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

Model of <i>svb</i> Regulation and Activity during Denticle Formation

<p>During epidermal differentiation, regulatory regions governing <i>svb</i> transcription integrate outputs from many signaling pathways (Wg, Hh, and DER) and positional cues to define the precise subset of epidermal cells that express <i>svb</i>. The Shavenbaby transcription factor triggers in turn the expression of different classes of genes encoding cellular effectors. They are directly involved in distinct aspects of trichome formation, including the reorganization of actin <i>(singed, forked, wasp,</i> and <i>shavenoid)</i>, extracellular matrix <i>(m)</i> and cuticle <i>(y)</i>, likely through modifying the activity of ubiquitous cellular machineries. Additional cytoskeletal factors or regulators (independent of <i>svb</i>) might be required for the fine sculpturing of each kind of trichome, characteristic of a given body region. Modifications of <i>svb cis</i>-regulatory regions thus provide a rich source of plasticity to evolve the trichome pattern and generate morphological diversification throughout species.</p

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

<i>m</i> Impinges on Denticle Formation

<div><p>(A) Consequences of alterations of <i>m</i> function and expression on denticle formation. The <i>m¹</i> mutation leads to morphological alteration of denticles, with a characteristic abnormal triangular shape. A similar phenotype is observed with <i>Df(1)m-MR,</i> a deficiency in which the entire <i>m</i> locus is deleted. Overexpression of <i>m,</i> by crossing the Ptc-Gal4 driver line to EP345-m, does not modify the cuticular phenotype in the corresponding regions (arrowheads). Re-expression of wild-type <i>m</i> products driven by the 0.4Kmin-gal4 transgenic lines, but not 0.4KminKO-Gal4 lines, rescues the characteristic denticles defects of <i>m¹</i> embryos. All pictures correspond to the A4 segment.</p> <p>(B) High magnification views of wild-type <i>(wt)</i> and <i>m¹</i> denticles (fourth rows of A4), as observed in either light (left) or scanning electron microscopy (right). Whereas the absence of <i>m</i> does not affect the denticle height (h), the width (w) is reduced, producing denticles of smaller area that display an abnormal shape lacking the median constriction characteristic of wild-type denticles. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040290#pbio-0040290-st002" target="_blank">Table S2</a> for quantification of these defects.</p></div

<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

Average area of endosomes analyzed in Garland cells from <i>wt, stam, hrs</i> and <i>hrs, stam</i> larvae.

<p>Average area of endosomes analyzed in Garland cells from <i>wt, stam, hrs</i> and <i>hrs, stam</i> larvae.</p

<i>stam</i> and <i>hrs</i> are required for tracheal cell migration in the air sac primordium.

<p>A. Schematic representation of the anterior part of a <i>Drosophila</i> third instar larva. The air sac primordium (ASP) (red) buds from the transverse connective branch (in grey) and is attached to the wing imaginal disc (orange). The tracheal system is drawn in grey and imaginal discs other than the wing disc are colored in yellow. B. Model for the formation of the air sac primordium during larval development. Tracheal cells divide and migrate during ASP formation. Migration occurs under the control of the FGFR signalling pathway. Tracheal cells at the distal tip of the primordium are extending filapodia in the direction of the FGF ligand source (blue). Double arrow indicates the position of ASP distal tip. C. Migration behaviour of <i>wild type</i>, <i>stam</i>, <i>hrs</i> and <i>stam hrs</i> mutant cells. Confocal micrographs of the ASP of a <i>Drosophila</i> third instar larva are shown. All tracheal cells are labelled in red (RFP-moesin) and MARCM clones are labelled in green (mCD8-GFP). The <i>FRT40A</i> chromosome was used as a wild-type control. MARCM clones were induced for <i>stam</i>, <i>hrs</i> and <i>stam</i>, <i>hrs</i>. Scale bar: 15 µm. White double arrows indicate the position of ASP distal tip. Percentages of distal clones are indicated for each genotype tested. Note the strong effect of mutations in <i>hrs</i> and <i>hrs, stam</i> on cell migration. For each genotype, more than 20 clones were scored.</p

Number of branching points of <i>hrs</i> and <i>stam</i> MARCM terminal cell clones in different FGFR signalling activity backgrounds.

<p>Number of branching points of <i>hrs</i> and <i>stam</i> MARCM terminal cell clones in different FGFR signalling activity backgrounds.</p