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. 2023 Sep 18;150(18):dev201708. doi: 10.1242/dev.201708

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© 2023. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 5. — Promoting ring constriction in the nephrocyte extra acytokinetic mitosis delays the assembly of slit diaphragms. (A-C′) Individual confocal sections (A,C), z-projection (B) and quantification (C′) of larval nephrocytes of the indicated genotypes stained with antibodies against slit diaphragm proteins. Arrowheads point to mononucleated nephrocytes (n=88 nephrocytes (N)/12 strings (S) out of 294N in A and n=8N/5S out of 67N in B], which at this stage exhibit a normal density of slit diaphragms [C, Duf staining, external views, quantified in C′ as percentage of nephrocyte surface area covered by slit diaphragms (SD); wt, n=16 regions of interest (ROIs), 9 cells, 5 strings; VT016847>sqh^DD, n=11 ROIs, 5 cells, 3 strings; VT016847>pbl, n=12 ROIs, 8 cells, 5 strings; mean±s.d.]. Box plot shows median values (horizontal bars), mean values (indicated by X) and first to third interquartile ranges (boxes); whiskers extend to the minimum and maximum data values. ns, not significant. (D-F‴) Z-projections (D-E″) and single sections (E‴-F‴) of embryonic nephrocytes of the indicated genotypes. Note in pbl-overexpressing nephrocytes the colocalisation of Pyd and Fas3 (yellow arrows in E-E″) at the cell membrane between adjacent mononucleated nephrocytes 1, 2 and 3 (arrowheads in E,E‴) and Pyd localisation at the outer membrane (white arrow in E,E′ and in a different plane in E‴) in binucleated cell 4 (n=13N/6S). In F-F‴ (n=7N/5S), arrowheads point to the colocalisation of Duf and Pyd with Fas3 at contact membranes abutting mononucleated nephrocytes overexpressing sqh^DD (arrow in F). (G-H‴) Ubiquitous overexpression of a constitutively active form of aPKC in embryonic nephrocytes delays the onset of the acytokinetic cell division to stage 15. At stage 14 nephrocytes are mononucleated (G, n=40N/5S) and accumulate slit diaphragm proteins at tricellular contacts (arrowheads). At stage 15 (H-H‴, n=20N/7S), slit diaphragm proteins accumulate at the equatorial cortex in binucleated cells (arrows). N, nucleus (E‴). Scale bars: 10 µm.