Expression of <i>Shh</i> and β-catenin Transcripts in Normal <i>(Apc^CKO/CKO)</i> and Mutant <i>(K14-cre; Apc^CKO/CKO)</i> Embryonic Skin

<p>(A–D) Section ISH with <i>Shh</i> probe in E14.5 normal (A), E14.5 mutant (B), E16.5 normal (C), and E16.5 mutant (D) skin. Broken lines indicate the interface between epithelium and mesenchyme. Scale bars: 50 μm. Whole mount in situ detection of β-catenin in E15.5 normal (E, G), mutant (F, H) embryos. Aberrant initiation of multiple hair placodes is evident at E14.5. Loss of <i>K14</i>-driven <i>Apc</i> loss caused aberrant pattern formation (F′) and formed ectopic hair placodes in normally hairless foot pads (H, arrows) which are absent in normal (G).</p

Tissue-Specific Detection and Expression of Deleted <i>Apc</i> Alleles

<div><p>(A) Tissue-specific genotyping PCR. Only genomic DNA samples from the skin (S) and thymus (T), but not liver (L) of mice positive for <i>K14-cre</i> show the presence of deleted <i>Apc^Δ580</i> allele.</p><p><b>(</b>B<b>)</b> Genotype- and tissue-specific expression of the truncated <i>Apc</i> transcripts. A representative gel of RT-PCR using primers F546 and R721, showing that only RNA from the skin and thymus but not liver of mice positive for <i>K14-cre</i> have transcripts from both wild-type (528 bp) and deleted (313 bp) <i>Apc</i> alleles.</p></div

Postnatal Mortality and Stunted Growth in <i>K14-cre; Apc^CKO/CKO</i> Mutant Mice

<div><p>Animals whose genotype is either heterozygous or homozygous for the wild-type <i>Apc</i> allele are referred to as normal (N); those whose genotype are <i>K14-cre; Apc^CKO/CKO</i> and show the presence of <i>K14-cre</i>–recombined mutant <i>Apc</i> allele are called mutant (M).</p><p>(A) Two P3 mutant mice, M1 and M2, and their normal littermates, showing size variation among mutants.</p><p>(B) P8 mutant mouse (right) and a normal littermate. Note sparseness of hair coat and abnormal ears.</p><p>(C–D) Vibrissae of whisker pads are short and oddly angled in a P12 mutant mouse (C), relative to control (D). Note the lack of incisors in the mutant.</p><p>(E) A P17 mutant mouse (right) with its littermate. Its bare forehead, dorsal median line, and abnormal ears are evident.</p><p>(F) Growth curve of mutants and normal littermates. Mutants exhibit stunted growth, which became more prominent as they aged, and weigh significantly less than littermates from P8 (<i>p</i> < 0.05).</p><p>(G) Comparison of mutant and normal thymus from P3 mice. The mutant thymus (left) is dramatically smaller for its age compared to the normal littermate (right). The scale bar equals 1 mm.</p><p>(H) Skeletal preparations of normal (left) and mutant (right), showing differences in development of both incisor (I) and molar (M) teeth.</p></div

Generation of the Conditional <i>Apc</i> Allele

<div><p>(A) Schematic diagram of exons 14 and 15 of the mouse <i>Apc</i> gene, the targeting vector, and the resulting conditional allele with 2 LoxP sites sandwiching the exon 14. The PGK-neomycin cassette was inserted within intron 14 by recombineering technique. This cassette is sandwiched by 2 FRT sites that could be removed by crossing to FLPe-expressing mice. Positions of PCR primers used for genotyping PCR (F2, R2, R4) and RT-PCR (F546 and R721) are indicated. Positions of probe used for Southern blot analysis with NdeI sites are also shown. Upon Cre-mediated recombination, exon 14 is removed and leads to truncated Apc protein, of which the first 580 aa correspond to the normal.</p><p>(B) Southern blot analysis of NdeI-digested genomic tail DNA isolated from F1 mice of various <i>Apc</i> mouse lines <i>(Apc^CKON, Apc^Δ580),</i> hybridized to a 600-bp probe. Tail genomic DNA from <i>Apc^CKON</i> F1 mice derived from a modified ES clone showed a 12-kb band for the <i>Apc^CKON</i> allele and a 10-kb band for the wild-type allele, whereas genomic DNA from the <i>Apc^Δ580</i> mouse was heterozygous for the <i>Apc^Δ580</i> allele (9.2-kb band).</p><p>(C) Kaplan-Meier survival plot of <i>Apc^CKO/+</i> mice (thin solid line, <i>n</i> = 39), <i>Apc^CKO/CKO</i> mice (thin dotted line, <i>n</i> = 57), <i>Apc^Δ580/+</i> mice (solid line, <i>n</i> = 51), and wild-type littermates (broken line, <i>n</i> = 21). Heterozygosity of the <i>Apc^Δ580</i> allele led to a significantly shortened survival (<i>p</i> < 0.0001), whereas those of heterozygous and homozygous <i>Apc^CKO</i> mice had no significant difference to that of wild-type littermates.</p></div

Histological and Immunochemical Examination of Thymus

<p>(A–D) P3 normal thymus. (E–G) Mild P3 mutant thymus. (H–K) Severe P3 mutant thymus. (L–O) P13 mutant thymus. Stained with H&E for histology (A, E, H, L), BrdU (B, I, M), β-catenin (C, F, J, N), and K14 (D, G, K, O). (B) Actively dividing thymocytes are visible at the superficial edge of cortex of normal P3 thymus. Note the progression of histological abnormalities in the mutant thymus from mild P3, severe P3 to P13 (A, E, H, L). Scale bars, 20 μm.</p

Deletion of <i>Msh2</i> in medium-spiny striatal neurons eliminates the majority of striatal <i>HTT</i> CAG expansions.

<p>GeneMapper traces of PCR-amplified <i>HTT</i> CAG repeats from striatum, cortex, liver and tail DNA of representative five-month <i>HdhQ111</i>/+ mice (<b>A</b>) or from striatum and tail of representative ten month <i>HdhQ111</i>/+ mice (<b>C</b>) with <i>Msh2</i>+/+, <i>Msh2</i>+/−, <i>Msh2Δ</i>/<i>Δ</i>, <i>Msh2Δ</i>/− and <i>Msh2</i>−/− genotypes. Constitutive CAG repeat lengths, as determined in tail DNA, are indicated. Instability indices were quantified from GeneMapper traces of PCR-amplified <i>HTT</i> CAG repeats from five-month striatum, cortex and liver (<b>B</b>) and ten-month striatum (<b>D</b>) of <i>HdhQ111</i>/+ mice with <i>Msh2</i>+/+, <i>Msh2</i>+/−, <i>Msh2Δ</i>/<i>Δ</i>, <i>Msh2Δ</i>/− and <i>Msh2</i>−/−genotypes. Five-month mice: <i>Msh2</i>+/+ (n = 6, CAG 113, 118, 119, 121, 123, 125), <i>Msh2</i>+/− (n = 4, CAG 114, 114, 120, 123), <i>Msh2Δ</i>/<i>Δ</i>(n = 5, CAG 113, 121, 121, 126, 129), <i>Msh2Δ</i>/−(n = 7, CAG 113, 121, 121, 122, 125, 125, 133) and <i>Msh2</i>−/− (n = 3, CAG 112, 120, 123). Ten-month mice: <i>Msh2</i>+/+ (n = 6, CAG 118, 121, 121, 123, 126, 134), <i>Msh2</i>+/− (n = 4, CAG 116, 118, 123, 131), <i>Msh2Δ</i>/<i>Δ</i> (n = 1, CAG 133), <i>Msh2Δ</i>/− (n = 7, CAG 115, 115, 117, 120, 121, 122, 123) and <i>Msh2</i>−/− (n = 1, CAG 132). Bars represent mean ± S.D. *** p<0.0001, * p<0.05 (Student’s t-test).</p

Conditional deletion of the <i>floxed Msh2</i> allele in the striatum. A.

<p>Genotyping for the conditional <i>Msh2</i> allele in genomic DNA extracted from striatum of <i>Msh2</i>+/+, <i>Msh2flox</i>/+, <i>Msh2flox</i>/+ <i>D9-Cre</i> and <i>Msh2flox</i>/flox <i>D9-Cre</i> mice. The deleted (Δ) <i>Msh2</i> allele is present only in mice harboring both the <i>Msh2flox</i> allele and the <i>D9-Cre</i> transgene. <b>B.</b> Genotyping for the conditional <i>Msh2</i> allele in genomic DNA extracted from five different tissues from a <i>Msh2flox</i>/+ <i>D9-Cre</i> mouse shows that the deletion is specific for the striatum. Mice were six weeks of age. <i>flox</i>: <i>Msh2</i> allele flanked by <i>loxP</i> sites; Δ:deleted <i>Msh2</i> allele; wt: wild-type <i>Msh2</i> allele.</p

Deletion of Msh2 in medium-spiny neurons delays nuclear huntingtin phenotypes. A, B.

<p>Nuclear mutant huntingtin immunostaining is decreased in the striata of five-month old <i>HdhQ111</i>/+ mice with deletion of <i>Msh2</i> in MSNs. <b>A.</b> Fluorescent micrographs of striata double-stained with anti-huntingtin mAb5374 and anti-histone H3 antibodies for three CAG repeat length-matched mice (<i>Msh2</i>+/+ CAG 113, <i>Msh2Δ</i>/<i>Δ</i> CAG 112, <i>Msh2−/−</i> CAG 113). <b>B.</b> Box plot showing upper and lower quartiles, median and range for the normalized mAb5374 immunostaining intensity (total mAb5374 staining intensity normalized to the number of H3-positive nuclei). Outlier (circle) is defined by a standard interquartile method and is included in the analysis. Multiple regression analysis was used to determine the effect of <i>Msh2</i> genotype on mAb5374 staining using normalized mAb5374 intensity (continuous variable) as a dependent variable and <i>Msh2</i> genotype (discrete variable), constitutive CAG length (continuous variable) and position (medial versus lateral, discrete variable) as independent variables. Both constitutive CAG length (P<0.05) and medial versus lateral position (P<0.001) were significantly associated with normalized mAb5374 intensity. Asterisks above the bars indicate a significant difference from <i>Msh2</i>+/+ at a p-value cut-off of p<0.05(*), p<0.01 (**), p<0.001 (***) in the regression analysis. <i>Msh2Δ</i>/− was not significantly different from <i>Msh2</i>+/− (p = 0.18). The five-month mice used in the quantitative analysis are as follows: <i>Msh2</i>+/+ (n = 6, CAG 113, 118, 119, 121, 123, 125), <i>Msh2</i>+/− (n = 4, CAG 114, 114, 120, 123), <i>Msh2Δ</i>/<i>Δ</i> (n = 5, CAG 113, 121, 121, 126, 129), <i>Msh2Δ</i>/− (n = 7, CAG 113, 121, 121, 122, 125, 125, 133) and <i>Msh2</i>−/− (n = 3, CAG 112, 120, 123). Note that the relatively “weak” effect of the <i>Msh2</i>−/− genotype likely reflects the small number of mice of this genotype and hence the least accurate estimate of the relationship of mAb5374 intensity to CAG length in the regression analysis. <b>C, D.</b> Intranuclear inclusions are decreased in the striata of ten-month old <i>HdhQ111</i>/+ mice with deletion of <i>Msh2</i> in MSNs. <b>C.</b> Fluorescent micrographs of striata stained with mAb5374 from mice with <i>Msh2</i>+/+ (CAG 121), <i>Msh2</i>+/− (CAG 123), <i>Msh2Δ</i>/<i>Δ</i> (CAG 133), <i>Msh2Δ</i>/− (CAG 123) and <i>Msh2</i>−/− (CAG 132) genotypes. <b>D.</b> Quantification of the percentage of cells containing an inclusion (more than one inclusion per cell was rarely observed). The total number of cells was determined by co-staining with histone H3 (not shown). The ten-month mice used in the quantitative analysis are as follows: <i>Msh2</i>+/+ (n = 6, CAG 118, 121, 121, 123, 126, 134), <i>Msh2</i>+/− (n = 4, CAG 116, 118, 123, 131), <i>Msh2Δ</i>/<i>Δ</i> (n = 1, CAG 133), <i>Msh2Δ</i>/− (n = 7, CAG 115, 115, 117, 120, 121, 122, 123) and <i>Msh2</i>−/− (n = 1, CAG 132). Bars represent mean ±S.D.</p

Elevation of components of the GLUT4 trafficking pathway during mouse skeletal muscle regeneration.

<p>A) VAMP2 and CHC22 levels during muscle regeneration after cardiotoxin injection on day 0 were compared between WT and CHC22-mice in muscle samples harvested on the day indicated. One typical set of immunoblots is shown at the left and quantification of VAMP2 relative to GAPDH signals from three experiments generated the ratios plotted at the right (upper plot). There was a statistically significant difference between WT and CHC22-mice in VAMP2 expression on day 14 after cardiotoxin injection (*<i>p</i><0.05), as determined by Student’s t test. Quantification of VAMP2 and CHC22 relative to GAPDH in the CHC22-mice shown at the left was plotted (right, bottom). B) GLUT4 levels during muscle regeneration for wild type and CHC22-mice (n = 3). One typical set of immunoblots is shown at the left and quantification of GLUT4 relative to GAPDH signals is plotted on the right. No significant difference between WT and CHC22-mice was detected. Molecular mass (kilodaltons) of the proteins detected is indicated at the right.</p

Delayed maturation and fiber type analysis of regenerating fibers in CHC22-transgenic mice.

<p>A) Hematoxylin and eosin staining of transverse muscle sections from WT or CHC22 transgenic mice after cardiotoxin injection at day 0, on the indicated days (D). Control (CTRL) sections were prepared from uninjured muscle. On day 28, some CHC22 myofibers were similar in cross-sectional area and diameter to WT myofibers; however, smaller myofibers (<40 µm diameter, marked by asterisks) were more frequently observed in the CHC22-mice compared to WT mice (scale bar, 20 µm; <i>n = </i>3). B) The mean cross-sectional area of myofibers with centrally located nuclei from WT (white bars) and CHC22-mice (black bars) was calculated and plotted for each indicated day after cardiotoxin injection. Control mice for each strain were not injected with cardiotoxin. There was a statistically significant difference in the average fiber cross-sectional area between WT mice and CHC22-mice on days 14 and 28 after injection (<i>n</i> = 3, evaluating 1200–2000 myofibers per mouse; **<i>p</i><0.01), as determined by Student’s t test. C) Transverse sections of muscle from CHC22-mice and WT mice, harvested 28 days after cardiotoxin injection, were stained for NADH-TR in order to determine the myofiber type. Oxidative (red) myofibers appear dark, glycolytic (white) myofibers appear light and intermediate (pink) myofibers appear intermediate in color. D) Fibers in 28-day regenerating muscle from CHC22-mice (black bars) and WT mice (white bars) were classified by type and their cross-sectional area measured in pixels using ImageJ (n = 3, evaluating ∼1500 fibers per mouse, *p<0.05, by Student’s t test). E) The percent of each fiber type in 28-day regenerating muscle from CHC22-mice (black bars) and WT mice (white bars) from the analysis in D is plotted.</p