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We have investigated the relationship between the function of the genehindsight(hnt), which is the Drosophila homolog ofRas Responsive Element Binding protein-1(RREB-1), and the EGFR signaling pathway. We report thathntmutant embryos are defective in EGFR signaling dependent processes, namely chordotonal organ recruitment and oenocyte specification. We also show the temperature sensitive hypomorphic allelehntpebbledis enhanced by the hypomorphic MAPK allelerolled(rl1). We find thathntoverexpression results in ectopicDPax2expression within the embryonic peripheral nervous system, and we show that this effect is EGFR-dependent. Finally, we show that the canonical U-shaped embryonic lethal phenotype ofhnt,which is associated with premature degeneration of the extraembyonic amnioserosa and a failure in germ band retraction, is rescued by expression of several components of the EGFR signaling pathway (sSpi,Ras85DV12,pntP1) as well as the caspase inhibitorp35. Based on this collection of corroborating evidence, we suggest that an overarching function ofhntinvolves the positive regulation of EGFR signaling.

Germline cells segregate from the soma to maintain their totipotency, but the cellular mechanisms of this segregation are unclear. The Drosophila melanogaster embryo forms a posterior group of primordial germline cells (PGCs) by their division from the syncytial soma. Extended plasma membrane furrows enclose the PGCs in response to the germ plasm protein Germ cell-less (Gcl) and Rho1–actomyosin activity. Recently, we found that loss of the Arf-GEF Steppke (Step) leads to similar Rho1-dependent plasma membrane extensions but from pseudocleavage furrows of the soma. Here, we report that the loss of step also leads to premature formation of a large cell group at the anterior pole of the embryo . These anterior cells lacked germ plasm, but budded and formed at the same time as posterior PGCs, and then divided asynchronously as PGCs also do. With genetic analyses we found that Step normally activates Arf small G proteins and antagonizes Rho1–actomyosin pathways to inhibit anterior cell formation. A uniform distribution of step mRNA around the one-cell embryo cortex suggested that Step restricts cell formation through a global control mechanism. Thus, we examined the effect of Step on PGC formation at the posterior pole. Reducing Gcl or Rho1 levels decreased PGC numbers, but additional step RNAi restored their numbers. Reciprocally, GFP–Step overexpression induced dosage- and Arf-GEF-dependent loss of PGCs, an effect worsened by reducing Gcl or actomyosin pathway activity. We propose that a global distribution of Step normally sets an inhibitory threshold for Rho1 activity to restrict early cell formation to the posterior.

Previous work has shown that many of the 18 family members of Drosophila nuclear receptor transcription factors function in a temporal hierarchy to coordinate developmental progression and growth with the rate limiting process of metabolism. To gain further insight into these interactions and processes, we have undertaken a whole-family analysis of nuclear receptor mRNA spatial expression patterns over the entire process of embryogenesis, as well as the 3rd instar wandering larva stage, by using high-resolution fluorescence in situ hybridization. Overall, the patterns of expression are remarkably consistent with previously mapped spatial activity profiles documented during the same time points, with similar hot spots and temporal profiles in endocrine and metabolically important tissues. Among the more remarkable of the findings is that the majority of mRNA expression patterns observed show striking subcellular distributions, indicating potentially critical roles in the control of protein synthesis and subsequent subcellular distributions. These patterns will serve as a useful reference for future studies on the tissue-specific roles and interactions of nuclear receptor proteins, partners, cofactors and ligands.

The early promoter of bacteriophage Mu has been identified and characterized by a nitrocellulose filter binding assay and analysis of RNA products transcribed in vitro and in vivo. A tight, heparin resistant, RNA polymerase-DNA binding site overlaps the left Mu Hind III site. In vitro [gamma 32P] ATP initiated transcription begins approximately 25 nucleotides to the right of the Hind III restriction site. Dinucleotide initiation with ApA and S1 mapping of in vitro and in vivo transcripts shows that transcription is initiated 1028 base pairs from the Mu genetic left end.