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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.

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 Ventral veins lacking (Vvl) 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.

In Drosophila, trunk visceral mesoderm, a derivative of dorsal mesoderm, gives rise to circular visceral muscles. It has been demonstrated that the trunk visceral mesoderm parasegment is subdivided into at least two domains by connectin expression, which is regulated by Hedgehog and Wingless emanating from the ectoderm. We now extend these findings by examining a greater number of visceral mesodermal genes, includinghedgehog and branchless. Each visceral mesodermal parasegment appears to be divided into five or six regions, based on differences in expression patterns of these genes. Ectodermal Hedgehog and Wingless differentially regulate the expression of these metameric targets in trunk visceral mesoderm. hedgehog expression in trunk visceral mesoderm is responsible for maintaining its own expression and conexpression. hedgehog expressed in visceral mesoderm parasegment 3 may also be required for normal decapentaplegic expression in this region and normal gastric caecum development. branchless expressed in each trunk visceral mesodermal parasegment serves as a guide for the initial budding of tracheal visceral branches. The metameric pattern of trunk visceral mesoderm, organized in response to ectodermal instructive signals, is thus maintained at a later time via autoregulation, is required for midgut morphogenesis and exerts feedback effect on trachea, ectodermal derivatives.

In contrast to planes, three-dimensional (3D) structures such as tubes are physically anisotropic. Tubular organs exhibit a striking orientation of landmarks according to the physical anisotropy of the 3D shape, in addition to planar cell polarization. However, the influence of 3D tissue topography on the constituting cells remains underexplored. Here, we identify a regulatory network polarizing cellular biochemistry according to the physical anisotropy of the 3D tube geometry (tube cell polarization) by a genome-wide, tissue-specific RNAi screen. During Drosophila airway remodelling, each apical cellular junction is equipotent to establish perpendicular actomyosin cables, irrespective of the longitudinal or transverse tube axis. A dynamic transverse enrichment of atypical protein kinase C (aPKC) shifts the balance and transiently targets activated small GTPase RhoA, myosin phosphorylation and Rab11 vesicle trafficking to longitudinal junctions. We propose that the PAR complex translates tube physical anisotropy into longitudinal junctional anisotropy, where cell-cell communication aligns the contractile cytoskeleton of neighbouring cells.

Background The structures and functions of promoter sequences of most genes have been analysed using in vitro transcription and/or cultured cell systems, neither possessing tissue-specific enhancers. Promoter–enhancer interactions in vivo, in particular, during ontogeny, are still poorly understood. Results We have established a new method for the assessment of promoter activity in cells that participate in fly body formation, using the UAS/GAL4 system. A functional analysis was then conducted on the promoter sequence of the engrailed gene in Drosophila embryos. A 38-bp-long sequence, terminating with an initiator or RNA start site and a downstream promoter element, was found to be capable of receiving activation signals from the engrailed stripe enhancer. Transcriptional efficiency was improved significantly by the presence of upstream promoting elements, most functionally replaceable with synthetic GAGA factor binding sites. Conclusions We identified the in vivo minimum promoter of engrailed and demonstrated that the GAGA factor binding sites serve primarily as quantitative elements which augment transcriptional efficiency. Evidence was also obtained that indicated that not only enhancer but also promoter sequences were involved in the determination of the tissue-specificity of gene expression.

The tubular networks of the Drosophila respiratory system and our vasculature show distinct branching patterns and tube shapes in different body regions. These local variations are crucial for organ function and organismal fitness. Organotypic patterns and tube geometries in branched networks are typically controlled by variations of extrinsic signaling but the impact of intrinsic factors on branch patterns and shapes is not well explored. Here, we show that the intersection of extrinsic hedgehog(hh) and WNT/wingless (wg) signaling with the tube-intrinsic Hox code of distinct segments specifies the tube pattern and shape of the Drosophila airways. In the cephalic part of the airways, hh signaling induces expression of the transcription factor (TF) knirps (kni) in the anterior dorsal trunk (DTa1). kni represses the expression of another TF spalt major (salm), making DTa1 a narrow and long tube. In DTa branches of more posterior metameres, Bithorax Complex (BX-C) Hox genes autonomously divert hh signaling from inducing kni, thereby allowing DTa branches to develop as salm-dependent thick and short tubes. Moreover, the differential expression of BX-C genes is partly responsible for the anterior-to-posterior gradual increase of the DT tube diameter through regulating the expression level of Salm, a transcriptional target of WNT/wg signaling. Thus, our results highlight how tube intrinsic differential competence can diversify tube morphology without changing availabilities of extrinsic factors., Author Summary Tubes are common structural elements of many internal organs, facilitating fluid flow and material exchange. To meet the local needs of diverse tissues, the branching patterns and tube shapes vary regionally. Diametric tapering and specialized branch targeting to the brain represent two common examples of variations with organismal benefits in the Drosophila airways and our vascular system. Several extrinsic signals instruct tube diversifications but the impact of intrinsic factors remains underexplored. Here, we show that the local, tube-intrinsic Hox code instructs the pattern and shape of the dorsal trunk (DT), the main Drosophila airway. In the cephalic part (DT1), where Bithorax Complex (BX-C) Hox genes are not expressed, the extrinsic Hedgehog signal is epistatic to WNT/Wingless signals. Hedgehog instructs anterior DT1 cells to take a long and narrow tube fate targeting the brain. In more posterior metameres, BX-C genes make the extrinsic WNT/Wingless signals epistatic over Hedgehog. There, WNT/Wingless instruct all DT cells to take the thick and short tube fate. Moreover, BX-C genes modulate the outputs of WNT/wingless signaling, making the DT tubes thicker in more posterior metameres. We provide a model for how intrinsic factors modify extrinsic signaling to control regional tube morphologies in a network.