Stages of epithelial tubule interconnection. (A) Diagram of the adult zebrafish kidney and stages of regeneration. Single lhx1a:eGFP+ mesenchymal cells (green) aggregate into dome-shaped rosettes on distal tubules (blue) and then proliferate to extend a new kidney nephron (red; glomerulus). Lumenal fusion (white) to the existing distal tubules enables new nephron function. (B) Prior to tubule fusion, phalloidin-stained (magenta) apical cell actin highlights a new tubule epithelial lumen (arrow indicates the direction of fusion) in the process of nephron fusion to the distal tubule (DT; dotted outline). Tg(lhx1a:egfp) (green) marks newly forming new nephrons adjacent to target distal tubules (DTs). Junctional nephron cells are constricted apically. Inset in B shows a higher-magnification view of a basal protrusion (arrowhead) with a phalloidin-positive (magenta) actin core. (C) After tubule fusion, phalloidin staining (magenta) outlines the apical cell actin, and lumen continuity between old and new nephron tubules (arrow indicates direction of fusion). DT, distal tubule (outlined). (D,E) New nephron cells in rosettes are apically constricted (D) and become basally constricted in the process of lumen fusion (E) (a, apical surface; b, basal surface). (F,G) Diagram of cell boundaries drawn from D and E (a, apical surface; b, basal surface). (H,I) Basal protrusions (H, arrowheads) invade the distal tubule (DT) at the point of tubule fusion; these cells are non-proliferating (I, arrowheads). (J,K) New nephrons lack laminin-positive basement membranes at their basal surface point of fusion, marked by Tg(lhx1a:egfp) expression (K, arrowheads). Blue fluorescence is Hoechst-stained nuclei. Arrows indicate the direction of fusion. Scale bars: 5 µm in C-K; 10 µm in B.
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