Fig. 6

Cdkn1a acts downstream of klf6a to negatively regulate erythroid cell maturation by inducing cell cycle arrest. (A) Scheme of cell sorting by FACS. The gata1:dsRed-positive cells were sorted from the transgenic line flk1:GFP/gata1:dsRed at 2 dpf. (B) qPCR showed cdkn1a upregulation in the sorted cell population of klf6a morphants (mean±SD, t-test, *P<0.05, n=3). (C) qPCR analysis showed that cdkn1a was decreased in klf6a mRNA-injected embryos at 36 hpf (mean±SD, t-test, **P<0.01, *P<0.05, n=3). (D) O-dianisidine staining showed that the number of mature RBCs was reduced in klf6a morphants and cdkn1a mRNA-injected embryos, and the defect in klf6a morphants was rescued by co-injection of cdkn1aMO. The arrowheads mark o-dianisidine-positive cells. (E) Analysis of Klf6a binding to the cdkn1a promoter by ChIP assay. The schematic of the cdkn1a promoter structure that was used in the reporter and ChIP assays. (F) ChIP-PCR. cdkn1a-NF/NR were negative control primers that were used to amplify the cdkn1a promoter region without the conserved Klf6a binding site. The cdkn1a-1F/1R primers were used to amplify the promoter region containing the conserved Klf6a binding site. (G) Reporter assay. Western blot analysis of overexpression of zebrafish Klf6a in HEK293T cells. (H) HEK293T cells were co-transfected with the promoter construct of cdkn1a containing the conserved Klf6 binding sites (GGGCGG) or mutated sites (TTTAAA) and the pCDNA-Klf6a plasmid. (I) BrdU and pH3 staining indicated the gata1:dsRed positive cells in S and M phases in CHT region of 2 dpf Tg (gata1:dsRed) embryos. The yellow arrowheads mark the erythrocytes in S/M phase. (J) The statistic graph showed the percentages of BrdU⁺gata1:dsRed⁺ and pH3⁺gata1:dsRed⁺ in the CHT region (mean±SD, t-test, *P<0.05, n=5).