Search

Photoreceptors in the crystalline Drosophila eye are recruited by receptor tyrosine kinase (RTK)/Ras signaling, mediated by the Epidermal Growth Factor receptor (EGFR) and Sevenless receptor. Analyses of an allelic deletion series of the mir-279/996 locus, along with a panel of modified genomic rescue transgenes, show that normal 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 de-represses 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 substantially requires miRNA control.

Grainy head (Grh) is a conserved transcription factor (TF) controlling epithelial differentiation and regeneration. To elucidate Grh functions, we identified embryonic Grh targets by ChIP-seq and gene expression analysis. We show that Grh controls hundreds of target genes. Repression or activation correlates with the distance of Grh binding sites to the transcription start sites of its targets. Analysis of 54 Grh-responsive enhancers during development and upon wounding suggests cooperation with distinct TFs in different contexts. In the airways, Grh repressed genes encode key TFs involved in branching and cell differentiation. Reduction of the POU-domain TF, Vvl, (ventral veins lacking) largely ameliorates the airway morphogenesis defects of grh mutants. Vvl and Grh proteins additionally interact with each other and regulate a set of common enhancers during epithelial morphogenesis. We conclude that Grh and Vvl participate in a regulatory network controlling epithelial maturation.

MicroRNAs (miRNAs) are regulators of global gene expression and function in a broad range of biological processes. Recent studies have suggested that miRNAs can function as tumor suppressors or oncogenes by modulating the activities of evolutionarily conserved signaling pathways that are commonly dysregulated in cancer. We report the identification of the miR-310 to miR-313 (miR-310/13) cluster as a novel antagonist of Wingless (Drosophila Wnt) pathway activity in a functional screen for Drosophila miRNAs. We demonstrate that miR-310/13 can modulate Armadillo (Arm; Drosophila β-catenin) expression and activity by directly targeting the 3′-UTRs of arm and pangolin (Drosophila TCF) in vivo. Notably, the miR-310/13-deficient flies exhibit abnormal germ and somatic cell differentiation in the male gonad, which can be rescued by reducing Arm protein levels or activity. Our results implicate a previously unrecognized function for miR-310/13 in dampening the activity of Arm in early somatic and germline progenitor cells, whereby inappropriate/sustained activation of Arm-mediated signaling or cell adhesion may impact normal differentiation in the Drosophila male gonad.

The activity of the Notch pathway revolves around a CSL-class transcription factor, which recruits distinct complexes that activate or repress target gene expression. The co-activator complex is deeply conserved and includes the cleaved Notch intracellular domain (NICD) and Mastermind. By contrast, numerous CSL co-repressor proteins have been identified, and these are mostly different between invertebrate and vertebrate systems. In this study, we demonstrate that mammalian BEND6 is a neural BEN-solo factor that shares many functional attributes with Drosophila Insensitive, a co-repressor for the Drosophila CSL factor. BEND6 binds the mammalian CSL protein CBF1 and antagonizes Notch-dependent target activation. In addition, its association with Notch- and CBF1-regulated enhancers is promoted by CBF1 and antagonized by activated Notch. In utero electroporation experiments showed that ectopic BEND6 inhibited Notch-mediated self-renewal of neocortical neural stem cells and promoted neurogenesis. Conversely, knockdown of BEND6 increased NSC self-renewal in wild-type neocortex, and exhibited genetic interactions with gain and loss of Notch pathway activity. We recapitulated all of these findings in cultured neurospheres, in which overexpression and depletion of BEND6 caused reciprocal effects on neural stem cell renewal and neurogenesis. These data reveal a novel mammalian CSL co-repressor in the nervous system, and show that the Notch-inhibitory activity of certain BEN-solo proteins is conserved between flies and mammals.

Drosophila Elav is the founding member of the conserved family of Hu RNA binding proteins (RBPs), which collectively play critical and diverse roles in post-transcriptional regulation. In particular, Elav has for >20 years served as the canonical neuronal marker, owing to the availability of specific monoclonal antibodies. Surprisingly, although Elav has a well-characterized neural cis-regulatory module, we find endogenous Elav is also ubiquitously transcribed and post-transcriptionally repressed in non-neural settings. In particular, mutant clones of multiple miRNA pathway components derepress ubiquitous Elav protein. Our re-annotation of the elav transcription unit shows that not only does it generate extended 3' UTR isoforms, its universal 3' UTR isoform is much longer than previously believed. This longer common 3' UTR region includes multiple conserved, high-affinity sites for the miR-279/996 family. Notably, out of several miRNA mutants tested, we find that endogenous Elav and a transgenic elav 3' UTR sensor are derepressed in mutant clones of mir-279/996. We also observe cross-repression of Elav by another RBP derepressed in non-neural miRNA pathway clones, namely Mei-P26. Finally, we demonstrate that ubiquitous Elav has regulatory capacity, since derepressed Elav can stabilize an Elav-responsive sensor. It is critical to restrict Elav outside of the nervous system as misexpression of Elav in non-neural territories has profoundly adverse consequences. Altogether, we define unexpected post-transcriptional mechanisms that direct appropriate cell-type specific expression of a conserved neural RBP.

Notch is a transmembrane receptor that mediates local cell-cell communication and coordinates a signaling cascade present in all animal species studied to date. Notch signaling is used widely to determine cell fates and to regulate pattern formation; its dysfunction results in a tremendous variety of developmental defects and adult pathologies. This primer describes the mechanism of Notch signal transduction and how it is used to control the formation of biological patterns.

Drosophila Elav is the founding member of the conserved family of Hu RNA-binding proteins (RBPs), which play crucial and diverse roles in post-transcriptional regulation. Elav has long served as the canonical neuronal marker. Surprisingly, although Elav has a well-characterized neural cis-regulatory module, we find endogenous Elav is also ubiquitously transcribed and post-transcriptionally repressed in non-neural settings. Mutant clones of multiple miRNA pathway components derepress ubiquitous Elav protein. Our re-annotation of the elav transcription unit shows not only that it generates extended 3′ UTR isoforms, but also that its universal 3′ UTR isoform is much longer than previously believed. This longer common 3′ UTR includes multiple conserved, high-affinity sites for the miR-279/996 family. Of several miRNA mutants tested, endogenous Elav and a transgenic elav 3′ UTR sensor are derepressed in mutant clones of mir-279/996. We also observe cross-repression of Elav by Mei-P26, another RBP derepressed in non-neural miRNA pathway clones. Ubiquitous Elav has regulatory capacity, since derepressed Elav can stabilize an Elav-responsive sensor. Repression of Elav in non-neural territories is crucial as misexpression here has profoundly adverse consequences. Altogether, we define unexpected post-transcriptional mechanisms that direct appropriate cell type-specific expression of a conserved neural RBP.

During Drosophila development, transcriptional activation of genes of the Enhancer of split Complex (E(spl)-C) is a major response to cell-cell signaling via the Notch (N) receptor. Although the structure and function of the E(spl)-C have been studied intensively during the past decade, these efforts have focused heavily on seven transcription units that encode basic helix-loop-helix (bHLH) repressors; the non-bHLH members of the complex have received comparatively little attention. In this report, we analyze the structure, regulation and activity of the m1, m2 and m6 genes of the E(spl)-C. We find that E(spl)m2 and E(spl)m6 encode divergent members of the Bearded (Brd) family of proteins, bringing to four (mα, m2, m4 and m6) the number of Brd family genes in the E(spl)-C. We demonstrate that the expression of both m2 and m6 is responsive to N receptor activity and that both genes are apparently direct targets of regulation by the N-activated transcription factor Suppressor of Hairless. Consistent with this, both are expressed specifically in multiple settings where N signaling takes place. Particularly noteworthy is our finding that m6 transcripts accumulate both in adult muscle founder cells in the embryo and in a subset of adepithelial (muscle precursor) cells associated with the wing imaginal disc. We show that overexpression of either m2 or m6 interferes with N-dependent cell fate decisions in adult PNS development. Surprisingly, while misexpression of m6 impairs lateral inhibition, overexpression of m2 potentiates it, suggesting functional diversification within the Brd protein family. Finally, we present our initial studies of the structure, expression and regulation of the newest member of the Brd gene family, Ocho, which is located in the recently identified Bearded Complex.

Cell-cell interactions mediated by the Notch receptor play an essential role in the development of the Drosophila adult peripheral nervous system (PNS). Transcriptional activation of multiple genes of the Enhancer of split Complex [E(spl)-C] is a key intracellular response to Notch receptor activity. Here we report that most E(spl)-C genes contain a novel sequence motif, the K box (TGTGAT), in their 3′ untranslated regions (3′ UTRs). We present three lines of evidence that demonstrate the importance of this element in the post-transcriptional regulation of E(spl)-C genes. First, K box sequences are specifically conserved in the orthologs of two structurally distinct E(spl)-C genes (m4 and m8) from a distantly related Drosophila species. Second, the wild-type m8 3′ UTR strongly reduces accumulation of heterologous transcripts in vivo, an activity that requires its K box sequences. Finally, m8 genomic DNA transgenes lacking these motifs cause mild gain-of-function PNS defects and can partially phenocopy the genetic interaction of E(spl)D with Notchspl. Although E(spl)-C genes are expressed in temporally and spatially specific patterns, we find that K box-mediated regulation is ubiquitous, implying that other targets of this activity may exist. In support of this, we present sequence analyses that implicate genes of the iroquois Complex (Iro-C) and engrailed as additional targets of K box-mediated regulation.

Gain-of-function alleles of the Drosophila gene Bearded (Brd) cause sensory organ multiplication and loss pheno-types indistinguishable at the cellular level from those caused by loss-of-function mutations in the genes of the Notch pathway (Leviten, M. W. and Posakony, J. W. (1996). Dev. Biol. 176, 264-283). We have carried out a molecular analysis of the structure and expression of both wild-type and mutant Brd transcription units. We find that the Brd transcript is truncated and accumulates to substantially higher levels in the gain-of-function mutants, due to the insertion of a transposable element of the blood family in the Brd 3′ untranslated region (UTR). The wild-type Brd 3′ UTR includes three copies of a 9-nucleotide sequence (CAGCTTTAA) that we refer to as the ‘Brd box’. Moreover, the 3′ UTRs of Brd and of the m4 transcription unit of the Enhancer of split gene complex [E(spl)-C] exhibit an unusually high degree of sequence identity that includes not only Brd box sequences but also a second motif we refer to as the ‘GY box’ (GTCTTCC). We find that both the Brd box and the GY box are also present in the 3′ UTRs of several basic helix-loop-helix repressor-encoding genes of the E(spl)-C, often in multiple copies, suggesting that a novel mode of post-transcriptional regulation applies to Brd and many E(spl)-C genes. The fact that the more abundant Brd mutant mRNA lacks the GY box and two of the Brd boxes present in wild-type Brd mRNA suggests that either or both of these elements may confer instability on transcripts that contain them. Finally, we find that Brd encodes a novel small protein of only 81 amino acids that is predicted to include a basic amphipathic α-helix. The deduced Brd protein shows sequence similarity to the E(spl)m4 protein, which is likewise expected to include a basic amphipathic α-helix, suggesting that the two proteins have related biochemical functions.