Depletion of Rcl1 causes hypoplastic digestive organs. (A) Schematic drawing shows the genomic DNA structure of the rcl1 gene and relative positions of the rcl1hi2452 (the viral vector insertion in intron 1) and rcl1zju1 (16bp insertion in the exon1) mutation sites. Primer pairs used for genotyping the two mutant alleles are indicated by different color arrowheads, and a gel photo of their corresponding PCR products is shown above the drawing. Yellow arrowheads, primer pair for checking the viral insertion in heterozygous (+/−) or homozygous (−/−) mutants; blue arrowheads, for checking the wild-type gene in wild-type sibling (+/+) or heterozygous mutant (+/−); red arrowheads, for genotyping the rcl1zju1 mutant by checking the PCR product digested by Dde1 (WT but not the mutant PCR product can be cleaved by Dde1). Lower panel: showing the DNA sequence changes in rcl1zju1 mutant (red letters, insertion; blue letters, substitution; capital letters, exon; small letters: intron). (B) Western blot of Rcl1 protein in embryos at 5dpf. Upper panels: WT and rcl1hi245; lower panels: WT and rcl1zju1. (C) Immunostaining of Rcl1 and Fibrillarin (Fib) in the liver of WT, rcl1hi2452 and rcl1zju1 mutant at 5dpf. Nuclei are stained with DAPI; Scale bars: 50 μm. (D) Comparison of overall morphology between rcl1hi2452and rcl1zju1embryos with their corresponding sibling at 5dpf. (E–G) WISH using the fabp10a, fabp2 and trypsin probes on 3dpf-old rcl1hi2452 and rcl1zju1 intercross embryos (E). WISH using the foxa3 and gata6 probes (F) and hhex and prox1 (G) on rcl1hi2452 intercross embryos at 30hpf and 48hpf, respectively. Denominator vs numerator: number of embryos exhibiting the phenotype vs number of embryos of corresponding genotype examined. Black arrow, liver bud; red arrow, exocrine pancreas. (H) Immunostaining of P-H3 (green) and Bhmt (red) on cryosections obtained from 2.5dpf-old WT and rcl1hi2452 mutant embryos (H). Nuclei are stained with DAPI; Nt: neural tube; Nc: notochord; Pd: pronephric duct; L: liver. (I) Quantitative analysis of the P-H3 positive cells in the liver and neural tube in WT and rcl1zju1 mutants from three embryos, respectively. **P < 0.01.
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