Figure 1.
Ctgf KO and Ctgf Lo-Hi allele.
A) Ctgf KO allele. Line 1 is the endogenous Ctgf locus. Line 2 is the Ctgf KO allele construct. Line 3 is the result following homologous recombination where exons 3–5 of the Ctgf gene were replaced with a neo cassette to generate the Ctgf KO allele. Black boxes indicate coding exons and a white box indicates the endogenous 3′UTR. B) Ctgf Lo-Hi allele. Line 1 is the endogenous Ctgf locus. The Ctgf Lo-Hi allele was generated by replacement of the endogenous Ctgf-3′UTR with a construct containing the c-fos-3′UTR and a Neo cassette flanked by loxP sites, followed by the bGH-3′UTR. In line 2, the modified Ctgf gene uses the less stable c-Fos-3′UTR which reduces gene expression (Ctgf Lo allele). In the bottom line, following Cre-mediated recombination, the c-Fos-3′UTR and Neo cassette are excised and the Ctgf gene uses the more stable bGH-3′UTR which increases gene expression (Ctgf Hi allele). TAA indicates a stop codon Restrcition sites are abbreviated B is BamH1, Bc is Bcl1, Nhe is Nhe1, Nsi is Nsi1, and Bg is Bgl2.
Figure 2.
Ctgf mRNA levels by quantitative RT-PCR.
A) Ctgf mRNA expression from tail samples of 10 day pups from a cross of Ctgf Lo/+ parents. All mice are siblings. The mean of Ctgf +/+ (wild type) expression is set to 100% expression LoLo: n = 5, Lo/+: n = 25, and +/+: n = 21. B) Ctgf mRNA expression from tissues of five month old mice. Ctgf Lo/+ mice are the control with Lo/+ expression set to 85% based on results from Figure 2A. For each organ Lo/+: n = 14 and Lo/KO: n = 8. C) Ctgf mRNA expression from whole mouse embryos at day points E11.5, normalized to Lo/+ the same as Figure 2B. Lo/+: n = 8 and Hi/+: n = 13 D) Ctgf mRNA expression from tissues of 10 month old mouse tissues normalized to a combined control of animals of the genotypes +/+;Cre-, Lo/+;Cre-, and +/+;Cre+. E) Neo gene copy number in embryos at day E11.5. Copy number is in arbitrary units and normalized to Lo/+ = 100%. Lo/+: n = 18 and Hi/+: n = 28. For all graphs the Lo/+ animals are heterozygous for the Ctgf Lo allele and do not have a Cre allele. The Hi/+ animals are heterozygous for the Ctgf Hi allele and have one EIIa-Cre allele.
Figure 3.
Embryos are from a cross between Ctgf Lo/+ and EIIa-Cre +/- parents. The number of embryos for each genotype classified as Normal, Small, or Small atypical. +/+ n = 9, Lo/+ n = 15, Cre+ n = 11, Hi/+ n = 7. The +/+ animals are wild type for Ctgf and do not have a Cre allele, Lo/+ animals are heterozygous for the Ctgf Lo allele and do not have a Cre allele. Cre+ are wild type for Ctgf and have one Cre allele. And Hi/+ are heterozygous for the Ctgf Hi allele and have one Cre allele.
Figure 4.
A) and E) are fixed embryos at E14.5 from a cross between Ctgf Lo/+ and EIIa-Cre +/− B6.129 mixed background parents. A) is a normal Lo/+ and E) is a small atypical Hi/+ at higher magnification. Top arrow denotes less developed eye in E) and the lower two arrows point to lateral and midline facial clefting, respectively. Embryos B) to D) and F) to H) are E10.5 embryos from a cross between Ctgf Lo/Lo and EIIa-Cre +/− 129/SvEv background parents. B), C), and F), G), are unfixed embryos. B), C) and D) are normal Ctgf Lo/+ embryos. F), G), and H) are Ctgf Hi/+ embryos. The top arrow in B) and F) points to the forebrain which appears smaller in F) and the lower arrow points to the mouth gape in F) also indicative of a small forebrain. The top arrow in C) and G) points to the eye and the bottom arrow points to the hindbrain which both appears abnormal in G). The pictures in D) and H) are from scanning electron microscopy. The arrows in D) and H) point to the first (top) and second (bottom) pharangeal (branchial) clefts. In D) there is normal closure of the clefts and in H) the clefts are abnormally patent (open). B) and C) are the same embryo and F) to H) are the same embryo. The bars in A), B), E), and F) represent 1000 µm and in C), D), G), and H) the bars represent 250 µm.
Figure 5.
Immunofluorescence of E11.5 Ctgf Lo and Hi embryos.
Panels A) – D) are stained with an anti-CTGF antibody and a Alexa 594 (Texas Red) secondary. Panels E) – H) are stained with DAPI to highlight nuclei. Panels I) – L) are a merge of the Red and the DAPI layer. Genotypes for each panel are as marked on the panel. The panels in each row are from the same section. All panels are at 5× magnification.
Figure 6.
Survivors of Ctgf Hi/+ embryonic lethality.
Gross morphology of high Ctgf-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 Ctgf Hi/+ mice (foreground) have small ears, shortened face and short body length. B) The short curly tail of the Hi/+ male. D) Short kinked tail and short body of the Hi/+ female (top) compared with a control sibling (bottom). All the animals shown are siblings from a cross between Ctgf Lo/+ and EIIa-Cre +/− animals on a mixed B6.129 background. The controls (background of A) and C) and bottom of D)) are of the genotype Ctgf Lo/+.
Table 1.
DEXA and necropsy measurements of Ctgf Hi/+ survivors and sibling controls.
Figure 7.
Ctgf mRNA levels in tamoxifen treated adults and CTGF protein abundance in plasma.
A) Ctgf mRNA determined by RT-PCR in tamoxifen treated mice from a cross of Ctgf Lo/Lo and CAG-Cre +/− animals on a B6.129 F1 background. All animals were treated with tamoxifen and Lo/+ is set to 85% as a control. Lo/+: n = 23 and Hi/+: n = 20. B) Quantification of CTGF protein abundance in plasma as calculated using ImageJ. Lo/Lo: n = 5, +/+: n = 5, and Hi/+: n = 7 animals for each genotype. C) A 330 bp band corresponds to the Ctgf Lo allele and a 240 bp band corresponds to the Ctgf Hi 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 Ctgf Hi 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 Ctgf Lo 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 Ctgf Lo/Lo mice, three WT mice and three Ctgf Hi/+ mice. In A) Lo/+ animals are heterozygous for the Ctgf Lo allele and do not have a Cre allele. In A), B), and D) Hi/+ animals are heterozygous for the Ctgf Hi allele, have one tamoxifen-inducible CAG-Cre. All animals have been treated with tamoxifen.
Table 2.
Strains in the Ctgf allelic series.