphology. We have identified the pb transcription unit by inducing and analyzing a series of pb null chromosomal breakpoints. In addition, we describe a variegating pb phenotype associated with two rearrangement breakpoints. Having identified the pb locus, we describe the expression of its RNA and protein products. Unlike the other homeotic genes of the ANT-C, pb has no obvious role in embryonic development. Nevertheless, pb protein is detected during embryogenesis in nuclei of the labial and maxillary lobes and in part of the mandibular segment. In this respect, pb expression parallels the early segment-specific expression of neighboring, embryonically essential homeotic genes. Accumulation of pb protein is also detected in mesodermal cells and in a unique subset of nuclei throughout the central nervous system. We also describe a transcription unit very close to pfc, which is expressed dorsally during embryogenesis in a pattern resembling that of the nearby zygotic lethal zerkniillt (zen) locus. This transcription unit has been shown to contain a homeo box and has been designated z2. Surprisingly, we find that individuals deleted for both the pb and z2 transcription units survive to adulthood and produce normal larval cuticular structures. Thus, pb and z2 show similarities to neighboring ANT-C genes in their expression patterns, yet these similarities are not manifested in comparable loss-of-function embryonic phenotypes.

The labial (lab) gene of Drosophila melanogaster is necessary for the proper development of the embryonic (larval) and adult head. We have identified the lab transcription unit within the proximal portion of the Antennapedia Complex (ANT-C) by mapping the molecular lesions associated with chromosomally rearranged lab alleles. We present its molecular structure, nucleotide sequence, and temporal pattern of expression. In addition, using antibodies generated against a fusion protein, we show that in the embryo the lab protein is distributed in neural and epidermal cells of the procephalic lobe; in a discrete loop of the midgut; and in specific progenitor sensory cells of the clypeolabrum, thoracic segments, and tail region. The regions of lab expression in the developing cephalon represent nonsegmented domains that are anterior to and largely nonoverlapping with the domains of expression of the Deformed (Dfd) and proboscipedia (pb) genes, two other homeotic loci of the ANT-C that also function to direct the development of head structures. Furthermore, lab head expression is associated with the complex cellular movements of head involution, a process that not only is defective in labembryos, but the failure of which appears to be largely responsible for the defects observed in mutant embryos.Finally, we suggest that lab head expression provides a molecular marker for an intercalary segment, an ancestral segment that has become morphologically indistinct during the evolution of the insect head.

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We present a molecular and genetic analysis which elucidates the role of deltex in the Notch signaling pathway. Using the yeast ‘interaction trap’ assay, we define the protein regions responsible for heterotypic interactions between Deltex and the intracellular domain of Notch as well as uncover homotypic interaction among Deltex molecules. The function of the Deltex-Notch interaction domains is examined by in vivo expression studies. Taken together, data from overexpression of Deltex fragments and from studies of physical interactions between Deltex and Notch, suggest that Deltex positively regulates the Notch pathway through interactions with the Notch ankyrin repeats. Experiments involving cell cultures indicate that the Deltex-Notch interaction prevents the cytoplasmic retention of the Suppressor of Hairless protein, which otherwise is sequestered in the cytoplasm via association with the Notch ankyrin repeats and translocates to the nucleus when Notch binds to its ligand Delta. On the basis of these findings, we propose a model wherein Deltex regulates Notch activity by antagonizing the interaction between Notch and Suppressor of Hairless.

Genetic data from Drosophila have suggested a functional relationship between the novel cytoplasmic protein encoded by the deltex locus and the transmembrane receptor encoded by Notch. We have demonstrated a direct interaction between these proteins from expression studies conducted in cultured cells, in yeast, and in the imaginal wing disc. deltex binds specifically to the Notch ankyrin repeats, a region that is crucial for Notch signaling and that constitutes the most conserved domain among Notch family members. In addition, we present a new Notch allele, Nsu42c, that is associated with a missense mutation within the fifth ankyrin repeat. In addition to representing a new class of viable Notch allele, this mutation behaves similarly to mutations of deltex and further implicates the ankyrin repeats in Notch function.

Prior genetic studies have suggested a functional relationship between the product of the deltex gene and those of three of the so-called "neurogenic" loci, Notch, Delta and mastermind. To gain further insight into this relationship, we have proceeded with a molecular characterization of deltex. We report that deltex encodes a maternally and zygotically expressed transcript that conceptually translates to a basic protein of novel sequence. Immunolocalization of the protein reveals an apparently ubiquitous distribution in embryonic and imaginal tissues. Because our detection methods also reveal a very low level of protein accumulation within the cytoplasm of cells, we have used transgenic flies to confirm this observation by ectopically expressing deltex under the control of a heat shock gene promoter. The resulting overexpression rescues deltex mutant defects but does not produce any obvious phenotypic abnormalities in otherwise wild-type flies. Finally, we examine genetically several Supressor of deltex mutations for evidence of functional integration with deltex and other neurogenic genes. We demonstrate that in addition to suppressing all adult morphological defects of deltex alleles, these suppressors also are capable of suppressing most synergistic effects involving deltex and Notch, Delta and mastermind.

Antibodies that specifically recognize proteins encoded by the homeotic genes: Sex combs reduced, Deformed, labial and proboscipedia, were used to follow the distribution of these gene products during embryogenesis. The position of engrailed-expressing cells was used as a reference and staining conditions were established that could distinguish, among cells expressing engrailed, one of the homeotic proteins or both. Our observations demonstrate two important facts about establishing identity in the head segments. First, in contrast to the overlapping pattern of homeotic gene expression in the trunk segments, we observe a non-overlapping pattern in the head for those homeotic proteins required during embryogenesis. In contrast, the spatial accumulation of the protein product of the non-vital proboscipedia locus overlaps partially with the distribution of the Deformed and Sex combs reduced proteins in the maxillary and labial segments, respectively. Second, two of the proteins, Sex combs reduced and Deformed, have different dorsal and ventral patterns of accumulation. Dorsally, these proteins are expressed in segmental domains while, within the ventral region, a parasegmental register is observed. The boundary where this change in pattern occurs coincides with the junction between the ventral neurogenic region and the dorsal epidermis. After contraction of the germ band, when the nerve cord has completely separated from the epidermis, the parasegmental pattern is observed only within the ventral nerve cord while a segmental register is maintained throughout the epidermis.