Phenotypes of supv3l1^GtT/GtT mutants. (A) Gross morphological defects at 5 dpf in supv3l1^Gt/Gt showing smaller eyes, lack of an inflated swim bladder, small and dark liver (arrowhead), and underdeveloped and dark intestine (arrow). (B) RT-PCR analysis showing undetectable supv3l1 mRNA in the mutant (Upper ) with normal β-actin mRNA levels (Lower). (C) Bar graph (mean ± SE) of whole-animal Western blot analysis of the levels of components of mitochondrial complexes showing a decrease in the levels of NDUFB6 (complex I), CoxVa (complex IV), and F-ATPase (complex V) in the mutants (n = 4). P < 0.05 for complex IV and ATPase. The difference for complex I did not reach statistical significance (P = 0.12). (D) Bar graph (mean ± SEM) showing a decrease in mitochondrial DNA relative to nuclear DNA (β-actin 2) (n = 4). P < 0.05 for both ND2 and ND6. n = 4.

Liver-specific expression of supv3l1 in supv3l1^GtT/GtT mutants rescues hepatocyte defects. (A–C) Images of Lipan (A), LiPan/supv3l1^GtT/GtT (B), LiPan/hepatocyte-Cre^+/-/supv3l1^GtT/GtT (C) showing liver agenesis in supv3l1^GtT/GtT and liver-specific rescue by hepatocyte-Cre. Other defects in supv3l1^GtT/GtT remain in hepatocyte-Cre^+/-/supv3l1^GtT/GtT. Each image is a merge of a bright-field image and a red fluorescent image of the same larva. (D–F) In situ hybridization analysis of transferrin a mRNA expression in 5-dpf larvae showing the specific expression in the liver of wild-type and heterozygous larva (90/114) (D), the absence in supv3l1^GtT/GtT (12/114) (E), and the restoration in the liver-specific rescue hepatocyte-Cre^+/-/supv3l1^GtT/GtT (12/114) (F). Eye size in E and F remains smaller than in D. Observed numbers are not different from expectation (χ² test, P = 0.623). (G and H) Liver H&E staining of wild-type (G), supv3l1^GtT/GtT (H), and hepatocyte-Cre^+/-/supv3l1^GtT/GtT (I) larvae at 5 dpf, indicating the poorly differentiated hepatocytes with vacuolated cytoplasm and weak nuclear staining in global mutants (H) is fully restored by hepatocyte-Cre (I). Arrowheads indicate hepatocytes.

Phenotype of liver-specific supv3l1 inactivation. (A–D) Confocal images of the Left (A and B) and Right (C and D) sides of 21-d-old LiPan (A and C) and LiPan/supv3l1^LKO (B and D) fish showing detached hepatocytes (white arrows) (B and D) and smaller right liver lobe (white arrow) in supv3l1^LKO fish (D). (E and F) Histological analysis of wild-type (E) and supv3l1^LKO (F) fish indicate disorganized liver with dead cells (arrowhead) but normal intestinal lumen (black arrows) in supv3l1^LKO fish (F). (F) Survival curve of supv3l1^LKO fish (lens YFP⁺) compared with supv3l1^GtNf/GtNf siblings (lens YFP^-) during the first 6 wk of life. One group was fed a larval diet until 28 dpf followed by a juvenile diet (red) and the other group was kept on the larval diet (blue). (G) Photograph of a wild-type and a supv3l1^LKO mutant at 5 wk of age showing the smaller size of the mutant.

Gene-trap cassette optimization. (A) Schematic of the mutagenicity reporters for candidate gene-trap cassettes. (B) Mutagenicity assay by semiquantitative RT-PCR analysis indicated that a cassette with three copies of BGH pA is more mutagenic than one with one copy. (C) Semiquantitative RT-PCR analysis indicated that a cassette with five copies of BGH pA is more mutagenic than one with three copies.

Gene-trap expression of mCherry in different mutagenized lines. (A) Image of fam49bbGtT/+ at 48 hpf. (B) Image of supv3l1^GtT/+ at 24 hpf. (C) Image of tfcp2l1^GtT/+ at 36 hpf.

Expression of mitochondrial complexes I and IV in supv3l1 mutant and sibling larvae. Larvae from a supv3l1^GtT/GtT incross were sorted by phenotype and analyzed individually by Western blot analysis for the levels of a complex I protein NDUFB6 using a monoclonal antibody (MS-108; MitoSciences) and the levels of a complex IV protein cytochrome C oxidase Va using a monoclonal antibody (MS-408; MitoSciences). Levels of β-actin were used as loading control.

Hepatocyte-specific Cre activity in hepatocyte-Cre embryos. (A) Double carriers of hepatocyte-Cre/Tg(eab2:[EGFP-T-mCherry]) were imaged at 5 dpf. Expression of the red fluorescent protein mCherry indicates Cre activity. (B–D) Hepatocyte-Cre does not rescue defects in intestinal epithelium. At 5 dpf, the intestinal epithelium in wild-type larvae is polarized and well organized with little cell death (B). In contrast, the intestinal epithelium in supv3l1^GtT/GtT mutants is disorganized with no apparent apical–basal polarity and abundant cell death (arrows) (C). Despite rescue of mature hepatocytes, the intestinal epithelium in supv3l1^LKO mutant is still disorganized with no apparent apical–basal polarity and abundant cell death (arrows) (D).

Additional evidence for hepatocyte-specific inactivation of supv3l1 in supv3l1^GtN/GtN, hepatocyte-Cre fish. (A) No mCherry was detected in 18-d-old supv3l1^GtN/GtN fish, including in the liver. (B) mCherry was detected only in hepatocytes of 18-d-old supv3l1^GtN/GtN, hepatocyte-Cre fish, but not in the 2F11+ ductal cells or in intestinal cells. GL, gut lumen; L, liver; K, kidney head. (C) Relative levels of supv3l1 transcripts in the liver of 18-d-old supv3l1^GtN/GtN and supv3l1^GtN/GtN, hepatocyte-Cre fish were determined by RT-PCR. Error bar = SEM. (D) Relative levels of supv3l1^GtT allele and supv3l1^GtN allele in the liver of 30-dold LiPan+//hepatocyte-Cre+// supv3l1^GtNf/+ and LiPan/supv3l1^LKO fish were determined by qPCR. Error bar = SD.