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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2001 Aug 14;98(17):9683–9687. doi: 10.1073/pnas.171283198

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

Narimon Honarpour *, Sandra L Gilbert †,‡, Bruce T Lahn †,‡, Xiaodong Wang §,¶, Joachim Herz *,‖
PMCID: PMC55512  PMID: 11504943

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. 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.

Keywords: spina bifida, neural development, apoptosis, caspase, cell death

Neural tube closure defects account for a significant portion of the congenital malformations that affect humans (1). Such defects include spina bifida, exencephaly, cranioschisis, and hydrocephaly. The cause and inheritance of these defects are complex, reflecting the complex developmental events required for neural tube formation and closure.

Neural plate formation, neural fold elevation, neural tube closure, and neural crest cell migration are just a few of the operations that are tightly regulated during neurulation. Many mouse models have been used to study neural tube defects (2). The mutants studied have either arisen spontaneously or have been generated by using targeted gene disruption. One such mouse model is the forebrain overgrowth (fog) mutant, which has a spontaneous mutation in an uncharacterized gene and is transmitted in an autosomal recessive manner (3).

Apoptotic cell death has been shown to play a critical role in neurogenesis. Specifically, proteins required for the cytochrome-c-mediated apoptotic pathway [such as apoptotic protease activating factor-1 (Apaf-1), caspase-9, and caspase-3] are important for neural tube closure in vivo (4–9). In this biochemical cascade, cytochrome c released from the mitochondria is recruited to Apaf-1. In the presence of dATP, the cytochrome-c/Apaf-1 complex is able to procure and promote activation of procaspase-9 to caspase-9. The activation of caspase-9 then results in the subsequent activation of caspase-3, a terminal event in apoptotic cell death. In mice, Apaf-1 deficiency results in a dramatic elevation in the number of neural progenitor cells resulting in the thickening of the neuroepithelium (9). Because morphogenetic forces play a critical role in neurulation (10), it is likely that the abundance of neural progenitors in Apaf-1 mutants profoundly affects the ability of the neural tube to close. Given that apoptotic cell death is crucial for appropriate neural development, uncharacterized mutant models that are used to study neurulation may possess defects in apoptotic programs. In this study, we show that mice with the fog mutation are functionally deficient in Apaf-1 protein. We find that Apaf-1 transcripts are aberrantly processed in homozygous fog mutants, resulting in reduced levels of the normal Apaf-1 message. In addition to the phenotypes that have been previously reported for the Apaf-1 knockouts generated by targeted gene disruption, the fog mutants frequently have spina bifida and some surviving adult animals have typical neural tube closure defects (3). These findings suggest that the degree of Apaf-1 expression is critical in the regulation of neural progenitor cell death, and that perturbations in this regulation result in neurodevelopmental pathology such as spina bifida.

Materials and Methods

Mapping of the Apaf-1 Allele.

Polymorphisms between the Apaf-1 alleles of C57BL/6J and SPRET mice were identified by sequencing the 3′ end of the locus. This revealed a 10-bp allelic size difference within the 3′ untranslated region (UTR). The following PCR primers were designed to generate a product that spans the polymorphism: Apaf-1-gap-F: 5′-CTATGAGCTAGTCATGTGTTAGA-3′ and Apaf1-gap-R: 5′-CCAATTCACAGACACTGACA-3′. Genomic DNA samples derived from 94 N2 animals from the BSS backcrosses (C57BL/6JEi × SPRET/Ei)F1 × SPRET/Ei, or the reciprocal BSB backcrosses (C57BL/6JEi × SPRET/Ei)F1 × C57BL/6JEi were analyzed by PCR for the 3′ UTR polymorphism. PCR was performed in a 20-μl volume that contained 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris (pH 9.0), 1% Triton X-100, 0.5 μM each primer, 0.1 mM dNTPs, 1 unit of Taq DNA polymerase, and 25 ng of genomic DNA. After denaturation at 95°C for 1.5 min, 30 cycles of PCR were performed where each cycle consisted of 1 min at 95°C, 45 sec at 60°C, and 45 sec at 72°C. Products were then resolved on 4% NuSieve agarose (FMC) gels. The results were then compared with The Jackson Laboratory Backcross Database (available at http://www.jax.org/resources/ documents/cmdata; see also http://www.informatics.jax.org/).

Generation of Apaf-1 Knockout Mice.

Mice carrying the disrupted Apaf-1 allele were generated as described (9). Mice carrying the fog mutation were obtained from The Jackson Laboratory (stock no. 002979). Embryos possessing one disrupted Apaf-1 allele and one fog mutant allele were obtained by crossing male mice heterozygous for the fog mutation with females heterozygous for a disrupted Apaf-1 allele.

Histology.

Specimens were fixed in 4% paraformaldehyde overnight and subsequently embedded in paraffin for sectioning. Sections were cut at 5-μm increments, and stained with hematoxylin/eosin.

Preparation of S-100 Extracts from Fibroblasts.

Mouse embryonic fibroblasts derived from day 16.5 embryos were prepared and maintained by using the procedure outlined (11). Primary fibroblast cultures were derived from the adult mouse tail. Pieces of the tissue were allowed to adhere to 25-cm2 flasks and were incubated in Dulbecco's modified media (high glucose with glutamine) supplemented with 20% FCS and penicillin/streptomycin. Fibroblasts were harvested and collected by centrifugation (1,800 × g for 15 min at 4°C). Cell pellets were washed once with ice-cold PBS and resuspended in 5 vol of ice-cold buffer A (20 mM Hepes, pH 7.5/10 mM KCl/1.5 mM MgCl2/1 mM NaEDTA/1 mM DTT/0.1 mM PMSF). After 15 min of incubation on ice, cells were homogenized with 20 strokes of a Dounce homogenizer. After centrifugation (2,000 × g, 5 min at 4°C) the supernatant was collected and centrifuged again (100,000 × g, 1 h at 4°C). The resulting supernatant (S-100 extract) was stored at −80°C.

Immunoblot Analysis.

Western blot analysis of Apaf-1 was performed as described (12). Immunoblot analysis was performed by using horseradish peroxidase-conjugated goat anti-rabbit IgG and enhanced chemiluminescence (ECL) Western blotting detection reagents (Amersham Pharmacia).

In Vitro Caspase-3 Activation Assay.

Procaspase-3 was translated in vitro and purified as described (13). A 1-μl aliquot of the in vitro translated procaspase-3 was incubated with 50 μg of cell extract in the presence or absence of 1 mM dATP and 1 mM MgCl2 at 30°C for 1 h. Before incubation, the volume was adjusted to 20 μl with buffer A. After incubation, 7 μl of 4× SDS sample buffer was added, samples were then boiled for 3 min, and then subjected to SDS/15% PAGE. The gel was transferred to a nitrocellulose filter, and 35S-labeled protein bands were visualized on a Fuji BAS-1500 phosphorimager.

Reverse Transcription (RT)-PCR.

Total RNA was purified from mouse embryonic fibroblasts by using the RNA-STAT-60 RNA isolation reagent. The total RNA was used to generate first-strand cDNA by using the SuperScript II reverse transcriptase kit and oligo(dT) primer available from Life Technologies. The first-strand cDNA was used for amplifying fragments of the Apaf-1 message. Oligonucleotides used in amplifying the aberrantly spliced Apaf-1 message from the first strand cDNA were the 1 and 13 primers, which have the sequence 5′-CATCAAGACATCCTACATCATGGATCACATG-3′ and 5′-GGTACTCCACCTTCACACAGCACTG-3′ respectively. Nearly full-length transcripts were amplified with the 1 primer and the 11 primer, 5′-CCGAGATTATCGACAGTCACATAGG-3′. The remainder of the coding region was amplified by using primers with the reverse complement sequence of 1 and 11 with the primers 5′-GCAATCTAGTCTCATAAGTGACCCTC-3′ and 5′-GGGTCAAGCTCAGTCGAGTATAACT-3′, respectively. PCR used to quantify the difference in Apaf-1 message between fog mutants and wild-type animals used primer pair 1 and 14, 5′-TCATGAGAAGCCCAGATTTGTCTTG-3′. β-Actin message was amplified by using primers provided with the RT-PCR Primer and Control Set kit from Life Technologies.

Northern Analysis.

Northern blots were performed as described (14). Total RNA obtained from cultured fibroblasts was eluted over an oligo(dT) column (mRNA purification kit, Amersham Pharmacia), and ≈5 μg of resulting RNA preparation was loaded per well. The blots were probed with a 32P-labeled fragment spanning 3,600 bp of the Apaf-1 cDNA (amplified by the 1 and 11 primer pair).

Results

The similarity in phenotype between the Apaf-1 −/− mice and fog mutant mice, as well as the proximity of both loci on mouse chromosome ten (3, 5), suggested Apaf-1 as a candidate gene for the fog mutation. To determine the chromosomal localization at a higher resolution, Apaf-1 was mapped by using two interspecific backcross mouse DNA panels from The Jackson Laboratory (15). Genomic DNA samples derived from 94 N2 animals from the BSS backcrosses (C57BL/6JEi × SPRET/Ei)F1 × SPRET/Ei were analyzed for a clear PCR-based polymorphic marker in the 3′ untranslated region of the Apaf-1 locus between the two species. The segregation pattern of the polymorphism was compared with The Jackson Laboratory Backcross Database, where the marker was found to cosegregate with D10Mit67 and closely segregate with several adjacent loci located on mouse chromosome 10 (Fig. 1). The mapping places the Apaf-1 locus at consensus position 45.7 on mouse chromosome 10. Similar data, albeit of lower resolution, were obtained from the reciprocal BSB backcross panel (C57BL/6JEi × SPRET/Ei)F1 × C57BL/6JEi (data not shown). Previous linkage analysis has shown that the fog mutant phenotype does not recombine with either D10Mit262 [47.5 centimorgan (cM)] or D10Mit230 (49.0 cM) (3). Thus, Apaf-1 maps within the minimal region to which the fog mutation has been localized (Fig. 1).

Figure 1.

Figure 1

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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.

To further evaluate the possibility that the Apaf-1 and fog alleles represent the same gene, female mice heterozygous for a disrupted Apaf-1 allele were crossed to male mice heterozygous for the fog mutation (obtained from The Jackson Laboratory). The pups from this cross were harvested at 16.5 days of gestation (Fig. 2). A variety of mutant phenotypes were observed, consistent with the defects reported for fog mutants (3) and with those seen in Apaf-1-deficient mice (Fig. 3). The fog/Apaf-1 mutant phenotype included exencephaly, cranioschesis, or either forebrain defect with lumbosacral defects. fog/fog embryos have been reported to have similar defects in addition to lumbosacral defects alone. The relative frequency of the different phenotypes was not assessed in this experiment; the frequency of these defects has been quantified in both fog mutants and Apaf-1 knockout mice (3, 6).

Figure 2.

Figure 2

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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

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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.

Western blot analysis of mouse embryonic fibroblasts derived from these mutant pups revealed an absence of Apaf-1 protein (Fig. 4A). Extracts prepared from these cells also failed to activate caspase-3 in vitro, consistent with the absence of Apaf-1 (Fig. 4B) (13). Furthermore, fibroblasts derived from fog/fog mutants had no detectable Apaf-1 transcript or protein by Northern (Fig. 4C Center) or Western blot analysis (Fig. 4C Left), respectively. Because the Apaf-1 message is normally expressed at low levels, there remained a possibility that even lower levels of Apaf-1 message (undetectable by Northern analysis) were present in the fog mutant. To address this, RT-PCR was performed on total RNA from wild-type and fog/fog mutant cultured fibroblasts. This analysis indicates that Apaf-1 transcripts are in fact present in the mutant, but are ≈10- to 20-fold less abundant (Fig. 4 C Right and D). The coding region (from start codon to stop codon) of the Apaf-1 cDNA was sequenced by using fragments amplified from fog/fog first-strand cDNA, and no mutations were found when compared with wild-type animals in the C3H/HeJ background on which the fog mutation originated (data not shown). Southern blot analysis of genomic DNA probed with 32P-labeled Apaf-1 cDNA revealed no differences in the gene structure of Apaf-1 between fog/fog and C3H/HeJ wild-type mice (data not shown). Additionally, no differences between the C3H/HeJ background and the fog mutant allele were found in 2,700 base pairs of the promoter region or the 3′ untranslated region (National Center for Biotechnology Information Nucleotide query, NM_009684) of the Apaf-1 gene. However, one primer pair that was used to amplify the coding sequence between exons 2 and 4 of Apaf-1 (containing the first exon of its ced-4 homology domain) from fog/fog first strand cDNA produced a predominant PCR fragment that was ≈1,750 bp larger than the predicted size (Fig. 5 A and B). The 2-kb fragment was sequenced and found to include the intron between exons 3 and 4 of the Apaf-1 gene (Fig. 5D). Because no mutations were found at the splice acceptor or donor sites flanking exons 3 and 4 or the intron between these two exons in the fog mutants; defective Apaf-1 mRNA processing appears to be the cause of the markedly reduced Apaf-1 message, protein levels, and activity in fog mutants (Fig. 4).

Figure 4.

Figure 4

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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

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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).

To address the possibility that a gene adjacent to Apaf-1 that regulates Apaf-1 expression was mutated in fog animals, mice carrying the fog mutation (which have a background similar to C3H/HeJ) were outcrossed to wild-type animals carrying Apaf-1 in the BL6 background. The aberrantly sized 2-kb RT-PCR fragment was amplified from the RNA of resulting progeny, which had one Apaf-1 allele from the fog mutant and one wild-type Apaf-1 allele from the BL6 background. Two separate PCRs, differing only in the downstream primer used to amplify a 340-bp fragment located within the aberrantly sized 2-kb fragment, were then used to determine the allelic origin of the 2-kb band. A 14-bp insertion present in the third intron of Apaf-1 gene of fog animals, but not in the BL6 wild-type mice, was used in the design of the downstream primers, such that one primer included the insertion and the other did not. The insertion found in the fog animals is also found in C3H/HeJ wild-type mice, indicating that the insertion is specific to the fog background but does not affect gene function. If the aberrant fragment resulted from a separate gene that affects Apaf-1 expression, then PCR products with primer pairs specific for the Apaf-1 gene in fog mutants and the Apaf-1 gene in BL6 wild-type mice would both be amplified. In Fig. 5C it is shown that only the primer including the insertion (specific to the Apaf-1 gene from fog mice) amplifies the 340-bp fragment located within the aberrant 2-kb band. This observation strongly suggests that the 2-kb band originates from the fog mutant allele and that the band is specific for the Apaf-1 gene rather than the result of a mutation in another gene affecting the expression of Apaf-1 in fog/fog mice.

Discussion

Residual Apaf-1 expression likely prevents normal neural tube closure in fog/fog mice. The fog mutation, although not yet defined on the nucleotide level, maps within the minimal region of mouse chromosome 10 containing Apaf-1, causes inefficient splicing of at least one intron in the Apaf-1 gene (the third intron) in a manner that is specific to the Apaf-1 gene in fog mutants, and results in dramatically reduced expression of Apaf-1 at the level of mRNA and protein. Furthermore, the fog mutation abolishes detectable Apaf-1 activity in vitro and is able to complement one disrupted Apaf-1 allele in producing a full Apaf-1 mutant phenotype in vivo. These results are most consistent with the fog mutation as single defect in the Apaf-1 gene and the critical neurodevelopmental role shown for Apaf-1.

Previous work has shown that modifier genes can vary the phenotype of Apaf-1-deficient mice, depending on their genetic background (9). Thus, the phenotypic differences between the fog mutant and the Apaf-1 mutants generated by targeted disruption are likely because of differences in the activity of modifier genes between the two mutant models and the low, residual Apaf-1 expression in the fog mutants that is not seen in Apaf-1 knockouts. In Fig. 4, fog mutants are shown to have markedly reduced levels of normal Apaf-1 message. This situation contrasts with the mutation in the Apaf-1 knockout, in which no normal message is produced. The enhanced survival of fog mutants over Apaf-1 knockout mice is best explained by the presence of residual Apaf-1 message. However, the frequent lumbosacral defects seen in fog mutants (derived in the C3H/HeJ background) but not in Apaf-1 knockouts (derived in the 129/BL6 background) is most likely the result of background differences. Apaf-1 mutants in a 129/BL6 mixed background possessing both forebrain defects and lumbosacral defects have been infrequently obtained (data not shown), although such defects in Apaf-1 knockouts derived in other strains have not been reported. Specifically, Apaf-1 mutant embryos in the 129/BL6 mixed background show exencephaly, cranioschesis, anencephaly, and lumbosacral defects in combination with exencephaly, whereas fog/fog mutant embryos have similar defects alone as well as in combination with exencephaly or cranioschesis. The common thread in all of the reported embryonic phenotypes is the failure of appropriate neural tube closure, which has been formally demonstrated in both Apaf-1 mutants and fog/fog mutants (3, 5, 8, 9). Given that both adult Apaf-1-deficient males and adult fog/fog males are infertile, the significant phenotypic overlap seen between Apaf-1 and fog/fog mutants suggests that fog mutants have a defect in Apaf-1 expression. However, the differences in the location of the neural tube closure defects between these two animal models may indicate either that an additional mutation exists in the fog/fog mice, which is revealed only in the presence of a particular genetic complement or reduced Apaf-1 expression, or that genetic modifiers on the background strain of the fog mutant enhance the effect of the Apaf-1 mutation on posterior neuropore closure thereby increasing the incidence of spina bifida. Based on the biochemical and genetic information presented, we conclude that the fog mutation is most likely a mutation in the Apaf-1 gene (identification of the mutation at the nucleotide level is required to make the conclusion with full certainty). The mutation affects the splicing of the third intron and the efficiency of Apaf-1 mRNA processing. Because an aberrantly sized 2-kb product was specifically amplified from the Apaf-1 gene in the fog mutant, we interpret the virtually absent expression of Apaf-1 mRNA found in fog/fog tissues as an indicator of significant Apaf-1 transcript degradation that is the result of the reduced splicing efficiency.

A recent report has shown that decreased Apaf-1 expression, as a result of DNA methylation, contributes to the pathogenesis of malignant melanoma (16). Here it is shown that reduced Apaf-1 expression because of aberrant Apaf-1 mRNA processing exists in fog mutant mice, which have spina bifida and survive to adulthood with neural tube closure defects. We also show that Apaf-1 deficiency can result in anencephaly. Thus, mutations affecting the expression levels of Apaf-1 or other proteins in the cytochrome-c-mediated apoptotic cascade (such as caspases-9 and -3) may play a fundamental role in the pathogenesis of spina bifida, because anencephaly and spina bifida are often present concurrently in human subjects (see Online Mendelian Inheritance in Man, Entry 182940, http://www3.ncbi.nlm.nih.gov/Omim/). As the cytochrome-c-mediated apoptotic pathway is critical for the death of neural progenitor cells (9), it may also be inferred that an excess of neural progenitors contributes to the pathogenesis of neural tube closure defects seen in humans.

The study of cytochrome-c-mediated apoptotic cell death in disease processes has been hampered by the embryonic lethality of the mutants deficient in any one of the proteins central to the cascade. Given that the fog mutant mice possess hypomorphic alleles of Apaf-1 and that adult mutants are readily available to investigators, examination of cell death in disease states occurring independently of the cytochrome-c-mediated cascade can be completed. Fields of investigation include (but are not limited to) immune dysfunction, ischemic cell death, and carcinogenesis.

Acknowledgments

We thank Jeffrey Stark, Kristi Cala, Daphne Davis, Jeff Cormier, Lorena Avila, and Carl Sidle for their technical assistance in completing this work. We also thank Mary Barter, Lucy Rowe, and Jennifer Johanson of The Jackson Laboratory for their expertise and help with the mapping analysis. This work was supported by National Institutes of Health Grants GM57158, HL 20948, and HL 63762, the Howard Hughes Medical Institute, the Welch Foundation, and the Alzheimer Association. J.H. was an Established Investigator of the American Heart Association and Parke–Davis.

Abbreviations

Apaf-1

apoptotic protease activating factor-1

RT

reverse transcription

cM

centimorgan

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