CARF facilitates Wnt/β-catenin signaling in zebrafish embryogenesis. (a) The whole mount in situ analysis of CARF expression in zebrafish embryogenesis. The embryos at indicated developmental stages were fixed for in situ hybridization with CARF probe. (b) CARF knockdown attenuates Wnt activation in Tg(tcf:egfp) line. (c, d) CARF^cas009 mutant or CARF morphants show reduced Wnt signaling activity via a p53-independet manner. Embryos injected with the indicated morpholinos or morpholino-mRNA mixture were fixed at the 60% epiboly stage and then analyzed by WISH with cdx4 (left) or tbx6 (right) probe. The relative expression of each marker was classified into two categories: normal (N) or weak (W). The number of the total embryos scored (n) is shown on the top of each bar.

Loss of CARF attenuates HSPC formation and caudal fin regeneration. (a–d) Knockdown CARF limits HSPC formation. Zebrafish embryos at 32 hpf were fixed for in situ hybridization with cmyb probe, and then classified into three categories: (a) Representative image for slightly increased (I), normal (N), decreased (D) or extremely low (E) level of c-myb WISH analysis. Knockdown CARF dramatically inhibits HSPC formation (b) which could not be rescued via p53 MO co-injection (c) and further validated by the live image of HSPC budding events in AGM of zebrafish Tg(flk1:mcherry;cmyb:egfp) line (d). (e–g) CARF^cas009 mutant exhibits reduced HSPC formation via a p53-independent manner. Injection of zebrafish CARF (zCARF) mRNA (f) but not p53 MO (g) restores decreased cmyb expression in CARF^cas009 mutants (e). (h) Schematic diagram of Tol2 transposase-mediated transient transgenesis of endothelial-specific promoter (flk1)-derived expression of constitutive activated β-catenin (ΔN β-catenin) and mCherry chimera protein in zebrafish embryos, which rescues hematopoietic defects in CARF^cas009 mutants while slightly increases HSC formation in wildtype zebrafish. (i, j) Delayed fin regeneration of CARF^cas009 mutants. Adult CARF^cas009 zebrafish were executed caudal fin amputation and then cultured for regeneration. The regenerated sections were cut down at 2 dpa for either WISH analysis with lef1 probes or length measurement.