Notch gain of function leads to blastema expansion and impaired fin regeneration. Wild-type and Tg(hsp70l:Gal4);Tg(UAS:myc-notch1a-intra) fish, referred to as Tg(UAS:NICD), were heat-shocked throughout the regeneration period. (A-B3) Live images of wild-type and Tg(UAS:NICD) fins at 1, 3 and 5 dpa. No phenotypic differences were observed at 1 (A,B) and 3 (A2,B2) dpa between wild-type and Tg(UAS:NICD). Regenerative outgrowth progresses in 5 dpa wild-type fish (A3) but is blocked in Tg(UAS:NICD) fish (B3). The blastema is swollen proximal to each fin radial (B3, arrow). (C) Mean length of the regenerate at 1, 3 and 6 dpa: regenerative outgrowth is significantly reduced in Tg(UAS:NICD) fish at 6 dpa; ***P<0.001. (D) Immunohistochemistry against the Myc tag on the NICD transgene (Myc-NICD) in 5 dpa Tg(UAS:NICD) fin sections. Expression of Myc-NICD is mosaic in the blastema, in two or three cell layers beneath the epidermis (arrowheads) and in intraray blastema cells (arrow). (E-F2) Haematoxylin and Eosin stained fin sections (5 dpa): the blastema of regenerated Tg(UAS:NICD) fins is broader than wild-type blastema (E,F, horizontal bars; supplementary material Fig. S4C). Higher magnification views of wild-type fins reveal densely packed distal blastema cells (E3, arrowhead), the looser organization in the central proximal region and the strict alignment in the periphery (E3, arrowhead). In Tg(UAS:NICD) fins, cells are densely packed and disorganized in both blastema regions (arrowheads, F,F2). This structure is similar to the blastema (E2,F,F2). (G-J) In situ hybridization at 5 dpa. her6 and her15 transcription expands into the proximal blastema region in Tg(UAS:NICD) fins (arrowheads, G-J). (K) qPCR analysis after a 10-hour heat-shock series (62-72 hpa). *P<0.05. Scale bars: 200 μm in B310 μm in F,F2,J.
|
