Sqh localization during retinal lumen formation.

<p>Confocal immunofluorescence micrographs of +/+; <i>sqh</i>-GFP/<i>sqh</i>-GFP (<i>sqh</i>-GFP in green) ommatidium co-stained with Chaoptin (Chp, magenta) at: (A) 48 h APF. (B) 72 h APF. (C) 96 h APF. Chaoptin labels the developing rhabdomeres.</p

Actomyosin contraction is required for increasing the width of the retinal lumen.

<p>(A) RNAi knockdown of Sqh in the flip-out clones. <i>hs</i>-<i>flp</i>/+; <i>GMR</i>><i>w</i>⁺ STOP>Gal4/+; UAS-<i>sqh</i> RNAi/UAS-<i>mCD8</i>-GFP. (C) Overexpression of a dominant-negative form of Zip in flip-out clones. <i>hs</i>-<i>flp</i>/+; <i>GMR</i>><i>w</i>⁺ STOP>Gal4/+; UAS-GFP-<i>zip</i>-Neck-Rod/UAS-<i>mCD8</i>-GFP. GFP (green) marks the cells expressing the RNAi or dominant negative constructs, EYS (red) labels the retinal lumen, and F-actin (blue) labels the rhabdomeres. (A, C) Low magnification view. (A′, C′) A magnified view of the highlighted areas. (B, D) Box and whisker plot of the distance between the R2 and the R4 rhabdomeres of the ommatidia shown in (A) (n = 31) and (C) (n = 18), respectively. Mosaic ommatidium containing both wildtype and mutant cells were not quantified for the box plot. Boxes extend from 25^th to 75^th percentile, with a line at the median. Whiskers extend to the most extreme values (Spear style). (E–G) Model for <i>Drosophila</i> retinal lumen formation. Arrows indicate direction of forces. Scale bar, 5 µm.</p

Defects in apical membrane separation are detected early and maintained throughout development.

<p>(A–C, E–G) Confocal immunofluorescence micrographs of (A–C) <i>eys</i>, <i>GMR</i>-Gal4/+ ommatidium and (E–G) <i>eys</i>, <i>GMR</i>-Gal4/+; UAS-<i>zip</i> RNAi/+ ommatidium co-stained with EYS (green) and F-actin (magenta) at (A,E) 48 h APF. (B,F) 72 h APF. (C,G) 96 h APF. (D,H) Transmission electron micrograph of (D,D′) <i>eys</i>, <i>GMR</i>-Gal4/+ ommatidium and (H,H′) <i>eys</i>, <i>GMR</i>-Gal4/+; UAS-<i>zip</i> RNAi/+ ommatidium at 72 h APF, (D′,H′) represents high magnification of boxed region in (D) or (H), respectively. Arrows denote juxtaposed apical rhabdomere membranes. Scale bar, (A–D, E–H) 2 µm, (D′,H′) 0.5 µm.</p

Temporal profile of the coordination of actomyosin contraction, steric hindrance of adhesion, and secretion of an extracellular matrix.

<p>(A–F) Confocal immunofluorescence micrographs of wild-type ommatidium. (A–C) Ommatidium stained with phospho-Sqh (pMRLC, green), EYS (red), and F-Actin (blue) at: (A) 24 h APF. (B) 45 h APF. (C) 48 h APF. (D–F) Ommatidium stained with Prominin (Prom, green), Chaoptin (Chp, red), and F-Actin (blue) at: (D) 24 h APF. (E) 45 h APF. (F) 48 h APF. Scale bar, 2 µm.</p

The Actomyosin Machinery Is Required for <i>Drosophila</i> Retinal Lumen Formation

<div><p>Multicellular tubes consist of polarized cells wrapped around a central lumen and are essential structures underlying many developmental and physiological functions. In <i>Drosophila</i> compound eyes, each ommatidium forms a luminal matrix, the inter-rhabdomeral space, to shape and separate the key phototransduction organelles, the rhabdomeres, for proper visual perception. In an enhancer screen to define mechanisms of retina lumen formation, we identified Actin5C as a key molecule. Our results demonstrate that the disruption of lumen formation upon the reduction of <i>Actin5C</i> is not linked to any discernible defect in microvillus formation, the rhabdomere terminal web (RTW), or the overall morphogenesis and basal extension of the rhabdomere. Second, the failure of proper lumen formation is not the result of previously identified processes of retinal lumen formation: Prominin localization, expansion of the apical membrane, or secretion of the luminal matrix. Rather, the phenotype observed with <i>Actin5C</i> is phenocopied upon the decrease of the individual components of non-muscle myosin II (MyoII) and its upstream activators. In photoreceptor cells MyoII localizes to the base of the rhabdomeres, overlapping with the actin filaments of the RTW. Consistent with the well-established roll of actomyosin-mediated cellular contraction, reduction of MyoII results in reduced distance between apical membranes as measured by a decrease in lumen diameter. Together, our results indicate the actomyosin machinery coordinates with the localization of apical membrane components and the secretion of an extracellular matrix to overcome apical membrane adhesion to initiate and expand the retinal lumen.</p></div

Regulators of Sqh are involved in retinal lumen formation.

<p>(A–D) Transmission electron micrographs of adult <i>Drosophila</i> ommatidia. (A) <i>eys</i>, <i>prom</i>, <i>GMR</i>-GAL4; UAS-<i>Rho1</i> RNAi/+. (B) <i>eys</i>, <i>prom</i>, <i>GMR</i>-GAL4/+; UAS-<i>rok</i> RNAi/+. (C) <i>eys</i>, <i>prom</i>, <i>GMR</i>-GAL4/+; UAS-<i>Mbs</i>^N300/+. (D) <i>eys</i>, <i>prom</i>, <i>GMR</i>-GAL4/+; UAS-<i>snail</i> RNAi/+. (E) Quantitative analysis of rhabdomere fusion seen in (A–D). Values represent mean ± SEM. *** <i>P</i><0.001, **<i>P</i><0.01 compared with <i>eys</i>, <i>prom</i>, <i>GMR</i>-Gal4/+. Arrows indicate the incomplete separation between rhabdomeres. Scale bar, 5 µm.</p

Reduction of <i>Act5C</i> dosage enhances rhabdomere adhesion.

<p>(A–D) Transmission electron micrographs of adult <i>Drosophila</i> ommatidia. (A) <i>w¹¹¹⁸</i>, wild type. (B) <i>eys</i>, <i>prom</i>/+. (C) Df(1)ED6829/+; <i>eys</i>, <i>prom</i>/+. The deficiency removes genomic region 5C7-5F3. (D) <i>Act5C</i>^G0245/+; <i>eys</i>, <i>prom</i>/+. (E) Quantitative analysis of rhabdomere fusion seen in (A–D). Values represent mean ± SEM. **<i>P</i><0.01 compared with <i>eys</i>, <i>prom</i>/+. Arrows indicate the incomplete separation between rhabdomeres. Scale bar, 5 µm.</p

Localization of Prominin, EYS, and Crumbs in the <i>Act5C</i>/+; EP-TH genetic background.

<p>Confocal immunofluorescence micrographs of wild type (A–C) and <i>Act5C</i>^G0245/+; <i>eys</i>, <i>prom</i>/+ (D–F) ommatidium at 72 h APF stained with: (A,D) EYS and F-actin. (B,E) Prominin (Prom) and F-actin. (C,F) Crumbs (Crb) and F-actin. Scale bar, 2 µm.</p

Non-muscle myosin II is involved in retinal lumen formation.

<p>(A–E′) Transmission electron micrographs of adult <i>Drosophila</i> ommatidia. (A) <i>eys</i>, <i>prom</i>, <i>GMR</i>-GAL4/+. (B) <i>eys</i>, <i>prom</i>, <i>GMR</i>-GAL4/+; UAS-<i>zip</i> RNAi/+. (C) <i>eys</i>, <i>prom</i>, <i>GMR</i>-GAL4/+; UAS-<i>sqh</i> RNAi/+. (D) <i>eys</i>, <i>prom</i>, <i>GMR</i>-GAL4/+; UAS-GFP-<i>zip</i>-Neck-Rod/+. (E and E′) <i>eys</i>, <i>prom</i>, <i>GMR</i>-GAL4/+; <i>Pph13</i>-Gal4/UAS-<i>sqh</i>^E20E21. (F) Quantitative analysis of rhabdomere fusion seen in (A–E′). Values represent mean ± SEM. *** <i>P</i><0.001, **<i>P</i><0.01 compared with <i>eys</i>, <i>prom</i>, <i>GMR</i>-Gal4/+. Arrows indicate adhesion between rhabdomeres. Scale bar, 5 µm.</p