MAMMALIAN GENETIC MODELS WITH MINIMAL OR COMPLEX PHENOTYPES

Hepatocyte Nuclear Factor 3β (Foxa2) Is Dispensable for Maintaining the Differentiated State of the Adult Hepatocyte

Newman J. Sund, Siew-Lan Ang, Sara Dutton Sackett, Wei Shen, Nathalie Daigle, Mark A. Magnuson, Klaus H. Kaestner
DOI: 10.1128/MCB.20.14.5175-5183.2000

Article Figures & Data

Figures

  • Fig. 1.
    Fig. 1.

    Cre-loxP-mediated targeting of the HNF3βgene and generation of homozygous HNF3βloxP/loxP mice. (A) Targeting vector for the HNF3βloxP allele. Primers β5′ and β3′ were used in PCR genotype analysis. (First line) Gene structure of the endogenous HNF3β locus. Exons are indicated as boxes, the striped box represents the winged helix domain, open triangles represent loxP sites, and arrow heads represent primer positions. (Second line) Targeting vector which introduces a cassette containing the neomycin-herpes simplex virus-thymidine kinase selection cassette (neo-tk) flanked by loxP sites downstream of exon 3. An additional loxP site was introduced in the intron upstream of exon 3. (Third line) Gene structure of homologous recombinants. The 3′ probe was used for the Southern blot analysis (data not shown). (Fourth line) Cre-mediated deletion results in either the HNF3β null allele (deletion of exon 3) or the HNF3βloxP allele (exon 3 flanked by loxP sites). Abbreviations: Bg,BglII; E, EcoRI; Xh, XhoI; Xba,XbaI. (B) HNF3βloxP/loxP mice are viable and healthy. Shown are the results of PCR genotype analysis of a litter from mating HNF3βloxP/+ heterozygotes inter se. TheHNF3βloxP allele segregates in expected Mendelian frequency. WT, wild type.

  • Fig. 2.
    Fig. 2.

    The HNF3βloxP allele is excised by the Alb.Cre transgene. (A to G) Immunohistochemical analysis of liver sections from 11.5 (A and B), 14.5 (C and D), and 18.5 (E and F) d.p.c. embryos and 10-week-old adult (G and H) mice stained with anti-HNF3β antibody. HNF3βloxP/loxP; Alb.Cre mice express HNF3β by 14.5 d.p.c. (B and D), but HNF-3β protein is reduced in 18.5-d.p.c. fetal livers and absent from adult livers (F and H). The arrowhead in panel H shows a rare HNF3β immunoreactive nucleus. Images were obtained using Nomarski optics of livers from wild-type control mice (A, C, E, and G) and mutant HNF3βloxP/loxP; Alb.Cre mice (B, D, G, and H). Images are shown at ×90 (A, B, G, and H) or ×180 (D to F) magnification.

  • Fig. 3.
    Fig. 3.

    Expression of hepatic transcription factors in adult livers from HNF3βloxP/loxP; Alb.Cre. mice. (A) Total RNA (30 μg) from livers of wild type control or HNF3βloxP/loxP; Alb.Cre mice was analyzed by RNase protection assay for mRNA levels of all HNF3 genes. HNF3β mRNA is no longer expressed in livers of HNF3βloxP/loxP; Alb.Cre mice, whereas HNF3α and HNF3γ steady-state mRNA levels are unchanged in HNF3βloxP/loxP; Alb.Cre mice compared to controls. (B) Total RNA (30 μg) from livers of wild-type control or HNF3βloxP/loxP; Alb.Cre mice was analyzed by RNase protection assay for mRNA levels of other liver-enriched HNFgenes. HNF1α and HNF1β steady-state mRNA concentrations are unaltered whereas HNF4α mRNA levels are slightly increased in HNF3βloxP/loxP; Alb.Cre mice compared to controls when quantitated using PhosphorImager analysis (data not shown). TATA box binding protein (TBP) served as the loading control. (C) Nuclear binding activities of HNF3α and HNF3γ are not increased in HNF3βloxP/loxP; Alb.Cre hepatocytes compared to controls when analyzed using an EMSA. A radioactive oligonucleotide probe for the albumin enhancer eG site was incubated with 2 μg of nuclear extract isolated from wild-type control or HNF3βloxP/loxP; Alb.Cre liver. An 80-fold molar excess of nonradioactive competitor oligonucleotide (Comp) was added as indicated: −, no indicated competitor added; eG, HNF3 binding site from albumin enhancer; C, non-specific competitor containing CCAAT site from albumin promoter. Specific HNF3 binding complexes are indicated. HNF3α and -β complexes from liver extracts migrate very closely together, as shown previously (9).

  • Fig. 4.
    Fig. 4.

    Analysis of steady-state mRNA levels and microarray expression profile of potential HNF3 targets in liver. (A) Total RNA (10 μg) from livers of wild-type control (Control) or HNF3βloxP/loxP; Alb.Cre mice was separated on denaturing agarose gels, blotted onto nylon membrane, and hybridized to the probes indicated (Apo, apolipoprotein; SDH, serine dehydrogenase; PEPCK, phosphoenolpyruvate carboxykinase; TAT, tyrosine amino transferase; GLUT2, glucose transporter 2). β-Actin (Actin) served as a loading control. PhosphorImager analysis did not reveal significant differences between the mRNA levels between the control and HNF3βloxP/loxP; Alb.Cre mice (data not shown). (B) Scatter plot analysis representing expression profiles between livers from wild-type control and HNF3βloxP/loxP; Alb.Cre mice using an oligonucleotide microarray representing 8,700 mouse cDNA and EST transcripts. (C) Reverse transcription-PCR analysis of total RNA from livers of wild-type control (Control) or HNF3βloxP/loxP; Alb.Cre mice using [α-32P]dATP. Steady-state mRNA levels of α-globin (accession no. AA109900) is decreased twofold, whereas deiodinase (accession no. AA212899) is induced twofold in livers of HNF3βloxP/loxP; Alb.Cre mice compared to controls. Signals were quantified using PhosphorImager analysis (data not shown). Clone A (accession no. AA267590) EST mRNA levels were not significantly different between wild-type and mutant livers, although the microarray hybridization had indicated a threefold difference.

  • Fig. 5.
    Fig. 5.

    Mice lacking hepatic HNF3β have normal glucose homeostasis. Glucose challenge of age-matched HNF3βloxP/loxP; Alb.Cre (□) and wild-type control (⧫) mice. Blood glucose levels are shown at indicated time points after intraperitoneal administration of glucose. Values are means ± standard errors of the means (error bars) of seven (control) or nine (HNF3βloxP/loxP; Alb.Cre) animals. There was no significant difference between experimental and control groups as determined by Students's t test for each time point.

Tables

  • Table 1.

    Circulating intermediates in plasma in HNF3βloxP/loxP; Alb.Cre and control micea

    IntermediateConcn in plasma in miceReference range (concn)
    ControlHNF3βloxP/loxP; Alb.Cre
    Creatine phosphokinase (U/liter)2,241 ± 1,4452,114 ± 5740 – 800
    Lactate dehydrogenase (U/liter)581 ± 120580 ± 86260 – 680
    AST-SGOTb(U/liter)150 ± 36139 ± 2172 – 288
    ALT-SGPTc(U/liter)60 ± 2932 ± 424 – 140
    Gamma glutamyl transferase (U/liter)0 ± 00.83 ± 0.500 – 2
    Amylase (U/liter)2,199 ± 1602,778 ± 303602 – 2,311
    Bilirubin, total (mg/dl)0.67 ± 0.450.27 ± 0.050.0 – 0.9
    Bilirubin, direct (mg/dl)0.13 ± 0.090.07 ± 0.030.0 – 0.2
    Uric acid (mg/dl)4.4 ± 1.91.9 ± 0.22.2 – 4.6
    Blood urea nitrogen (mg/dl)21.3 ± 1.419.8 ± 1.59.0 – 28.0
    Creatinine (mg/dl)0.38 ± 0.030.33 ± 0.030.2 – 0.7
    Albumin (g/dl)2.9 ± 0.52.3 ± 0.42.6 – 4.6
    Protein, total (g/dl)4.7 ± 0.23.9 ± 0.44.0 – 6.2

    • ↵a Metabolic parameters were experimentally determined or analyzed by Ani Lytics, Inc., as described under Materials and Methods. Values are represented as means ± standard errors of the means. Comparisons were made among mice of similar age and sex (n = 6 animals for each group).

    • ↵b Aspartate aminotransferase-serum glutamic oxalacetic transaminase.

    • ↵c Alanine aminotransferase-serum glutamic pyruvic transaminase.

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