Fig. 4

Gata6 and Hhex synergistically activates lrh-1 transcription. (A) Immuno- and dye staining of livers in the siblings (n = 6) and lrh-1 mutants (n = 6) at 7.5 dpf. 2F11 (green), intrahepatic bile duct cells; Phalloidin (red) and Mdr1 (cyan), bile canaliculi. (Scale bar, 50 μm.) (B) Quantification of the length of bile canaliculi in the figure (A). Few bile canaliculi could be observed in the lrh-1 mutant liver. (C) The lrh-1 expression revealed by WISH in gata6 and hhex mutants at 30 hpf. Red arrowhead, liver bud. (D) An illustration of the −3 kb promoter region of zebrafish lrh-1 gene highlighted with nine putative GATA binding sites (black square). The putative GATA binding sites were predicted on the website https://jaspar.genereg.net/. (E) Luciferase assays of the zebrafish −3 kb lrh-1 promoter in HepG2 cells. (F) Luciferase assays of the zebrafish −1.5 kb and −3 kb lrh-1 promoters with Gata6 in HepG2 cells. (G) ChIP-qPCR analysis of Gata6 occupancy on the lrh-1 promoter in Tg(pIDM:HA-gata6) larvae. As per the platform https://jaspar.genereg.net/, the three binding sites (A, B, and C in D) with the higher scores were selected. The coding region of lrh-1 in exon 7 was used as a negative control. (H) ChIP-seq reads across lrh-1 promoter loci with Tg(pIDM:HA-gata6) larvae upon DOX treatment. Gata6 bound to −1.6 kb of promoter loci of lrh-1 in zebrafish. (I) Luciferase assays of the zebrafish −3 kb lrh-1 promoter with Hhex and wild type and mutated variants Gata6 in HepG2 cells. (J) The expression of prox1 in gata6−/−; hhex−/− double mutants with the overexpression of lrh-1 by Tg(hsp70:HAM-lrh-1) heat-shocked at 14 hpf. Red arrowhead, liver bud. Data are mean ± SD, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, nonsignificant, two-tailed unpaired t test.