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We have screened a collection of transposable-element-induced mutations for those which dominantly modify the extra R7 phenotype of a hypomorphic yan mutation. The members of one of the identified complementation groups correspond to disruptions of the tramtrack (ttk) gene. As heterozygotes, ttk alleles increase the percentage of R7 cells in yan mutant eyes. Just as yan mutations increase ectopic R7 cell formation, homozygous ttk mutant eye clones also contain supernumerary R7 cells. However, in contrast to yan, the formation of these cells in ttk mutant eye tissue is not necessarily dependent on the activity of the sina gene. Furthermore, although yan mutations dominantly interact with mutations in the Ras1, Draf, Dsor1, and rolled (rl) genes to influence R7 cell development, ttk mutations only interact with yan and rl gene mutations to affect this signaling pathway. Our data suggest that yan and ttk both function to repress inappropriate R7 cell development but that their mechanisms of action differ. In particular, TTK activity appears to be autonomously required to regulate a sina-independent mechanism of R7 determination.

phyllopod (phyl) encodes a novel protein required for fate determination of photoreceptors R1, R6, and R7, the last three photoreceptors to be recruited into the ommatidia of the developing Drosophila eye. Genetic data suggests that phyl acts downstream of Ras1, raf, and yan to promote neuronal differentiation in this subset of photoreceptors. Ectopic expression of phyl in the cone cell precursors mimics the effect of ectopic activation of Ras1, suggesting that phyl expression is regulated by Ras1. phyl is also required for embryonic nervous system and sensory bristle development.

During Drosophila third instar larval development, one or more pulses of the steroid hormone ecdysone activate three temporally distinct sets of genes in the salivary glands, represented by puffs in the polytene chromosomes. The intermolt genes are induced first, in midthird instar larvae; these genes encode a protein glue used by the animal to adhere itself to a solid substrate for metamorphosis. The intermolt genes are repressed at puparium formation as a high titer ecdysone pulse directly induces a small set of early regulatory genes. The early genes both repress their own expression and activate more than 100 late secondary-response genes. The Broad-Complex (BR-C) is an early ecdysone-inducible gene that encodes a family of DNA binding proteins defined by at least three lethal complementation groups: br, rbp, and l(1)2Bc. We have found that the BR-C is critical for the appropriate regulation of all three classes of ecdysone-inducible genes. Both rbp and l(1)2Bc are required for glue gene induction in mid-third instar larvae. In addition, the l(1)2Bc function is required for glue gene repression in prepupae; in l(1)2Bc mutants the glue genes are re-induced by the late prepupal ecdysone pulse, recapitulating a mid-third instar regulatory response at an inappropriate stage in development. The l(1)2Bc function is also required for the complete ecdysone induction of some early mRNAs (E74A, E75A, and BR-C) and efficient repression of most early mRNAs in prepupae. Like the intermolt secondary-response genes, the late secondary-response genes are absolutely dependent on rbp for their induction. An effect of l(1)2Bc mutations on late gene activity can also be detected, but is most likely a secondary consequence of the submaximal ecdysone-induction of a subset of early regulatory products. Our results indicate that the BR-C plays a key role in dictating the stage-specificity of the ecdysone response. In addition, the ecdysone-receptor protein complex alone is not sufficient for appropriate induction of the early primary-response genes, but requires the prior expression of BR-C proteins. These studies define the BR-C as a key regulator of gene activity at the onset of metamorphosis in Drosophila.

Pulses of the steroid hormone ecdysone function as temporal signals to coordinate the development of both larval and adult tissues in Drosophila. Ecdysone acts by triggering a genetic regulatory hierarchy that can be visualized as puffs in the larval polytene chromosomes. In an effort to understand how the ecdysone signal is transduced to result in sequential gene activation, we are studying the transcriptional control of E74, an early gene that appears to play a regulatory role in the hierarchy. Northern blot analysis of RNA isolated from staged animals or cultured organs was used to characterize the effects of ecdysone on E74 transcription. Ecdysone directly activates both E74A and E74B promoters. E74B mRNA precedes that of E74A, each mRNA appearing with delay times that agree with their primary transcript lengths and our previous transcription elongation rate measurement of approximately 1.1 kb/min. The earlier appearance of E74B transcripts is enhanced by its activation at an approximately 25-fold lower ecdysone concentration than E74A. E74B is further distinguished from E74A by its repression at a significantly higher ecdysone concentration than that required for its induction, close to the concentration required for E74A activation. These regulatory properties lead to an ecdysone-induced switch in E74 expression, with an initial burst of E74B transcription followed by a burst of E74A transcription. We also show that the patterns of ecdysone-induced E74A and E74B transcription vary in four ecdysone target tissues. These studies provide a means to translate the profile of a hormone pulse into different amounts and times of regulatory gene expression that, in turn, could direct different developmental responses in a temporally and spatially regulated manner.

The Sevenless receptor tyrosine kinase is required for the development of the R7 photoreceptor cell in the Drosophila eye. Several components of the Sevenless signal transduction pathway have been identified in genetic screens for enhancers/suppressors of the sevenless phenotype. These studies suggest that activation of Sevenless leads to stimulation of Ras1 activity, whereas Gap1 appears to act as a negative regulator of the pathway. Inactivation of the Gap1 locus causes transformation of non-neuronal cone cells into supernumerary R7 cells. This same mutant phenotype is observed when activated Ras1 is expressed under the control of the sevenless promoter. While studies in other organisms have demonstrated a role for ras gene products in signal transduction, the effectors of Ras activity have not yet been identified. We are carrying out genetic screens for enhancers and suppressors of the Gap1 and activated Ras1 phenotypes in the hope of identifying genes encoding some of these effectors. We are conducting chemical mutagenesis screens and have also screened existing collections of P element lines. A molecular characterization of the most promising mutations is in progress.

Protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine phosphatase present in most tissues and cell types, has been implicated in the regulation of cell cycle progression, DNA replication, transcription, and translation. Here we present genetic evidence suggesting that PP2A functions downstream of Ras1 in the Sevenless receptor tyrosine kinase (RTK) signal transduction pathway that specifies R7 photoreceptor cell fate in the developing Drosophila eye. Ras1 and downstream cytoplasmic kinases, Raf, MEK, and MAPK, comprise an evolutionarily conserved cascade that mediates the transmission of signals from RTKs at the plasma membrane to specific factors in the nucleus. Using transgenic flies expressing constitutively activated Ras1 or Raf proteins that function independently of upstream signaling events, we show that a reduction in the dose of the gene encoding the catalytic subunit of PP2A stimulates signaling from Ras1 but impairs signaling from Raf. This suggests that PP2A both negatively and positively regulates the Ras1 cascade by dephosphorylating factors that function at different steps in the cascade.

FTZ-F1, a member of the steroid receptor superfamily, has been implicated in the activation of the homeobox segmentation gene fushi tarazu early in Drosophila embryogenesis. We have cloned a developmental isoform of FTZ-F1 and found that it is expressed as a product of the previously identified, midprepupal chromosome puff at 75CD. The 75CD puff occurs in the midst of a period of intense puffing activity that is triggered in response to the steroid hormone ecdysone at the onset of metamorphosis. Indirect immunofluorescent staining for FTZ-F1 on Drosophila polytene chromosomes reveals binding to over 150 chromosomal targets, which include 75CD itself and prominent late prepupal puffs that are predicted to be regulated by midprepupal puff proteins. These results suggest a role for FTZ-F1 as a regulator of insect metamorphosis and underscore the repeated utilization of a regulatory protein for widely separate developmental pathways.

The recent determination of the site-specific DNA binding properties of several proteins related to the ets oncoprotein has allowed the definition of a novel DNA binding domain, designated the ETS domain. In Drosophila, an ETS domain is present in the early ecdysone-induced E74A protein, which binds DNA in a site-specific manner and interacts with many ecdysone-induced polytene chromosome puffs at the onset of metamorphosis. As a first step toward determining the function of ETS-domain proteins during Drosophila development, we have used PCR amplification with degenerate oligonucleotides to isolate five other ets-related genes. Two of these genes, D-ets-2 and D-elg, have been previously identified. The proteins encoded by these genes are highly related to one another and to the seven identified vertebrate ETS-domain proteins, within the approximately 85-amino-acid DNA binding domain. In situ hybridization to polytene chromosomes revealed that these ets-related genes are not clustered in the genome and that only E74 corresponds to an ecdysone-inducible puff locus. These five ets-related genes are distinguished further from E74 in that they are transcribed through most of development, suggesting that they do not perform a stage-specific function. They are, however, expressed in a variety of patterns in early embryos, suggesting roles in the development of specific cell types. D-ets-2 is expressed in a complex pattern that changes dynamically during early embryogenesis. D-ets-3 and D-ets-6 are expressed in the ventral nervous system. The expression of D-ets-3 is higher in the three thoracic segments and lower in the abdominal segments. The high levels of expression in the thoracic segments are dependent on the presence of the bithorax complex. D-ets-4 and D-elg are expressed at their highest levels in the pole cells, suggesting a role in the development of the germline. This study represents the first effort in any organism to systematically isolate members of the ets gene family. The identification of six independent ets-related genes demonstrates that the ETS-domain proteins constitute a new family of potential transcriptional regulators encoded by the Drosophila genome.

We have isolated an ecdysone-inducible gene, E74 , from the early puff at position 74EF in the Drosophila polytene chromosomes. We show that E74 consists of three nested transcription units that derive from unique promoters but share a single polyadenylation site. The 60 kb E74A unit is directly induced by ecdysone and leads to the synthesis of a 6.0 kb mRNA that contains an unusually long 5′ leader (1891 nucleotides) with 17 short ORFs. Within the fifth of the seven E74A introns are two E74B promoters that direct the synthesis of 4.8 and 5.1 kb mRNAs. The nested arrangement of these transcription units leads to the formation of two E74 proteins, each with a unique N-terminal domain joined to a common C-terminal domain. The unique N-terminal domains contain regions rich in acidic amino acids while the C-terminal domain is rich in basic amino acids and is very similar to proteins encoded by the ets proto-oncogene superfamily.

Cell-fate specification of the R7 photoreceptor cell is controlled by the sevenless receptor tyrosine kinase (SevRTK) and Ras1, the Drosophila homologue of mammalian H-ras, K-ras and N-ras oncogenes. An activated form of Ras1 expressed under control of the sevenless enhancer/promoter (sev-Ras1v12) induces production of supernumerary R7 photoreceptor cells, which causes the eye to become rough in appearance. To isolate mutations in genes functioning downstream of Ras1, we carried out a screen for dominant suppressors and enhancers of this rough eye phenotype. Approximately 850,000 mutagenized flies were screened, and 282 dominant suppressors and 577 dominant enhancers were isolated. Mutations in the Drosophila homologues of Raf, MEK, MAPK, type I Geranylgeranyl Transferase and Protein Phosphatase 2A were isolated, as were mutations in several novel signaling genes. Some of these mutant genes appear to be general signaling factors that function in other Ras1 pathways, while one seems to be more specific for photoreceptor development. At least two suppressors appear to function either between Ras1 and Raf or in parallel to Raf.