Dominant Drop mutants are gain-of-function alleles of the muscle segment homeobox gene (msh) whose overexpression leads to the arrest of eye development.

Dominant Drop (Dr) mutations are nearly eyeless and have additional recessive phenotypes including lethality and patterning defects in eye and sensory bristles due to cis-regulatory lesions in the cell cycle regulator string (stg). Genetic analysis demonstrates that the dominant small eye phenotype is the result of separate gain-of-function mutations in the closely linked muscle segment homeobox (msh) gene, encoding a homeodomain transcription factor required for patterning of muscle and nervous system. Reversion of the Dr(Mio) allele was coincident with the generation of lethal loss-of-function mutations in msh in cis, suggesting that the dominant eye phenotype is the result of ectopic expression. Molecular genetic analysis revealed that two dominant Dr alleles contain lesions upstream of the msh transcription start site. In the Dr(Mio) mutant, a 3S18 retrotransposon insertion is the target of second-site mutations (P-element insertions or deletions) which suppress the dominant eye phenotype following reversion. The pattern of 3S18 expression and the absence of msh in eye imaginal discs suggest that transcriptional activation of the msh promoter accounts for ectopic expression. Dr dominant mutations arrest eye development by blocking the progression of the morphogenetic furrow leading to photoreceptor cell loss via apoptosis. Gal4-mediated ubiquitous expression of msh in third-instar larvae was sufficient to arrest the morphogenetic furrow in the eye imaginal disc and resulted in lethality prior to eclosion. Dominant mutations in the human msx2 gene, one of the vertebrate homologs of msh, are associated with craniosynostosis, a disease affecting cranial development. The Dr mutations are the first example of gain-of-function mutations in the msh/msx gene family identified in a genetically tractible model organism and may serve as a useful tool to identify additional genes that regulate this class of homeodomain proteins.

Pattern formation in the absence of cell proliferation: tissue-specific regulation of cell cycle progression by string (stg) during Drosophila eye development.

During Drosophila eye development, the posterior-to-anterior movement of the morphogenetic furrow coordinates cell cycle progression with the early events of pattern formation. The cdc25 phosphatase string (stg) has been proposed to contribute to the synchronization of retinal precursors anterior to the furrow by driving cells in G(2) through mitosis and into a subsequent G(1). Genetic and molecular analysis of Drop (Dr) mutations suggests that they represent novel cis-regulatory alleles of stg that inactivate expression in eye. Retinal precursors anterior to the furrow lacking stg arrest in G(2) and fail to enter mitosis, while cells within the furrow accumulate high levels of cyclins A and B. Although G(2)-arrested cells initiate normal pattern formation, the absence of stg results in retinal patterning defects due to the recruitment of extra photoreceptor cells. These results demonstrate a requirement for stg in cell cycle regulation and cell fate determination during eye development.

Ingrowth by photoreceptor axons induces transcription of a retrotransposon in the developing Drosophila brain.

The development of the lamina, the first optic ganglion of the fly visual system, depends on inductive cues from the innervating photoreceptor axons. lacZ expression from a P-element insertion, A72, occurs in the anlage of the lamina coincident with axon ingrowth from the eye imaginal disc. In eyeless mutants lacking photoreceptor axons, lacZ expression did not occur. The P-element was found to have inserted within the 3' long terminal repeat (LTR) of a '17.6' type retrotransposon. The expression pattern of 17.6 transcripts in the brain in wild-type and eyeless mutants paralleled the expression of the lacZ reporter. Analysis of 17.6 cis-regulatory sequences indicates that the lamina-specific expression is due to the combined action of an enhancer element in the LTR and a repressor element within the internal body of the retrotransposon. The regulation of the 17.6 retrotransposon provides a model for the study of innervation-dependent gene expression in postsynaptic cells during neurogenesis.

The trithorax gene, a trans-acting regulator of the bithorax complex in Drosophila, encodes a protein with zinc-binding domains.

The trithorax (trx) gene functions in segment determination in Drosophila through interaction with genes of the bithorax complex and Antennapedia complex. Genetic evidence suggests that trx may be considered a positive regulator of homeotic genes. Sequencing of cDNAs corresponding to the entire trx transcription unit revealed the existence of an unusually long open reading frame encoding 3759 amino acids. The main features of the predicted trx protein are several cysteine-rich regions which can be folded into zinc finger-like domains. Cysteine-rich portions expressed from trx cDNAs in Escherichia coli are capable of zinc binding in vitro, suggesting a possible function for the trx product as a metal-dependent DNA-binding protein. Analysis of trx mutant embryos with antibody to the Ultrabithorax (Ubx) gene product showed decreased staining in parasegment 6 of the ventral nerve cord of late embryos. However, expression of Ubx was not affected in embryos carrying the lethal mutation trxE3, in which one of the putative zinc finger-like domains of the trx protein is deleted. This differential effect of the E3 mutation suggests that trx exhibits other function(s) besides those involved in the regulation of Ubx expression in the ventral nerve cord of the embryo.

The Drosophila fsh locus, a maternal effect homeotic gene, encodes apparent membrane proteins.

The maternal effect gene fsh is involved in the establishment of segments and the specification of their identities; the progeny of mutant females are missing portions of thoracic and abdominal segments, and may have homeotic transformations of third thoracic segments to second thoracic segments. The fsh locus interacts synergistically with loci such as Ubx and trx in the production of homeotic transformations. We have characterized cDNA clones corresponding to the major fsh transcripts expressed in ovaries and early embryos, and to a pupal transcript. The expression of fsh transcripts in ovaries is restricted to the germline; in developing embryos, transcripts are found throughout the cytoplasm. The different ovarian/embryonic transcripts (7.6 and 5.9 kb) are generated by use of alternative polyadenylation and splice sites. These transcripts encode two large predicted proteins of 110 and 205 kDa that have unusual amino acid compositions: 40% of the residues are glycine, alanine, or serine, and there are several regions of homopolymers and simple sequence repeats. Hydropathy analysis indicates that these proteins span the membrane. We suggest that the expression of fsh proteins in the membrane of the embryo is required for proper functioning of genes such as Ubx in the specification of segmental identity.

Cloning and molecular characterization of the trithorax locus of Drosophila melanogaster.

The trithorax (trx) locus of Drosophila melanogaster affects segment determination primarily in the thoracic region. Mutant flies show transformations of the third and, to a lesser extent, first thoracic segment toward the second thoracic segment; abdominal transformations also occur. Prior genetic evidence suggested that these effects are based on interactions between trx and genes of the bithorax complex and Antennapedia complex. Further, interactions between the maternal effect locus female sterile homeotic (fsh) and trx have been observed. To aid in a molecular analysis of trx function, we have cloned the locus by a P-element transposon tagging approach. Five insertion mutations have been mapped within a region of about 10 kilobases; one of these mutations reverted coincident with the loss of the insertion. Transcription mapping suggests that two RNAs of about 12 and 15 kilobases are the major transcripts of the trx locus and that the transcription unit comprises a region of about 25 kilobases. Transcripts from the trx locus are distributed uniformly in early embryos, but at 14-16 hr after fertilization the ventral nerve cord contains a higher concentration of trx RNA than other regions of the embryo.

pen repeat sequences are GGN clusters and encode a glycine-rich domain in a Drosophila cDNA homologous to the rat helix destabilizing protein.

Several cDNA clones that contain the pen repeat have been isolated and sequenced; pen consists of clusters of GGN triplets, where N can be any nucleotide. Some of the pen repeat sequences are found within long open reading frames in which they encode oligoglycine stretches. For one of the clones, the deduced amino acid sequence of the entire open reading frame, especially in the region preceding the glycine-rich domain, shows strong homology to the rat helix destabilizing protein [Cobianchi, F., SenGupta, D. N., Zmudzka, B. Z. & Wilson, S. H. (1986) J. Biol. Chem. 261, 3536-3543]. The rat protein and homologs in other organisms are single-stranded nucleic acid binding proteins, some of which are major components of heterogeneous nuclear ribonucleoprotein particles. We suggest that we have cloned a cDNA encoding a Drosophila single-stranded nucleic acid binding protein.

Genetic and molecular analysis of fs(1)h, a maternal effect homeotic gene in Drosophila.

Mutations at the Drosophila melanogaster locus female sterile (1) homeotic (fs(1)h) result in segmental abnormalities including missing organs and homeotic transformations in the progeny of mutant mothers. Homeotic transformations are enhanced when the zygotes carry one of several third chromosome mutations, specifically alleles or deficiencies of the trithorax (trx) locus, also called Regulator-of-bithorax, and some alleles of bithorax complex (BX-C) genes. These observations suggest that maternally derived fs(1)h+ product is required, in interaction with trx and BX-C genes, for normal segment specification. The fs(1)h gene and an adjacent gene, lethal (1) myospheroid (l(1)mys), have been cloned by chromosomal walking. Mutations of fs(1)h were found within a 13-kb stretch of DNA. Poly(A)+ RNAs migrating as a doublet at 7.6 kb and a single band at 5.9 kb, which are homologous to the fs(1)h+ chromosomal region, are found in ovaries and early embryos. The largest RNAs are derived from a 20-kb chromosomal region encompassing the sites of all mapped fs(1)h alleles.