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. 2001 Aug 14;98(17):9683-7.
doi: 10.1073/pnas.171283198.

Apaf-1 deficiency and neural tube closure defects are found in fog mice

N Honarpour  1 , S L Gilbert, B T Lahn, X Wang, J Herz
Affiliations

V体育平台登录 - Apaf-1 deficiency and neural tube closure defects are found in fog mice

N Honarpour et al. Proc Natl Acad Sci U S A. .

Abstract

The forebrain overgrowth mutation (fog) was originally described as a spontaneous autosomal recessive mutation mapping to mouse chromosome 10 that produces forebrain defects, facial defects, and spina bifida. Although the fog mutant has been characterized and available to investigators for several years, the underlying mutation causing the pathology has not been known. Because of its phenotypic resemblance to apoptotic protease activating factor-1 (Apaf-1) knockout mice, we have investigated the possibility that the fog mutation is in the Apaf-1 gene. Allelic complementation, Western blot analysis, and caspase activation assays indicate that fog mutant mice lack Apaf-1 activity VSports手机版. Northern blot and reverse transcription-PCR analysis show that Apaf-1 mRNA is aberrantly processed, resulting in greatly reduced expression levels of normal Apaf-1 mRNA. These findings are strongly suggestive of the fog mutation being a hypomorphic Apaf-1 defect and implicate neural progenitor cell death in the pathogenesis of spina bifida-a common human congenital malformation. Because a complete deficiency in Apaf-1 usually results in perinatal lethality and fog/fog mice more readily survive into adulthood, these mutants serve as a valuable model with which apoptotic cell death can be studied in vivo. .

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Figures

Figure 1
Figure 1
Partial chromosomal linkage map showing mouse chromosome 10 with loci linked to Apaf-1. (A) Apaf-1 maps 45.7 centimorgan (cM) distal to the centromere of mouse chromosome 10 by using the Jackson Laboratory interspecific backcross panel BSS. Ninety-four of the (C57BL/6JEi × SPRET/Ei)F1 × SPRET/Ei progeny were typed for inheritance of the Mus domesticus or Mus spretus alleles. The Jackson BSS map is depicted with the centromere toward the top. A 10 cM scale bar is shown to the left of the figure. Loci that map to the same position are listed in alphabetical order. (B) The loci linked to Apaf-1 are listed in order with the most proximal at the top. Black boxes represent the M. domesticus allele, and white boxes depict the M. spretus allele. For each haplotype, the number of animals in the BSS panel displaying that haplotype is listed below each column of boxes. The percent recombination between adjacent loci is listed in cM to the right of the figure, along with the SE. Raw data from The Jackson Laboratory were obtained from the World Wide Web address http://www.jax.org/resources/documents/cmdata.
Figure 2
Figure 2
Embryonic day 16.5 pups derived from Apaf-1 +/−; fog/+ crosses possess a mutant phenotype. (A) The phenotype of a wild-type pup. (B and C) The phenotypes of the mutant pups obtained. The phenotypes seen in the head region closely resemble those seen in fog/fog and Apaf-1 −/− newborns. (D) A wild-type embryo at a level analogous to that shown for the mutants. (E and F) Histological sections from the mutant pups shown in B and C. Note that the histological findings in these animals mimic those of the Apaf-1 knockout. olf, Olfactory bulb; br, poorly organized brain tissue in a mutant with cranioschesis; ex, exencephalic brain tissue; p, palate; r, retina.
Figure 3
Figure 3
Phenotypic variation in Apaf-1-deficient newborns closely resembles that seen in fog embryos. (A) A wild-type littermate. In the mutant shown in B, the generalized hypercellularity of the forebrain and basal ganglionic region results in exencephaly with compression and narrowing of the ventricles. Midline closure defects involving the palate and nasal septum are also seen in Apaf-1 mutant pups, and in extreme cases cranioschesis (as seen in C) occurs. (D) An anencephalic Apaf-1 mutant newborn. The telencephalon, mesencephalon, and diencephalon are absent, but remnants of the olfactory nerves remain. fb, Forebrain; olf, olfactory bulbs; p, palate; r, retina; br, poorly organized brain tissue.
Figure 4
Figure 4
Biochemical analysis of fibroblasts derived from mutant pups and fog/fog fibroblasts. (A) Fibroblast extracts derived from the mutant pups of the Apaf-1 +/−, fog/+ cross are compared with extracts derived from wild-type and Apaf-1 knockout mouse embryonic fibroblasts for reactivity to an antibody directed against Apaf-1. No detectable Apaf-1 (130 kDa in mass) is seen in the lane loaded with extracts from the fibroblasts of the mutant pup or from the Apaf-1-deficient pup. (B) The same extracts from A are tested for the ability to cleave 35S-labeled caspase-3 in vitro. Although extracts from wild-type fibroblasts are able to cleave procaspase-3, extracts from the mutant fibroblasts and Apaf-1-deficient fibroblasts fail to cleave and activate procaspase-3. (C) Protein and RNA prepared from fibroblasts of wild-type mice (WT) and mice homozygous for the fog mutation (fog/fog) were analyzed by using Western blot analysis (left), Northern blot analysis (center), and RT-PCR (right). No detectable Apaf-1 protein (130 kDa) or mRNA (7.5 kb) is present in fog/fog samples. Cyclophilin mRNA was probed on the same blot and used as a loading control (1.4 kb). Primer pairs used for RT-PCR (spanning all but 70 bp of the Apaf-1 cDNA) amplify a 3,600-bp fragment in the fog/fog sample but at a reduced level. This indicates that Apaf-1 message is present at significantly reduced quantities in fog mutants (the β-actin product running at 350 bp is shown as a positive control). (D) RT-PCR is used to amplify a fragment (primer pair 1 and 14) spanning exons 2 through 4 of Apaf-1 from fog/fog and wild-type first-strand cDNA. The product is less abundant and appears in higher PCR cycle numbers in fog/fog samples relative to wild-type samples.
Figure 5
Figure 5
Aberrant processing of the Apaf-1 transcript in fibroblasts derived from fog/fog mice. When primers flanking exons 2 and 4 (primers 1 and 13) in Apaf-1 are used, an aberrantly sized RT-PCR product is amplified (2 kb) in the fog/fog mutant. The 350-bp product is a β-actin positive control (shown in A). This fragment is smaller than the genomic fragment amplified by the same primers from wild-type or fog/fog DNA (shown in B) because it is mRNA that is partially processed. (C) Total RNA was prepared from the abdominal wall of mice carrying one fog allele (originated from the C3H/HeJ background) and one Apaf-1 allele in the BL6 background. The genetic background of the fog/fog mutant possesses a 14-bp insertion in the intron between exons 3 and 4 of Apaf-1 that is absent from the BL6 background. A common upstream primer (F, 5′-GCAAGGACACAGATGGTGGAATAAC-3′) and a pair of downstream primers (C3H/Hej, 5′-AGTTTGAGGACAGCCAGGTC-3′ or BL6, 5′-CTACAGAGTGAGTTTGTACAC-3′) designed to amplify an internal fragment within the 2-kb product were used in a PCR distinguishing the origin of the band, based on the presence or absence of the insertion. Left shows that the primer pair designed to include the insertion in the C3H/HeJ background preferentially amplifies the fragment. This indicates that the 2-kb band is produced by the Apaf-1 allele in the fog/fog mutant, and not a separate gene that regulates Apaf-1 expression. Right shows the same bands amplified from background specific DNA templates in each PCR (C3H/HeJ template in the left lane and BL6 template in the right lane). Thus, the two primer pairs are equally capable of amplifying the 340-bp fragment. (D) The diagram illustrates that the fog/fog fibroblasts splice out the intron between exon 2 and exon 3 (shown in blue), but fail to splice out the intron between exon 3 and exon 4 of Apaf-1 (shown in red, with an asterisk).
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