Ni et al., 2012 - Conditional control of gene function by an invertible gene trap in zebrafish. Proceedings of the National Academy of Sciences of the United States of America   109(38):15389-15394 Full text @ Proc. Natl. Acad. Sci. USA

Fig. 3 Phenotypes of supv3l1GtT/GtT mutants. (A) Gross morphological defects at 5 dpf in supv3l1Gt/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.

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Stage: Day 5
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Fig. 4 Liver-specific expression of supv3l1 in supv3l1GtT/GtT mutants rescues hepatocyte defects. (AC) Images of Lipan (A), LiPan/supv3l1GtT/GtT (B), LiPan/hepatocyte-Cre+/-/supv3l1GtT/GtT (C) showing liver agenesis in supv3l1GtT/GtT and liver-specific rescue by hepatocyte-Cre. Other defects in supv3l1GtT/GtT remain in hepatocyte-Cre+/-/supv3l1GtT/GtT. Each image is a merge of a bright-field image and a red fluorescent image of the same larva. (DF) 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 supv3l1GtT/GtT (12/114) (E), and the restoration in the liver-specific rescue hepatocyte-Cre+/-/supv3l1GtT/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), supv3l1GtT/GtT (H), and hepatocyte-Cre+/-/supv3l1GtT/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.

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Fig. 5 Phenotype of liver-specific supv3l1 inactivation. (AD) Confocal images of the Left (A and B) and Right (C and D) sides of 21-d-old LiPan (A and C) and LiPan/supv3l1LKO (B and D) fish showing detached hepatocytes (white arrows) (B and D) and smaller right liver lobe (white arrow) in supv3l1LKO fish (D). (E and F) Histological analysis of wild-type (E) and supv3l1LKO (F) fish indicate disorganized liver with dead cells (arrowhead) but normal intestinal lumen (black arrows) in supv3l1LKO fish (F). (F) Survival curve of supv3l1LKO fish (lens YFP+) compared with supv3l1GtNf/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 supv3l1LKO mutant at 5 wk of age showing the smaller size of the mutant.

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Stage: Days 21-29

Fig. S1 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.

Fig. S2 Gene-trap expression of mCherry in different mutagenized lines. (A) Image of fam49bbGtT/+ at 48 hpf. (B) Image of supv3l1GtT/+ at 24 hpf. (C) Image of tfcp2l1GtT/+ at 36 hpf.

Fig. S3 Expression of mitochondrial complexes I and IV in supv3l1 mutant and sibling larvae. Larvae from a supv3l1GtT/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.

Fig. S4 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 supv3l1GtT/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 supv3l1LKO mutant is still disorganized with no apparent apical–basal polarity and abundant cell death (arrows) (D).

Fig. S5 Additional evidence for hepatocyte-specific inactivation of supv3l1 in supv3l1GtN/GtN, hepatocyte-Cre fish. (A) No mCherry was detected in 18-d-old supv3l1GtN/GtN fish, including in the liver. (B) mCherry was detected only in hepatocytes of 18-d-old supv3l1GtN/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 supv3l1GtN/GtN and supv3l1GtN/GtN, hepatocyte-Cre fish were determined by RT-PCR. Error bar = SEM. (D) Relative levels of supv3l1GtT allele and supv3l1GtN allele in the liver of 30-dold LiPan+//hepatocyte-Cre+// supv3l1GtNf/+ and LiPan/supv3l1LKO fish were determined by qPCR. Error bar = SD.

Acknowledgments:
ZFIN wishes to thank the journal Proceedings of the National Academy of Sciences of the United States of America for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Proc. Natl. Acad. Sci. USA