The amd1-skp2 cascade regulates hepatocyte proliferation during liver development and HCC progression in zebrafish. (A) Comparison of liver size from 2 dpf to 7 dpf using in situ hybridization and live Tg(fabp10:GFP) transgenic embryos. Lateral view. (B) Schedule for sorting different staged hepatocytes and bulk RNA sequencing. (C) Among the top 25 genes of liver-enriched genes at 3 dpf, 56% were reported to be enriched in the embryonic liver in the Zfin database, 8% were reported in early literature, and 36% were uncharacterized. (D) Four genes were randomly selected from those enriched and highly expressed in hepatocytes at 3 dpf. Their expression levels were higher in hepatocytes at 3 dpf compared with adult hepatocytes. (E) The relative expression of gatm (0.63%), nos2b (9.7%), nos1 (1.0%), and amd1 (3.0%) in 3 dpf hepatocytes was much higher than in adult hepatocytes. (F) Single staining (amd1 as probe) and double staining (uox for fast red, amd1 for blue) at 4 dpf. Uox and amd1 were colocalized in the liver. (G) From 2 dpf to 5 dpf, liver size was smaller in amd1^7−/− embryos than in controls. (H) At 4.5 dpf, the number of proliferating hepatocytes stained with H3P in amd1^7−/− embryos was lower than in control embryos. (I) RNA sequencing data analysis showed that in amd1^7−/− embryos, 94 genes were up-regulated, and 104 genes were down-regulated; there was no significant difference in the expression of 28,439 genes. (J)In situ experiments showed that skp2 was down-regulated in the liver and gut in amd1^7−/− embryos at 4 dpf. (K) Liver size in skp2 mutants (k2, k4) was smaller than in controls (k1, k3). (L) The number of proliferating hepatocytes stained with EdU in skp2 mutants (l2) is smaller than in controls (l1). (M) Quantitative reverse transcription PCR results showed that the expression of rho and ahcy was significantly down-regulated in embryos treated with 80 μM SMIP004. (N) In situ experiments showed that 100% of embryos treated with 80 μM SMIP004 (Nn2) displayed smaller livers than controls (Nn1). Inhibiting skp2 activity using SMIP004 resulted in 91.6% of amd1^7−/− embryos (Nn4) displaying much smaller livers than controls (Nn3). (O) Cell proliferation was evaluated using H₃P staining. After treatment with SMIP004, the number of H₃P-stained hepatocytes was decreased compared with controls (control, n = 5; SMIP treatment, n = 6; p = 0.0004). SMIP004 treatment further decreased the number of H₃P-stained hepatocytes in amd1^7−/− embryos (n = 6; p = 0.171). (P) Compared with normal liver, the expression of amd1 (5.39-fold increase; p = 0.0162) and skp2 (7.93-fold increase; p = 0.0035) was significantly increased. (Q) Dox-induced overexpression of Kras increased liver growth in the HCC model (Qq2; p ≤ 0.0001), amd1 loss of function decreased the size of the liver in the HCC model (Qq3; p ≤ 0.0001), and simultaneous inhibition of skp2 and amd1 resulted in much smaller livers in the HCC model (Qq4; p = 0.0012). (R) Compared with controls, amd1 loss of function decreased the number of H₃P-stained hepatocytes (Rr5; p ≤ 0.0001), and simultaneous inhibition of skp2 and amd1 further decreased the number of H₃P-stained hepatocytes (Rr8; p = 0.0005). Values are reported as mean ± standard error of the mean. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. HCC, hepatocellular carcinoma; dpf, days post fertilization.