Drosophila distal-less and rotund bind a single enhancer ensuring reliable and robust bric-a-brac2 expression in distinct limb morphogenetic fields

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.Most identified Drosophila appendage-patterning genes encode DNA-binding proteins, whose cross-regulatory interactions remain to be better characterized at the molecular level, notably by studying their direct binding to tissue-specific transcriptional enhancers. A fine-tuned spatio-temporal expression of bric-a-brac2 (bab2) along concentric rings is essential for proper proximo-distal (P-D) differentiation of legs and antennae. However, within the genetic interaction landscape governing limb development, no transcription factor directly controlling bab2 expression has been identified to date. Using site-targeted GFP reporter assay and BAC recombineering, we show here that restricted bab2 expression in leg and antennal imaginal discs relies on a single 567-bp-long cis-regulatory module (CRM), termed LAE (for leg and antennal enhancer). We show that this CRM (i) is necessary and sufficient to ensure normal bab2 activity in developing leg and antenna, and (ii) is structurally and functionally conserved among Drosophilidae. Through deletion and site-directed mutagenesis approaches, we identified within the LAE essential sequence motifs required in both leg and antennal tissues. Using genetic and biochemical tests, we establish that in the LAE (i) a key TAAT-rich activator motif interacts with the homeodomain P-D protein Distal-less (Dll) and (ii) a single T-rich activator motif binds the C2H2 zinc-finger P-D protein Rotund (Rn), leading to bab2 up-regulation respectively in all or specifically in the proximal-most ring(s), both in leg and antenna. Joint ectopic expression of Dll and Rn is sufficient to cell-autonomously activate endogenous bab2 and LAE-driven reporter expression in wing and haltere cells. Our findings indicate that accuracy, reliability and robustness of developmental gene expression do not necessarily require cis-regulatory information redundancy. © 2013 Baanannou et al.This work was supported by grants from the French governmental agency for Research (CNRS), the Paul Sabatier University (UPS) and the “Association pour la Recherche sur le Cancer” (ARC). AB was supported by the CNRS and the Midi Pyrenees Region. LHM-V was supported by the Mexican CONACYT.Peer Reviewe

Étude de la régulation transcriptionnelle des gènes bab au cours du développement des appendices chez la drosophile

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Rôles distincts des sous-unités du module CDK8 du complexe médiateur de la transcription au cours du développement de la drosophile

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Specific tracheal migration is mediated by complementary expression of cell surface proteins

Migration of the Drosophila tracheal cells relies on cues provided by nearby cells; however, little is known about how these signals specify a migratory path. Here we investigate the role of cell surface proteins in the definition of such a pathway. We have found that the PS1 integrin is required in the tracheal cells of the visceral branch, whereas the PS2 integrin is required in the visceral mesoderm; both integrins are necessary for the spreading of the visceral branch over its substratum. This is the first identification of a cell surface molecule with expression restricted to a subset of tracheal cells that all migrate in a given direction. We have also found that expression of PS1 in the visceral branch is regulated by the genes that direct tracheal cell migration, showing that integrin expression is part of the cell-fate program that they specify. These results support a model in which signal transduction determines the tracheal migratory pathways by regulating the expression of cell surface proteins, which in turn interact with surface molecules on the surrounding cell population

Inducible degradation of the Drosophila Mediator subunit Med19 reveals its role in regulating developmental but not constitutively-expressed genes

The multi-subunit Mediator complex plays a critical role in gene expression by bridging enhancer-bound transcription factors and the RNA polymerase II machinery. Although experimental case studies suggest differential roles of Mediator subunits, a comprehensive view of the specific set of genes regulated by individual subunits in a developing tissue is still missing. Here we address this fundamental question by focusing on the Med19 subunit and using the Drosophila wing imaginal disc as a developmental model. By coupling auxin-inducible degradation of endogenous Med19 in vivo with RNA-seq, we got access to the early consequences of Med19 elimination on gene expression. Differential gene expression analysis reveals that Med19 is not globally required for mRNA transcription but specifically regulates positively or negatively less than a quarter of the expressed genes. By crossing our transcriptomic data with those of Drosophila gene expression profile database, we found that Med19-dependent genes are highly enriched with spatially-regulated genes while the expression of most constitutively expressed genes is not affected upon Med19 loss. Whereas globally downregulation does not exceed upregulation, we identified a functional class of genes encoding spatially-regulated transcription factors, and more generally developmental regulators, responding unidirectionally to Med19 loss with an expression collapse. Moreover, we show in vivo that the Notch-responsive wingless and the E(spl)-C genes require Med19 for their expression. Combined with experimental evidences suggesting that Med19 could function as a direct transcriptional effector of Notch signaling, our data support a model in which Med19 plays a critical role in the transcriptional activation of developmental genes in response to cell signaling pathways

Mercury chloride exposure induces DNA damage, reduces fertility, and alters somatic and germline cells in Drosophila melanogaster ovaries

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Scheme depicting the CRISPR/Cas9-based strategy used to generate <i>Med19^AID</i>, a <i>Med19</i> degradable and fluorescent allele.

The donner construct (middle) highlights the nature of the homology arms used to introduce the AID-GFP cassette by homologous recombination. Position of the PCR primers used to characterize the wt vs 19AID allele is shown. (TIF)</p

Degradation of Med19AID in the posterior compartment of the wing imaginal disc using the <i>engrailed</i> driver (<i>en</i>>).

(A) Confocal images of wing discs dissected from en>TIR1 Med19AID larvae either fed for one hour with NAA (1H), or mock treated (no auxin). (B) Box plot showing the Med19AID depletion level calculated as the posterior to anterior GFP intensity ratio in the wing pouch of en>TIR1; Med19AID wing imaginal discs (n = 5) obtained and imaged as in (A). (TIF)</p

List of canonical housekeeping genes.

(TIF)</p