Subpopulation of GD17 fetuses exhibiting severe craniofacial phenotypes.

<p>Included in the study population were 9 fetuses with phenotypes not typically observed in wildtype C57BL/6J mice exposed to the employed ethanol exposure paradigm (A-I). Single allele mutations in <i>Shh</i> or <i>Gli2</i> were detected in 8 of 9 fetuses in this severely affected subpopulation. In addition to varying degrees of upper midfacial deficiency, other notable defects included exencephaly (A), iridial coloboma and microphthalmia (A-D), apparent anophthalmia (E, G, I), agnathia (E), micrognathia (A-D, F-I), and proboscis (I). Median cleft lip was also observed (B, C). Within this subpopulation, fetuses were assigned dysmorphology scores as follows: 2 (A), 3 (B-F), 4 (G-I).</p

Facial dysmorphology scores by treatment and genotype.

<p>Facial dysmorphology scores by treatment and genotype.</p

Effect of treatment and genotype on facial dysmorphology.

<p>To avoid litter bias, the average dysmorphology score from each genotypic group was determined for each litter in the study population. Values represent the mean plus the standard error of litter averages for each genotype and treatment. Brackets indicate p values of ≤ 0.05 as determined by a one-tailed student’s t-test.</p

Facial dysmorphology predicts medial forebrain deficiency.

<p>Superior views of dissected brains are shown for a normal fetus (A) and for representative examples of each category of facial dysmorphology (B-E). Medial facial deficiency was associated with increasing hypoplasia of the cerebral cortices (B-E), increasing hypoplasia (B, C) or absence of the olfactory bulbs (D, E), and incomplete division of the forebrain (D, E).</p

Facial dysmorphology rating scale.

<p>Illustrated are a GD 17 fetus having normal facial morphology and 4 fetuses with varying degrees of medial facial deficiency. Numbers assigned to each image (0–4) are scores representing differing degrees of severity of facial dysmorphology. As compared to normal fetuses, those receiving a score of 1 had a notably diminished area of pigmentation between the nostrils (solid arrow) accompanied by reduction in the depth of the normally present median central notch of the upper lip (dashed arrow). A score of 2 was assigned to fetuses that had lost the median lip notch, but still had some remaining pigment at the tip of the nose. Individuals presenting with a single central nostril were assigned a score of 3 and those given a score of 4 had no nostrils.</p

Phenotype of E13.5 embryos.

<p>Embryos are from a cross between <i>Ctgf Lo/+</i> and <i>EIIa-Cre +/-</i> parents. The number of embryos for each genotype classified as Normal, Small, or Small atypical. <i>+/+</i> n = 9, <i>Lo/+</i> n = 15, <i>Cre⁺</i> n = 11, <i>Hi/+</i> n = 7. The +/+ animals are wild type for <i>Ctgf</i> and do not have a Cre allele, <i>Lo/+</i> animals are heterozygous for the <i>Ctgf Lo</i> allele and do not have a Cre allele. <i>Cre⁺</i> are wild type for <i>Ctgf</i> and have one Cre allele. And <i>Hi/+</i> are heterozygous for the <i>Ctgf Hi</i> allele and have one Cre allele.</p

<i>Ctgf</i> mRNA levels by quantitative RT-PCR.

<p>A) <i>Ctgf</i> mRNA expression from tail samples of 10 day pups from a cross of <i>Ctgf Lo/+</i> parents. All mice are siblings. The mean of <i>Ctgf</i> +/+ (wild type) expression is set to 100% expression <i>LoLo</i>: n = 5, <i>Lo/+</i>: n = 25, and <i>+/+</i>: n = 21. B) <i>Ctgf</i> mRNA expression from tissues of five month old mice. <i>Ctgf Lo/+</i> mice are the control with <i>Lo/+</i> expression set to 85% based on results from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012909#pone-0012909-g002" target="_blank">Figure 2A</a>. For each organ <i>Lo/+</i>: n = 14 and <i>Lo/KO</i>: n = 8. C) <i>Ctgf</i> mRNA expression from whole mouse embryos at day points E11.5, normalized to <i>Lo/+</i> the same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012909#pone-0012909-g002" target="_blank">Figure 2B</a>. <i>Lo/+</i>: n = 8 and <i>Hi/+</i>: n = 13 D) <i>Ctgf</i> mRNA expression from tissues of 10 month old mouse tissues normalized to a combined control of animals of the genotypes <i>+/+;Cre-</i>, <i>Lo/+;Cre-,</i> and <i>+/+;Cre⁺</i>. E) Neo gene copy number in embryos at day E11.5. Copy number is in arbitrary units and normalized to <i>Lo/+</i> = 100%. <i>Lo/+</i>: n = 18 and <i>Hi/+</i>: n = 28. For all graphs the <i>Lo/+</i> animals are heterozygous for the <i>Ctgf Lo</i> allele and do not have a Cre allele. The <i>Hi/+</i> animals are heterozygous for the <i>Ctgf Hi</i> allele and have one EIIa-Cre allele.</p

<i>Ctgf</i> mRNA levels in tamoxifen treated adults and CTGF protein abundance in plasma.

<p>A) <i>Ctgf</i> mRNA determined by RT-PCR in tamoxifen treated mice from a cross of <i>Ctgf Lo/Lo</i> and <i>CAG-Cre +/−</i> animals on a B6.129 F1 background. All animals were treated with tamoxifen and <i>Lo/+</i> is set to 85% as a control. <i>Lo/+</i>: n = 23 and <i>Hi/+</i>: n = 20. B) Quantification of CTGF protein abundance in plasma as calculated using ImageJ. <i>Lo/Lo</i>: n = 5, <i>+/+</i>: n = 5, and <i>Hi/+</i>: n = 7 animals for each genotype. C) A 330 bp band corresponds to the <i>Ctgf Lo</i> allele and a 240 bp band corresponds to the <i>Ctgf Hi</i> allele. The PCR reaction is not quantitative but the presence of the 240 bp band indicates that Cre-mediated excision of DNA flanked by the loxP sequences has taken place. Presence of 330 bp band in animals with <i>Ctgf Hi</i> allele (Hi) indicates that the excision is not complete. DNA from the wild type animal (WT) does not amplify with these primers indicating the absence of the <i>Ctgf Lo</i> allele. The lane of size markers is indicated by M. D) A representative western showing a doublet band for CTGF just above 31 kDa for three <i>Ctgf Lo/Lo</i> mice, three WT mice and three <i>Ctgf Hi/+</i> mice. In A) <i>Lo/+</i> animals are heterozygous for the <i>Ctgf Lo</i> allele and do not have a Cre allele. In A), B), and D) <i>Hi/+</i> animals are heterozygous for the <i>Ctgf Hi</i> allele, have one tamoxifen-inducible CAG-Cre. All animals have been treated with tamoxifen.</p

DEXA and necropsy measurements of <i>Ctgf Hi/+</i> survivors and sibling controls.

<p>The top half of table is DEXA measurements of live mice at 10 months of age and the bottom is necropsy data. Mice were euthanized at 10 months of age and total body weight and organ weights were measured. Males and females were pooled. The p-value for each measurement is from Student T-test. Statistically significant p-values are shown in black. Controls: n = 9 and <i>Hi/+</i>: n = 4. Genotype of controls: <i>Lo/+</i>: n = 4, <i>Cre⁺</i>: n = 3, and <i>+/+</i>: n = 2. <i>Cre⁺</i> animals are wild type for <i>Ctgf</i> and <i>+/+</i> animals are wild type for <i>Ctgf</i> and do not have a Cre allele. BMD  =  bone mineral density and BMC  =  bone mineral content.</p

Survivors of <i>Ctgf Hi/+</i> embryonic lethality.

<p>Gross morphology of high <i>Ctgf</i>-expressing survivors at 10 months old. Males are shown in A) and B) and females are shown in C) and D). In A) and B) the <i>Ctgf Hi/+</i> mice (foreground) have small ears, shortened face and short body length. B) The short curly tail of the <i>Hi/+</i> male. D) Short kinked tail and short body of the <i>Hi/+</i> female (top) compared with a control sibling (bottom). All the animals shown are siblings from a cross between <i>Ctgf Lo/+</i> and <i>EIIa-Cre +/−</i> animals on a mixed B6.129 background. The controls (background of A) and C) and bottom of D)) are of the genotype <i>Ctgf Lo/+</i>.</p