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. 1999 Oct 12;96(21):11946-51.
doi: 10.1073/pnas.96.21.11946.

Disruption of the murine nuclear factor I-A gene (Nfia) results in perinatal lethality, hydrocephalus, and agenesis of the corpus callosum

L das Neves  1 , C S Duchala, F Tolentino-Silva, M A Haxhiu, C Colmenares, W B Macklin, C E Campbell, K G Butz, R M Gronostajski
Affiliations

Disruption of the murine nuclear factor I-A gene (Nfia) results in perinatal lethality, hydrocephalus, and agenesis of the corpus callosum

L das Neves et al. Proc Natl Acad Sci U S A. .

Erratum in

  • Proc Natl Acad Sci U S A 2001 Mar 27;98(7):4276. Godinho F [corrected to Tolentino-Silva F]

Abstract

The phylogenetically conserved nuclear factor I (NFI) family of transcription/replication proteins is essential both for adenoviral DNA replication and for the transcription of many cellular genes. We showed previously that the four murine NFI genes (Nfia, Nfib, Nfic, and Nfix) are expressed in unique but overlapping patterns during mouse development and in adult tissues. Here we show that disruption of the Nfia gene causes perinatal lethality, with >95% of homozygous Nfia(-/-) animals dying within 2 weeks after birth. Newborn Nfia(-/-) animals lack a corpus callosum and show ventricular dilation indicating early hydrocephalus. Rare surviving homozygous Nfia(-/-) mice lack a corpus callosum, show severe communicating hydrocephalus, a full-axial tremor indicative of neurological defects, male-sterility, low female fertility, but near normal life spans. These findings indicate that while the Nfia gene appears nonessential for cell viability and DNA replication in embryonic stem cells and fibroblasts, loss of Nfia function causes severe developmental defects. This finding of an NFI gene required for a developmental process suggests that the four NFI genes may have distinct roles in vertebrate development.

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Figures

Figure 1
Figure 1
Disruption of the murine Nfia gene and demonstration of “exon-skipping.” (A) The structure of a portion of the wild-type Nfia gene is shown with exons 1a and 2 as black boxes, EcoRI sites marked E, and the 7.0-kb HindIII fragment shown with a bracket. The targeting vector was constructed as described in Materials and Methods, and recombination within the 5′ and 3′ regions with the wild-type gene would yield the disrupted Nfia gene missing most of exon 2. (B) Clones of ES cells electroporated with the targeting vector were lysed and DNA was isolated, digested with HindIII, transferred to membranes, and probed with the probe shown as P in A. This probe lies outside of the targeting vector and detects a 7.0-kb band from the wild-type Nfia allele and a 5.0-kb band from the disrupted allele. Lanes 1–5, 7, and 8 show a wild-type pattern, while lane 6 shows both the wild-type and disrupted alleles. (C) Genomic DNA from the tails of +/+, +/−, and −/− animals was digested with HindIII and analyzed as in B. (D) The nested primers shown as arrows below the disrupted gene in A were used to amplify genomic DNA of the progeny of a mouse heterozygous for the disrupted Nfia− allele. Lanes 2, 3, 6, and 8 are positive for the 2.3-kb PCR fragment, while lanes 1, 4, 5, and 7 are negative. Size markers of HindIII-digested λ DNA are shown in the leftmost lane, marked λ. The rightmost set of nested primers from A lies outside of the targeting construct. (E) RNA from the brains of wild-type (lanes 3 and 4), heterozygous (+/−, lanes 5 and 6), and homozygous Nfia− mice (−/−, lanes 7–10) was isolated, reverse transcribed, and PCR amplified by using primers in exon 3 and either exon 1a or 1b of Nfia as denoted above the lanes. The products were analyzed on a 2% agarose gel along with 123-bp ladder markers (lane M). The arrows E1b-E2-E3 and E1a-E2-E3 show the positions of correctly spliced NFI-A mRNAs, while the arrows E1b-E3 and E1a-E3 show the positions of the products lacking exon 2. Lanes 1 and 2 contained no reverse transcribed product.
Figure 2
Figure 2
Hydrocephalus in homozygous Nfia− mice. (A) A homozygous Nfia− pup (−/−) and a wild-type littermate (+/+) are shown. Note the smaller size and foreshortened head of the −/− animal. (B) Enlargement of A showing the characteristic “dome head” of the Nfia− (−/−) animal compared with the more wedge-shaped head of wild-type animal. (C and D) Cresyl violet stained coronal sections through the brains of 6-month-old Nfia−/− (−/−) and wild-type (+/+) animals. Note the dilation of the ventricles (arrows in C). (Bar in D = 2 mm.)
Figure 3
Figure 3
Absence of corpus callosum in Nfia−/− mice. Cresyl violet-stained coronal sections thorough the brains of C57BL/6 fetuses 18 days post coitus. (A and B) Wide-field pictures of the brains of −/− and +/+ littermates with the region surrounding the corpus callosum boxed. (C and D) Expansion of A and B showing the corpus callosum in the +/+ animal (labeled cc in D) and the absence of the corpus callosum in the −/− animal. Serial sections throughout this region showed a complete absence of callosal development in −/− animals. (Bars at the bottom right of B and D equal 2 and 1 mm, respectively.)
Figure 4
Figure 4
Reduced GFAP expression in Nfia−/− mice. Coronal cryostat sections of the brains of 3-month-old Black Swiss Nfia−/− (−/−) and wild-type mice (+/+) were fixed and stained with antibodies against PLP and GFAP. (A and B) PLP expression in Nfia−/− (A, −/−) and wild-type (B, +/+) mouse brains. Note expanded ventricles indicating hydrocephalus and absence of the corpus callosum (tract lying directly above the hippocampus spanning the two hemispheres) in A vs. B. Expression of PLP appears normal in both animals in the tracts of the fimbria (dark sac-like regions on lower left and right of the hippocampus). (C and D) GFAP expression in Nfia−/− (C, −/−) and wild type (D, +/+) mouse brains. Note the severe reduction in GFAP staining in the cortex and hippocampus (as assessed by reduced levels of punctate cellular brown precipitate), the expanded ventricles, and the absence of corpus callosum in C vs. D. Loss of GFAP staining is regional with reduction of staining in the cortex and hypothalamus but retention of staining with some altered morphology in the fimbria (sacs below the hippocampus) and thalamic nuclei (not shown). (E and F) Higher magnification of GFAP expression in the left dentate gyrus regions of C and D. Note distorted dentate gyrus appearance (probably secondary to hydrocephalus) and reduced GFAP expression in the dentate gyrus in E vs. F. (Bars = 1 mm.)
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