|
Figure 5
(A) qPCR relative quantification of lef1 mRNA in kidney tissue of WT and cox2a-/- harvested at 7 dpi. Gene was normalized to the mean expression level in uninjured (Un-Inj) kidneys, which was set to 1. **p<0.01, ***p<0.001; ns, no significant difference (n = 3). (B) FACS-related RT-PCR analysis of wnt4a expression in Tg(lhx1a:DsRed) labeled RPCs at 5 dpi, and wnt4a was expressed in these cells. (C) The wnt4a mRNA levels were assessed by RT-PCR in uninjured or injured kidneys. (D) lhx1a mRNA levels were evaluated by RT-PCR in wnt4a-/- and WT zebrafish kidneys at 7 dpi. β-actin was used as a sample control. (E–H) lhx1a whole-mount in situ hybridization (WISH) showing the trunk kidney region at 7 dpi. (G) the number of lhx1a+ cell aggregates in wnt4a-/- was less than that in WT. (H) Injection of dmPGE2 could rescue the influence of Wnt4a deficiency. (I) lhx1a+ cell aggregates of whole kidney were calculated using ImageJ. n = 5–7 fish for each condition. Data were analyzed by ANOVA, ***p<0.001; ns, no significant difference. (J, K) Gentamicin induced lhx1a+ new nephrons with proliferating EdU+ nuclei at 5 dpi (n = 5). (L, M) The proliferation of lhx1a+ cells in wnt4a-/- was significantly less than that in WT (n = 6). (N, O) dmPGE2 could rescue the effect of Wnt4a deficiency and recover the proliferation of lhx1a+ cells in wnt4a-/- (n = 6). (K, M, O) show the higher-magnification images of the boxed areas showed in (J, L, N). Scale bar in (J–O), 100 μm. (G) Proliferation ratio of lhx1a+ RPCs in (J–O) was calculated using ImageJ. n = 5–7 in each condition. Data were analyzed by ANOVA, ***p<0.001; ns, no significant difference.
PGE2–EP4 signaling promotes nephron regeneration through PGE2/Wnt interaction.