Tenectin is a novel alphaPS2betaPS integrin ligand required for wing morphogenesis and male genital looping in Drosophila.

International audienceMorphogenesis of the adult structures of holometabolous insects is regulated by ecdysteroids and juvenile hormones and involves cell-cell interactions mediated in part by the cell surface integrin receptors and their extracellular matrix (ECM) ligands. These adhesion molecules and their regulation by hormones are not well characterized. We describe the gene structure of a newly described ECM molecule, tenectin, and demonstrate that it is a hormonally regulated ECM protein required for proper morphogenesis of the adult wing and male genitalia. Tenectin's function as a new ligand of the PS2 integrins is demonstrated by both genetic interactions in the fly and by cell spreading and cell adhesion assays in cultured cells. Its interaction with the PS2 integrins is dependent on RGD and RGD-like motifs. Tenectin's function in looping morphogenesis in the development of the male genitalia led to experiments that demonstrate a role for PS integrins in the execution of left-right asymmetry

A luminal glycoprotein drives dose-dependent diameter expansion of the Drosophila melanogaster hindgut tube.

International audienceAn important step in epithelial organ development is size maturation of the organ lumen to attain correct dimensions. Here we show that the regulated expression of Tenectin (Tnc) is critical to shape the Drosophila melanogaster hindgut tube. Tnc is a secreted protein that fills the embryonic hindgut lumen during tube diameter expansion. Inside the lumen, Tnc contributes to detectable O-Glycans and forms a dense striated matrix. Loss of tnc causes a narrow hindgut tube, while Tnc over-expression drives tube dilation in a dose-dependent manner. Cellular analyses show that luminal accumulation of Tnc causes an increase in inner and outer tube diameter, and cell flattening within the tube wall, similar to the effects of a hydrostatic pressure in other systems. When Tnc expression is induced only in cells at one side of the tube wall, Tnc fills the lumen and equally affects all cells at the lumen perimeter, arguing that Tnc acts non-cell-autonomously. Moreover, when Tnc expression is directed to a segment of a tube, its luminal accumulation is restricted to this segment and affects the surrounding cells to promote a corresponding local diameter expansion. These findings suggest that deposition of Tnc into the lumen might contribute to expansion of the lumen volume, and thereby to stretching of the tube wall. Consistent with such an idea, ectopic expression of Tnc in different developing epithelial tubes is sufficient to cause dilation, while epidermal Tnc expression has no effect on morphology. Together, the results show that epithelial tube diameter can be modelled by regulating the levels and pattern of expression of a single luminal glycoprotein

The endogenous siRNA pathway is involved in heterochromatin formation in Drosophila

A new class of small RNAs (endo-siRNAs) produced from endogenous double-stranded RNA (dsRNA) precursors was recently shown to mediate transposable element (TE) silencing in the Drosophila soma. These endo-siRNAs might play a role in heterochromatin formation, as has been shown in S. pombe for siRNAs derived from repetitive sequences in chromosome pericentromeres. To address this possibility, we used the viral suppressors of RNA silencing B2 and P19. These proteins normally counteract the RNAi host defense by blocking the biogenesis or activity of virus-derived siRNAs. We hypothesized that both proteins would similarly block endo-siRNA processing or function, thereby revealing the contribution of endo-siRNA to heterochromatin formation. Accordingly, P19 as well as a nuclear form of P19 expressed in Drosophila somatic cells were found to sequester TE-derived siRNAs whereas B2 predominantly bound their longer precursors. Strikingly, B2 or the nuclear form of P19, but not P19, suppressed silencing of heterochromatin gene markers in adult flies, and altered histone H3-K9 methylation as well as chromosomal distribution of histone methyl transferase Su(var)3–9 and Heterochromatin Protein 1 in larvae. Similar effects were observed in dcr2, r2d2, and ago2 mutants. Our findings provide evidence that a nuclear pool of TE-derived endo-siRNAs is involved in heterochromatin formation in somatic tissues in Drosophila

Loss of <i>tnc</i> causes reduced apical cell circumferences, altered cell arrangement, and a smaller outer tube diameter.

<p>(A–H) Embryos were labelled with anti-DECad, and serial z-stacked images spanning the upper half of the hindgut tube were merged to reveal apical cell circumferences. Arrows point to the Si/Li border. The hindgut of a wild type and <i>tnc</i> mutant embryo at dorsal view (A and B) is shown together with high magnifications of respective Si (in C and D) and posterior Li (in E and F). Identically sized squares (in C and D) span ∼10 cells of the wild type Si and ∼18 cells of the mutant Si. Brackets of identical size (in E and F) illustrate that cells are more elongated along the lumen perimeter in the wild type compared to the mutant. In the dorsal Li (G and H, ventral view), identically sized brackets span 9 or 11 cells along the border cells in the wild type and mutant hindgut, respectively. The mutant hindgut also has fewer cells at the dorsal lumen perimeter. (I and J) The outer hindgut diameter, visualized by labelling for Dg (green) and merging serial z-stacked images that span the entire hindgut, is reduced in the mutants. Stippled and full lines represent wild type Si and Li diameter, respectively. (K and L) Labelling with anti-Fas3 (green) reveal comparable level and distribution of Fas3 (bracket) in the hindgut epithelium of wild type (K) and <i>tnc</i> mutant (L) embryos. Co-staining for Tnc (magenta) shows the absence of Tnc in the mutant hindgut. All images are of stage 16 embryos. Scale bars: 10 µm in A (A and B), 10 µm in C (C–H), 10 µm in I (I and J), 10 µm in K (K and L).</p

Tnc is a glycosylated intralumial matrix-component.

<p>(A) Tnc from wild type and <i>tnc^13c</i> mutant embryos and larvae (l) were detected on western blot and resided as high-molecular weight species in the stacking gel (stippled line indicates end of stacking gel). Anti-α-Tubulin was used as loading control. (B) Protein extracts from stage 16 wild type embryos were subjected to deglycosylation (no enz = no enzyme, N-Gly = N-Glycanase (PNGase F), O-Gly = O-Glycanase, O-Gly+ = O-Glycanase+Sialidase+β(1-4) Galactosidase+β-N-Acetylglucosaminidase). Addition of O-glycanase caused slightly faster migration of Tnc. (C–H) Embryos were co-labelled for the Tn antigen (green) and Crb (magenta) (C, D, F and G) or with VVA (E and H). Anti-Tn stains the wild type lumen with highest intensity in Si at stages 15 and 16 (C and D). The staining is reduced in mutant embryos (F and G). Arrows point to the Si/Li border. VVA also labels the wild type lumen (E) stronger than the mutant lumen (H). The embryos were processed in parallel and the hindgut was imaged at similar views with identical confocal settings. (I) Wild type embryos were prepared with Clark's fixation and stained for Tnc (I, green) and the Tn antigen (I′, magenta). The merged image (I″) shows partial overlap of the staining. Arrow points to the Si/Li border. (J) High magnification of the hindgut in (I), showing a striated pattern of Tnc-staining. Scale bars: 10 µm in I, 5 µm in J.</p

Tnc is required for hindgut lumen diameter expansion.

<p>(A–C) Wild type embryos were labelled for Crb (magenta) and Dg (green) to visualize the apical and basal surfaces of the hindgut epithelium. Arrows point to anterior and posterior Li borders, and stippled lines mark outer tube diameter. Crb-staining only is shown in A′–C′. At stage 13 (A, lateral view) the hindgut is narrow with an anterior hook pointing ventral. By stage 14, (B, dorsal view) the anterior hook has turned right and Crb-expressing border cells demarcate hindgut subdomains. From stage 14 to stage 16 (C, dorsal view) the hindgut expands in diameter and length. (D–F) <i>tnc^13c</i> mutant embryos stained for Crb revealed a normal hindgut lumen at stage 13 (D), slight reduction in lumen diameter at stage 14 (E) and a clear reduction in lumen diameter at stage 16 (F), compared to the wild type. White and open arrowheads in (C′) and (F) illustrate lumen diameter of Li and Si, respectively. (G) Drawings of the hindgut lumen at stages 14 and 16 with Si in red and Li in blue. Border cells (black lines) mark anterior and posterior boundaries of Li and separates dorsal Li (dLi) and ventral Li (vLi). (H) The graphs show mean lumen diameter of Si and Li at stages 14 and 16 in the wild type and at stage 16 in <i>tnc^13c</i> mutants. Li expands in diameter from 6.3 (+/−0.09) µm to 8.8 (+/−0.16) µm, and Si from 8.9 (+/−0.52) µm to 17.3 (+/−0.56) µm. * = P-value<0.05. Bars represent standard error of mean (n>5). Scale bar: 10 µm. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002850#pgen.1002850.s002" target="_blank">Figures S2</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002850#pgen.1002850.s003" target="_blank">S3</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002850#pgen.1002850.s004" target="_blank">S4</a>.</p

Tnc acts non-cell-autonomously.

<p>(A) <i>enGAL4</i> drives expression of <i>UAS-GFP</i> in the dorsal Li (bracket), as seen by labelling with anti-GFP (green) and anti-Crb (magenta). (B and C) Stage 16 embryos stained for DECad and Crb show that <i>enGAL4</i>-driven expression of <i>tnc</i> in dorsal Li (bracket) caused enlarged apical cell circumferences in both dorsal and ventral Li (C), when compared to embryos that only express <i>enGAL4</i> (B). (D–G) <i>enGAL4</i> drives expression of <i>UAS-GFP</i> (green) in a cluster of cells in the anterior salivary gland (D, stage 13). <i>enGAL4</i>-driven expression of Tnc in salivary glands resulted in local tube dilation (E). By merging serial z-stacked images, the apical cell circumferences were visualized (F). Note that <i>enGAL4</i> drives expression in one side of the tube, but all cells at the perimeter show enlarged apical cell circumference. Co-staining for Tnc (green) and Crb (magenta) shows that luminal Tnc localizes to the dilated part of the salivary gland lumen (G). (H) A possible model for the function of Tnc during lumen dilation. Expression of <i>tnc</i> in the tubular epithelium causes tube dilation according to the level of expression (“low" and “high") and causes differential dilation along the tube. Once inside the lumen, Tnc acts on surrounding cells, possibly by generating a mechanical pressure, to expand the tube wall.</p