Fig. 3

Cellular proliferation and survival are impaired in krm1^nl10 pLLP. (A-B2) BrdU incorporation in WT and krm1^nl10 primordia between 32.5 and 34hpf. (C) Quantification of cells that have incorporated BrdU, showing a significant decrease in krm1^nl10 mutant embryos versus WT (n=10 WT and 9 mutant embryos; **P<0.001, Student′s t-test). (D-E2) Confocal projections of pLLP at 40hpf labeled with TUNEL (red) in Tg(cldnB:GFP)+ (green) in WT (D,D2) and krm1^nl10 (E,E2) embryos. (F) TUNEL-positive fractions of pLLPs. Significantly more krm1^nl10 mutant pLLPs contained TUNEL-positive puncta (n=35 WT and 40 mutant embryos; *P<0.03, Fisher′s Exact Test). (G-J2) Confocal projections of live Tg(cldnB:GFP) (green) embryos at 24hpf labeled with nuclear-localized Kaede before (green) and after (red) photoconversion. At 24hpf, both WT (G,G2) and krm1^nl10 (H,H2) embryos contain photoconverted Kaede cells in the leading zone of the pLLP. At 48hpf, photoconverted Kaede cells in the WT embryo have divided and are localized in the tc NMs (I,I2). In the krm1^nl10 embryo, photoconverted cells have not proliferated (J,J2). (K) Quantification of photoconverted Kaede cells at 24 and 48hpf; there are significantly fewer converted cells at 48hpf in krm1^nl10 mutant pLLP compared with WT (n=24 WT and 15 krm1^nl10 mutant embryos; **P<0.001, Student′s t-test). Scale bars: 20μm.