Shavenbaby Couples Patterning to Epidermal Cell Shape Control

It is well established that developmental programs act during embryogenesis to determine animal morphogenesis. How these developmental cues produce specific cell shape during morphogenesis, however, has remained elusive. We addressed this question by studying the morphological differentiation of the Drosophila epidermis, governed by a well-known circuit of regulators leading to a stereotyped pattern of smooth cells and cells forming actin-rich extensions (trichomes). It was shown that the transcription factor Shavenbaby plays a pivotal role in the formation of trichomes and underlies all examined cases of the evolutionary diversification of their pattern. To gain insight into the mechanisms of morphological differentiation, we sought to identify shavenbaby's downstream targets. We show here that Shavenbaby controls epidermal cell shape, through the transcriptional activation of different classes of cellular effectors, directly contributing to the organization of actin filaments, regulation of the extracellular matrix, and modification of the cuticle. Individual inactivation of shavenbaby's targets produces distinct trichome defects and only their simultaneous inactivation prevent trichome formation. Our data show that shavenbaby governs an evolutionarily conserved developmental module consisting of a set of genes collectively responsible for trichome formation, shedding new light on molecular mechanisms acting during morphogenesis and the way they can influence evolution of animal forms

The Hrs/Stam Complex Acts as a Positive and Negative Regulator of RTK Signaling during Drosophila Development

BACKGROUND: Endocytosis is a key regulatory step of diverse signalling pathways, including receptor tyrosine kinase (RTK) signalling. Hrs and Stam constitute the ESCRT-0 complex that controls the initial selection of ubiquitinated proteins, which will subsequently be degraded in lysosomes. It has been well established ex vivo and during Drosophila embryogenesis that Hrs promotes EGFR down regulation. We have recently isolated the first mutations of stam in flies and shown that Stam is required for air sac morphogenesis, a larval respiratory structure whose formation critically depends on finely tuned levels of FGFR activity. This suggest that Stam, putatively within the ESCRT-0 complex, modulates FGF signalling, a possibility that has not been examined in Drosophila yet. PRINCIPAL FINDINGS: Here, we assessed the role of the Hrs/Stam complex in the regulation of signalling activity during Drosophila development. We show that stam and hrs are required for efficient FGFR signalling in the tracheal system, both during cell migration in the air sac primordium and during the formation of fine cytoplasmic extensions in terminal cells. We find that stam and hrs mutant cells display altered FGFR/Btl localisation, likely contributing to impaired signalling levels. Electron microscopy analyses indicate that endosome maturation is impaired at distinct steps by hrs and stam mutations. These somewhat unexpected results prompted us to further explore the function of stam and hrs in EGFR signalling. We show that while stam and hrs together downregulate EGFR signalling in the embryo, they are required for full activation of EGFR signalling during wing development. CONCLUSIONS/SIGNIFICANCE: Our study shows that the ESCRT-0 complex differentially regulates RTK signalling, either positively or negatively depending on tissues and developmental stages, further highlighting the importance of endocytosis in modulating signalling pathways during development

The Collagen V Homotrimer [α1(V)]3 Production Is Unexpectedly Favored over the Heterotrimer [α1(V)]2α2(V) in Recombinant Expression Systems

Collagen V, a fibrillar collagen with important functions in tissues, assembles into distinct chain associations. The most abundant and ubiquitous molecular form is the heterotrimer [α1(V)]2α2(V). In the attempt to produce high levels of recombinant collagen V heterotrimer for biomedical device uses, and to identify key factors that drive heterotrimeric chain association, several cell expression systems (yeast, insect, and mammalian cells) have been assayed by cotransfecting the human proα1(V) and proα2(V) chain cDNAs. Suprisingly, in all recombinant expression systems, the formation of [α1(V)]3 homotrimers was considerably favored over the heterotrimer. In addition, pepsin-sensitive proα2(V) chains were found in HEK-293 cell media indicating that these cells lack quality control proteins preventing collagen monomer secretion. Additional transfection with Hsp47 cDNA, encoding the collagen-specific chaperone Hsp47, did not increase heterotrimer production. Double immunofluorescence with antibodies against collagen V α-chains showed that, contrary to fibroblasts, collagen V α-chains did not colocalized intracellularly in transfected cells. Monensin treatment had no effect on the heterotrimer production. The heterotrimer production seems to require specific machinery proteins, which are not endogenously expressed in the expression systems. The different constructs and transfected cells we have generated represent useful tools to further investigate the mechanisms of collagen trimer assembly

Le syndrome d’Ehlers-Danlos: l’architecture matricielle en question

Le syndrome d’Ehlers-Danlos constitue un groupe hétérogène de maladies génétiques du tissu conjonctif. Il est caractérisé par une peau hyper-extensible, des articulations anormalement mobiles et des vaisseaux fragiles. Les anomalies moléculaires responsables de cette maladie portent souvent sur les collagènes et les enzymes assurant leur maturation. La forme classique du syndrome, qui sera principalement discutée dans cet article, est majoritairement due à des mutations du collagène V, un collagène fibrillaire présent en petite quantité dans les tissus affectés. Cependant, des anomalies moléculaires du collagène I ou de la ténascine peuvent aussi être responsables de ce syndrome. De plus, chez la souris, l’invalidation de gènes codant pour d’autres molécules matricielles (SPARC, thrombospondine, petits protéoglycanes riches en leucine) conduit à des phénotypes mimant ce syndrome et suggère que ces molécules pourraient donc être impliquées. Comme les anomalies du collagène V restent à ce jour principalement responsables de cette affection, nous discuterons son rôle physiologique à la lumière des observations cliniques et fondamentales. Nous tenterons de comprendre comment le collagène V interagit avec les autres molécules pour déterminer les caractéristiques tissulaires.Ehlers-Danlos syndrome (EDS) is a heterogeneous heritable connective tissue disorder characterized by hyperextensible skin, hypermobile joints and fragile vessels. The molecular causes of this disorder are often, although not strictly, related to collagens and to the enzymes that process these proteins. The classical form of the syndrome, which will be principally discussed in this review, can be due to mutations on collagen V, a fibrillar collagen present in small amounts in affected tissues. However, collagen I and tenascin have also been demonstrated to be involved in the same type of EDS. Moreover gene disruption of several other matrix molecules (thrombospondin, SPARC, small leucine rich proteoglycans...) in mice, lead to phenotypes that mimic EDS and these molecules have thus emerged as new players. As collagen V remains the prime candidate, we discuss, based on fundamental and clinical observations, its physiological role. We also explore its potential interactions with other matrix molecules to determine tissue properties

Development of a Functional Skin Matrix Requires Deposition of Collagen V Heterotrimers

Collagen V is a minor component of the heterotypic I/III/V collagen fibrils and the defective product in most cases of classical Ehlers Danlos syndrome (EDS). The present study was undertaken to elucidate the impact of collagen V mutations on skin development, the most severely affected EDS tissues, using mice harboring a targeted deletion of the α2(V) collagen gene (Col5a2). Contrary to the original report, our studies indicate that the Col5a2 deletion (a.k.a. the pN allele) represents a functionally null mutation that affects matrix assembly through a complex sequence of events. First the mutation impairs assembly and/or secretion of the α1(V)(2)α2(V) heterotrimer with the result that the α1(V) homotrimer is the predominant species deposited into the matrix. Second, the α1(V) homotrimer is excluded from incorporation into the heterotypic collagen fibrils and this in turn severely impairs matrix organization. Third, the mutant matrix stimulates a compensatory loop by the α1(V) collagen gene that leads to additional deposition of α1(V) homotrimers. These data therefore underscore the importance of the collagen V heterotrimer in dermal fibrillogenesis. Furthermore, reduced thickness of the basement membranes underlying the epidermis and increased apoptosis of the stromal fibroblasts in pN/pN skin strongly indicate additional roles of collagen V in the development of a functional skin matrix

<i>svb</i> Downstream Targets Act Collectively in the Formation of Both Denticle and Dorsal Hairs

<div><p>(A) Denticle defects resulting from the combinations of individual mutations of <i>sn³, f^36a, m¹, sha¹,</i> and <i>wsp³</i> (views of the ventral region of the A4 segment). Embryos triple mutant for <i>m, sn,</i> and <i>f</i> display an aggravated phenotype with respect to each simple mutant or double mutant. This leads to poorly differentiated denticles, which display an extremely thin tip and a small triangular base. In addition, the lateral spacing of mutant denticles is reduced, a consequence of denticle splitting with two tiny extensions side by side. The combined inactivation of <i>sn, f, m,</i> and <i>wsp</i> further increases the severity of mutant phenotypes, producing very small and highly abnormal denticles. Similarly, in embryos simultaneously lacking <i>sn, f, m,</i> and <i>sha,</i> the few remaining extensions are atrophic, and numerous denticles are replaced by naked cuticle.</p> <p>(B) <i>svb</i> downstream targets are required for dorsal hair formation. The dorsal region of a wild-type abdominal segment displays a stereotyped arrangement of epidermal extensions presenting a specific morphology: a row of large trichomes pointing anteriorly, a stripe of naked cuticle, three rows of extensions of intermediate size, and several rows of thin hairs. The cumulated inactivation of <i>svb</i> targets profoundly impairs dorsal hair formation. Hairs that display the superimposition of single mutant phenotypes are thickened <i>(m)</i>, crooked <i>(sn, f)</i> and split <i>(f, wsp, sha)</i>. In <i>sn³, f^36a, m¹</i> triple mutants, dorsal hairs are severely reduced in size and, in several cases, the formation of dorsal hair is abrogated, leaving abnormal naked regions, as best seen in the first row of trichomes. These phenotypes are even more pronounced following the combination with <i>wsp</i> mutation, and culminate in embryos lacking <i>m, sn, f,</i> and <i>sha,</i> where most dorsal hairs are absent and replaced by naked cuticle. In some cases, atrophic dorsal hairs are seen as duplicated spots, revealing hair splitting as observed in <i>sha</i> and <i>f</i> embryos. All pictures correspond to the A4 segment.</p></div

Control of <i>m</i> Expression by <i>svb</i> in the Embryonic Epidermis

<div><p>(A) Schematic representation of the signaling pathways that control morphological differentiation of the ventral embryonic epidermis, at stage 12 (top) and at the end of embryogenesis (bottom); anterior is to the left. In addition to <i>engrailed (en),</i> posterior cells express <i>Hedgehog (Hh),</i> and <i>patched (ptc)</i> is expressed in a two-cell-wide stripe on each side of the <i>Hh</i>-expressing cells<i>. Hh,</i> together with <i>serrate (Ser),</i> activates <i>rhomboid (Rho)</i> expression in a three-cell wide stripe. The Rhomboid protease activates the ligand of the d-EGF receptor (DER), whose activation triggers the expression of <i>svb,</i> resulting in the formation of six to seven rows of denticles. Wingless, which is expressed in the posterior-most row of anterior cells, diffuses asymmetrically and represses <i>shavenbaby</i> transcription to form naked cuticle.</p> <p>(B) Whole-mount in situ hybridization of <i>svb</i> (top) and <i>m</i> (bottom) mRNA; cuticles are shown in the middle panels. Inactivation of <i>svb</i> prevents the formation of most trichomes and abolishes <i>m</i> epidermal expression. <i>m</i> expression foreshadows the pattern of trichomes in D. melanogaster and <i>D. sechellia</i> larvae. Yellow brackets outline two dorsal segments.</p> <p>(C) Close-up of the cuticle region corresponding to the third (A3) and fourth (A4) abdominal segments (top) and <i>m</i> mRNA distribution (bottom), in wild-type D. melanogaster embryos (left), ptc-Gal4-driven expression of UAS-OvoA (middle) and expression of UAS-<i>svb</i> under the control of wg-Gal4 (right). Whereas in wild type, <i>m</i> is expressed in each segment in a five to seven–cell-wide stripe, the expression of OvoA prevents the formation of denticle rows 2–3 and represses <i>m</i> expression in the corresponding cells (red lines). Reciprocally, ectopic expression of <i>svb</i> in <i>wg</i> cells triggers the formation of supernumerary denticles and ectopic expression of <i>m</i> (arrowheads).</p></div

<i>svb</i> Directs the Expression of Genes Encoding Actin-Remodeling Proteins Required for Denticle Formation

<div><p>(A) Cuticle preparations showing denticle morphology in <i>f^36a sn³ sha¹</i> and <i>wsp³</i> mutants. All views correspond to the same region, i.e., the ventral-most region of the A4 segment. Close-ups are scanning electron microscopy magnification of a representative denticle from the fourth row of denticles.</p> <p>(B) mRNA expression of <i>forked, singed, shavenoid/kojak,</i> and <i>wasp</i> in the ventral embryonic abdomen (A2–A6) of wild-type, <i>svb</i> mutants, and embryos expressing <i>svb</i> in <i>wg</i> cells, as observed from in situ hybridization. Arrowheads point to <i>wg</i> cells. Anterior is to the left in all pictures.</p></div

<i>svb</i> Target Genes Are Involved in Separate Features of Denticle Edification

<div><p>(A) Subcellular localization of Singed, Forked, and Miniature in the epidermis of stage 15 wild-type embryos. Distribution of α-catenin, a component of adherens junctions that underlines the cell contour, was observed in embryos expressing α-catenin-GFP driven by E22C-Gal4. Red indicates F-actin, and green indicates α-catenin, Singed, Forked, and Miniature.</p> <p>(B) Transmission electron microscopy analysis of denticle cells from wild-type <i>(wt)</i>, <i>m¹,</i> and <i>sn³, f^36a</i> double mutant embryos. As in smooth cells, the flat region of the apical cell face organizes microvilli that contact cuticle layers only at the apex. In contrast, the plasma membrane is in close contact with cuticle along the entire wild-type denticle contour. Although the <i>m¹</i> mutation alters this membrane/cuticle contact, no defects are visible in <i>sn³, f^36a</i> embryos. Close-up pictures show a region of the growing extension. Scale bar represents 0.25 μm.</p></div

An Evolutionarily Conserved Enhancer Directs the <i>svb</i>-Dependent Expression of <i>m</i>

<div><p>(A) Evolutionary conservation of the <i>m</i> locus and summary of transgenic reporter constructs. Transcribed regions of <i>m</i> span more than 15kb and harbor an unrelated gene, <i>CG9360,</i> transcribed from the complementary stand. Histograms plot the level of sequence conservation between D. melanogaster and D. pseudoobscura (top) or D. virilis (bottom), as represented from the Vista Genome Browser package. Red, light blue, and dark blue peaks correspond to conserved sequences in non-coding regions, 3′ and 5′ UTR, and translated sequences, respectively. Genomic regions displaying high evolutionary conservation were fused with reporter LacZ genes, encoding either a cytoplasmic (6Kmin and 3Kmin) or nuclear (0.4Kmin) β-gal enzyme, and used to generate transgenic lines.</p> <p>(B and C) Compared to <i>m</i> mRNA (B), 6Kmin constructs reproduce endogenous <i>m</i> expression (C).</p> <p>(D) Deletion of intronic sequences leads to a strong reduction of staining in 3Kmin constructs.</p> <p>(E) The 0.4Kmin construct drives <i>m</i>-like epidermal expression at a high level. The white box indicates the ventral region selected for the close-up presented in panel (H).</p> <p>(F and G) Like the endogenous gene, this enhancer is responsive to <i>svb,</i> since staining is absent in <i>svb</i> mutants (F) and additional stripes are produced after <i>svb</i> ectopic expression (arrowheads) (G).</p> <p>(H) Close up of the ventral region (segments A3–A5) of a 0.4Kmin embryo, showing that the β-gal reporter (red) is co-expressed with endogenous Miniature protein (green) in epidermal cells.</p> <p>(I and J) Electrophoresis mobility shift assays show the specific binding of the Svb protein to wild-type <i>m</i> enhancer. Introduction of 2 point mutations prevents in vitro binding (I) and leads to an inactive enhancer when assayed in vivo (J). Sequence of the Svb binding site (capital letters) and introduced mutations (red) are indicated.</p></div