The transcription factor Nfix is essential for normal brain development

Christine E Campbell 1 2 Michael Piper 3 Cline Plachez 4 Yu-Ting Yeh 1 Joan S Baizer 0 1 Jason M Osinski 2 E David Litwack 4 Linda J Richards 3 Richard M Gronostajski 1 2 0 Dept. of Physiology and Biophysics, State University of New York at Buffalo , 3435 Main St., Buffalo, NY 14214 , USA 1 Program in Neuroscience, State University of New York at Buffalo , 3435 Main St., Buffalo, NY 14214 , USA 2 Dept. of Biochemistry and New York State Center of Excellence in Bioinformatics and Life Sciences, State University of New York at Buffalo , 3435 Main St., Buffalo, NY 14214 , USA 3 The School of Biomedical Sciences and The Queensland Brain Institute, The University of Queensland , Otto Hirschfeld Building, St Lucia, Queensland, 4072 , Australia 4 Dept. of Anatomy and Neurobiology and the Program in Neuroscience, University of Maryland , Baltimore , School of Medicine, HSF II , S251, 20 Penn St., Baltimore, MD 21201 , USA Background: The Nuclear Factor I (NFI) multi-gene family encodes site-specific transcription factors essential for the development of a number of organ systems. We showed previously that Nfia-deficient mice exhibit agenesis of the corpus callosum and other forebrain defects; Nfib-deficient mice have defects in lung maturation and show callosal agenesis and forebrain defects resembling those seen in Nfia-deficient animals, while Nficdeficient mice have defects in tooth root formation. Recently the Nfix gene has been disrupted and these studies indicated that there were largely uncharacterized defects in brain and skeletal development in Nfix-deficient mice. Results: Here we show that disruption of Nfix by Cre-recombinase mediated excision of the 2nd exon results in defects in brain development that differ from those seen in Nfia and Nfib KO mice. In particular, complete callosal agenesis is not seen in Nfix-/- mice but rather there appears to be an overabundance of aberrant Pax6and doublecortin-positive cells in the lateral ventricles of Nfix-/- mice, increased brain weight, expansion of the cingulate cortex and entire brain along the dorsal ventral axis, and aberrant formation of the hippocampus. On standard lab chow Nfix-/- animals show a decreased growth rate from ~P8 to P14, lose weight from ~P14 to P22 and die at ~P22. If their food is supplemented with a soft dough chow from P10, Nfix-/- animals show a lag in weight gain from P8 to P20 but then increase their growth rate. A fraction of the animals survive to adulthood and are fertile. The weight loss correlates with delayed eye and ear canal opening and suggests a delay in the development of several epithelial structures in Nfix-/- animals. Conclusion: These data show that Nfix is essential for normal brain development and may be required for neural stem cell homeostasis. The delays seen in eye and ear opening and the brain morphology defects appear independent of the nutritional deprivation, as rescue of perinatal lethality with soft dough does not eliminate these defects. - Background Nuclear Factor I (NFI) transcription/replication proteins are essential for both Adenoviral DNA replication [1-3] and for the regulation of transcription throughout development [4]. There are 4 NFI genes in mammals (Nfia, Nfib, Nfic and Nfix) and single NFI genes in Drosophila, C. elegans, Anopheles, Sea Urchin and other simple animals [4-6]. No NFI genes have been found in plants, bacteria or single cell eukaryotes. In mammals, NFI proteins form homo- or heterodimers and the 4 NFI genes are expressed in complex, overlapping patterns during embryogenesis [7,8]. NFI proteins bind to a dyad-symmetric binding site (TTGGCN5GCCAA) with high affinity [9,10], and NFI proteins have been shown to either activate or repress gene expression depending on the promoter and cellular context [4,11]. The presence of 4 NFI genes with possibly overlapping functions in mammals makes it a challenge to identify in vivo targets of individual NFI proteins and the roles of NFI genes in development. Previous studies showed that NFI genes are essential for normal prenatal and postnatal mouse development. Disruption of Nfia causes multiple neuroanatomical defects including agenesis of the corpus callosum and hydrocephalus along with perinatal lethality [12]. More recent studies indicate that some of these brain defects may be due to altered formation of midline glial structures [13]. We also showed recently that NFIA and NFIB regulate oligodendrocyte differentiation in developing spinal cord [14] and that loss of Nfia can cause defects in kidney development [15]. Targeted disruption of Nfib also results in callosal agenesis and other neuroanatomical defects, but Nfib-/- animals die at birth, apparently due to a severe delay in lung maturation [16]. In contrast, loss of Nfic has a more modest phenotype with no observed brain defects but major defects in postnatal tooth development, including aberrant incisor formation and greatly reduced molar root formation [17]. Nfic-/- animals survive to adulthood and are fertile if their diet is supplemented with soft dough which presumably reduces the need for chewing. Here we have generated a conditional Nfix knockout (KO) mouse and analyzed the effect of germline loss of Nfix. As in Nfia and Nfib KO mice, loss of Nfix causes severe neuroanatomical defects including expansion of the cingulate cortex and the entire brain along the dorsal ventral axis, aberrant hippocampal development and the generation of excessive Pax6-expressing ventricular cells. These defects suggest that Nfix may function in the repression of neural stem cell proliferation or in cell migration. In addition, loss of Nfix causes delays in eye and ear opening and a decreased weight gain that appear related to the postnatal lethality seen in Nfix-/- mice. Results Growth phenotype of Nfix-/- animals Initial breedings indicated that mice homozygous or heterozygous for the targeted Conditional Allele (Fig. 1AD) had no obvious phenotypes (data not shown). Therefore the knockout allele (KO) was generated by Cre-mediated recombination and mice containing the KO allele were analyzed (Fig. 1E). Nfix transcripts extending from exon 1 to exon 3 were detected in both wild-type (WT) and heterozygous Nfix-/+ mice, but no intact transcripts were detected in Nfix-/- mice (Fig. 1E). Since exons 2 and 3 are in different protein reading frames, no functional proteins should be generated from transcripts lacking exon 2. Mice heterozygous for the knockout allele had no obvious phenotype and upon breeding their progeny were born at a Mendelian ratio (1:2:1,+/+:-/+:-/-, 52:127:51, 32 litters). However, by postnatal day 8 (P8) Nfix-/- (KO) animals began to gain less weight per day than their Nfix -/+ (HET) and +/+ (WT) littermates and by P14 the Nfix-/- animals began to lose weight (Fig. 2A). In addition, Nfix-/- animals opened their eyelids and developed open ear canals ~3 days later than +/+ or Nfix-/+ animals (Fig. 2C &2D), suggesting a general delay i (...truncated)