reg6 function is required for plexus formation. In the temperature up-shift experiment, wild-type TG(fli1:EGFP)y1 (A–C) and reg6;TG(fli1:EGFP)y1 (D–F) fish were allowed to complete anastomosis at the permissive temperature (20°C) and were then shifted to the restrictive temperature (33°C). In wild-type animals, the regenerating vessels form anastomotic bridges at the time of shift (A). By 12 h postshift (hps), multiple distally extending sprouts grow out from the distal face of the anastomotic bridges (B). The regenerating vessels in wild-type animals develop normal plexuses 48 h after shift to the restrictive temperature (C). In contrast, although the regenerating vessels in reg6 mutants had successfully completed anastomosis at the time of shift (D, regenerates shifted at 77 hpa are shown), they developed swollen vessels within 2 days at the restrictive temperature (F). hps, hour post shift. Red arrows, amputation plane; yellow arrows, anastomotic bridges; yellow arrowheads, swollen vessels; double-headed arrow, plexus. Scale bar, 100 μm.
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