Joshua Kavaler

Frequent Unanticipated Alleles of lethal giant larvae in Drosophila Second Chromosome Stocks

Forty years ago, a high frequency of lethal giant larvae (lgl) alleles in wild populations of Drosophila melanogaster was reported. This locus has been intensively studied for its roles in epithelial polarity, asymmetric neural divisions, and restriction of tissue proliferation. Here, we identify a high frequency of lgl alleles in the Bloomington second chromosome deficiency kit and the University of California at Los Angeles Bruinfly FRT40A-lethal P collection. These unrecognized aberrations confound the use of these workhorse collections for phenotypic screening or genetic mapping. In addition, we determined that independent alleles of insensitive, reported to affect asymmetric cell divisions during sensory organ development, carry lgl deletions that are responsible for the observed phenotypes. Taken together, these results encourage the routine testing of second chromosome stocks for second-site alleles of lgl.

Insensitive is a corepressor for Suppressor of Hairless and regulates Notch signalling during neural development

The Notch intracellular domain functions as a co‐activator for the DNA‐binding protein Suppressor of Hairless (Su(H)) to mediate myriad cell fate decisions. Notch pathway activity is balanced by transcriptional repression, mediated by Su(H) in concert with its Drosophila corepressor Hairless. We demonstrate that the Drosophila neural BEN‐solo protein Insensitive (Insv) is a nuclear factor that inhibits Notch signalling during multiple peripheral nervous system cell fate decisions. Endogenous Insv was particularly critical when repressor activity of Su(H) was compromised. Reciprocally, ectopic Insv generated several Notch loss‐of‐function phenotypes, repressed most Notch targets in the E(spl)‐C, and opposed Notch‐mediated activation of an E(spl)m3‐luc reporter. A direct role for Insv in transcriptional repression was indicated by binding of Insv to Su(H), and by strong chromatin immunoprecipitation of endogenous Insv to most E(spl)‐C loci. Strikingly, ectopic Insv fully rescued sensory organ precursors in Hairless null clones, indicating that Insv can antagonize Notch independently of Hairless. These data shed first light on the in vivo function for a BEN‐solo protein as an Su(H) corepressor in the Notch pathway regulating neural development.

The transcription factor D-Pax2 regulates crystallin production during eye development in Drosophila melanogaster

The generation of a functioning Drosophila eye requires the coordinated differentiation of multiple cell types and the morphogenesis of eye‐specific structures. Here we show that D‐Pax2 plays a significant role in lens development through regulation of the Crystallin gene and because Crystallin is also expressed in D‐Pax2+ cells in the external sensory organs. Loss of D‐Pax2 function leads to loss of Crystallin expression in both eyes and bristles. A 2.3 kilobase (kb) upstream region of the Crystallin gene can drive GFP expression in the eye and is dependent on D‐Pax2. In addition, D‐Pax2 binds to an evolutionarily conserved site in this region that, by itself, is sufficient to drive GFP expression in the eye. However, mutation of this site does not greatly affect the regulatory regions function. The data indicate that D‐Pax2 acts to promote lens development by controlling the production of the major protein component of the lens. Whether this control is direct or indirect remains unresolved. Developmental Dynamics 238:2530–2539, 2009.

Discrete regulatory regions control early and late expression of D-Pax2 during external sensory organ development.

The transcription factor D‐Pax2 is required for the correct differentiation of several cell types in Drosophila sensory systems. While the regulation of its expression in the developing eye has been well studied, little is known about the mechanisms by which the dynamic pattern of D‐Pax2 expression in the external sensory organs is achieved. Here we demonstrate that early activation of D‐Pax2 in the sensory organ lineage and its maintenance in the trichogen and thecogen cells are governed by separate enhancers. Furthermore, the initial activation is controlled in part by proneural proteins whereas the later maintenance expression is regulated by a positive feedback loop. Developmental Dynamics 240:1769–1778, 2011.

miRNA suppression of a Notch repressor directs non-neuronal fate in Drosophila mechanosensory organs

Although there is abundant evidence that individual microRNA (miRNA) loci repress large cohorts of targets, large-scale knockout studies suggest that most miRNAs are phenotypically dispensable. Here, we identify a rare case of developmental cell specification that is highly dependent on miRNA control of an individual target. We observe that binary cell fate choice in the Drosophila melanogaster peripheral sensory organ lineage is controlled by the non-neuronally expressed mir-279/996 cluster, with a majority of notum sensory organs exhibiting transformation of sheath cells into ectopic neurons. The mir-279/996 defect phenocopies Notch loss of function during the sheath–neuron cell fate decision, suggesting the miRNAs facilitate Notch signaling. Consistent with this, mir-279/996 knockouts are strongly enhanced by Notch heterozygosity, and activated nuclear Notch is impaired in the miRNA mutant. Although Hairless (H) is the canonical nuclear Notch pathway inhibitor, and H heterozygotes exhibit bristle cell fate phenotypes reflecting gain-of-Notch signaling, H/+ does not rescue mir-279/996 mutants. Instead, we identify Insensible (Insb), another neural nuclear Notch pathway inhibitor, as a critical direct miR-279/996 target. Insb is posttranscriptionally restricted to neurons by these miRNAs, and its heterozygosity strongly suppresses ectopic peripheral nervous system neurons in mir-279/996 mutants. Thus, proper assembly of multicellular mechanosensory organs requires a double-negative circuit involving miRNA-mediated suppression of a Notch repressor to assign non-neuronal cell fate.

The mir-279/996 cluster represses receptor tyrosine kinase signaling to determine cell fates in the Drosophila eye

ABSTRACT Photoreceptors in the crystalline Drosophila eye are recruited by receptor tyrosine kinase (RTK)/Ras signaling mediated by Epidermal growth factor receptor (EGFR) and the Sevenless (Sev) receptor. Analyses of an allelic deletion series of the mir-279/996 locus, along with a panel of modified genomic rescue transgenes, show that Drosophila eye patterning depends on both miRNAs. Transcriptional reporter and activity sensor transgenes reveal expression and function of miR-279/996 in non-neural cells of the developing eye. Moreover, mir-279/996 mutants exhibit substantial numbers of ectopic photoreceptors, particularly of R7, and cone cell loss. These miRNAs restrict RTK signaling in the eye, since mir-279/996 nulls are dominantly suppressed by positive components of the EGFR pathway and enhanced by heterozygosity for an EGFR repressor. miR-279/996 limit photoreceptor recruitment by targeting multiple positive RTK/Ras signaling components that promote photoreceptor/R7 specification. Strikingly, deletion of mir-279/996 sufficiently derepresses RTK/Ras signaling so as to rescue a population of R7 cells in R7-specific RTK null mutants boss and sev, which otherwise completely lack this cell fate. Altogether, we reveal a rare setting of developmental cell specification that involves substantial miRNA control. Summary: miR-279/996 are deployed in non-neuronal cells of the developing Drosophila eye to restrict RTK/Ras signaling and directly target the rho, ru and boss 3′ UTRs, with miR-279/996 loss yielding ectopic R7 photoreceptors and cone cell depletion.