The transcription factor Nfixis essential for normal brain development
© Campbell et al; licensee BioMed Central Ltd. 2008
Received: 09 August 2007
Accepted: 13 May 2008
Published: 13 May 2008
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 Nfic-deficient 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.
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 Pax6- and 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.
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.
Nuclear Factor I (NFI) transcription/replication proteins are essential for both Adenoviral DNA replication [1–3] and for the regulation of transcription throughout development . 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 . More recent studies indicate that some of these brain defects may be due to altered formation of midline glial structures . We also showed recently that NFIA and NFIB regulate oligodendrocyte differentiation in developing spinal cord  and that loss of Nfia can cause defects in kidney development . 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 . 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 . 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.
Growth phenotype of Nfix-/- animals
Aberrant brain development in Nfix-/- animals: Cortex and aberrant ventricular cells
To assess whether some of the aberrant cells were early neuronal cells, sections of +/+ and Nfix-/- brains were stained with antibodies directed against doublecortin (DCX), a marker of migrating neurons . A few DCX expressing cells are seen in the +/+ brain, possibly representing migrating neurons destined for the rostral migratory stream (Fig. 3E, panels 1–2, arrows) . In contrast, 50–100% of the aberrant cells stained with α-DCX (Fig. 3E, panels 3–4), indicating that they express this marker of migrating neurons. In the regions surrounding the aberrant ventricular cells there also appear to be a greater number of DCX-positive cells that had the morphology of migrating neurons (i.e. a stained cell body with a distinct putative leading process, arrows in Fig. 3E, panel 4). These data indicate that a fraction of these aberrant ventricular cells express markers of migratory neurons. Because of this increase in DCX positive cells, neural stem cell homeostasis in Nfix-/- mice is currently being investigated.
Increased midline DV depth of cortex and subcortical regions in Nfix-/- mice.
DV ctx mm
DV brain mm
DV ctx mm
DV brain mm
Ratio -/- / +/+
Increase in brain wet weights in Nfix-/- mice.
Fixed Brain wt. (g)
Aberrant brain development in Nfix-/- animals: Hippocampus
Examination of serial coronal sections also demonstrated severe distortion of the hippocampus, particularly evident in sections at the level of the dentate gyrus. At P28 there were several major forebrain abnormalities. Dorsal views (Fig. 5A &5E) of the brains of P28 WT (+/+) (Fig. 5A) and Nfix-/- mice (Fig. 5E) show that the overall shape of the two brains is different with the Nfix-/- cortex more elongated in the anterior-posterior direction (A-P) compared to the +/+ brain. The extent of the exposed cerebellum is shorter in the Nfix-/- brain. As noted above in Fig. 4, the Nfix-/- brain is elongated in the dorso-ventral (D-V) direction; (compare Figs. 5B vs. 5F, 5C vs. 5G and 5D vs. 5H). The shape of the corpus callosum at this level is also different; it is more "u-shaped" in the Nfix-/- brain (Fig. 5B vs. 5F) and the angle of junction of the fimbria and hippocampus is more oblique in the WT than in the Nfix-/- mouse. The shape of the hippocampus is also distorted in the Nfix-/- mouse, with a dorsal enlargement in CA1 (Fig. 5C &5G, arrows). The dentate gyrus is shorter in the Nfix-/- brains (arrowheads in Fig. 5B &5F, 5C &5G and 5D &5H), and the angle of the "arrowhead" of the gyrus is larger. In addition, in some brains a slightly "wavy" appearance of the CA3/CA4 fields in posterior sections of the hippocampus are seen (e.g. Fig. 5H), possibly indicating a change in cytoarchitecture of the pyramidal cell layer here. Finally, the A-P extent of the corpus callosum appears somewhat shorter in the Nfix-/- than in the +/+ brain; it has just ended in 5D in the +/+ compared to 5G in the -/- brain. Such a change is consistent with the partial callosal agenesis noted in a previous study ; however, as discussed below these data are subject to interpretation due to the acallosal phenotype seen in 129 strain mice.
Nfixexpression in developing brain
Nfixexpression at the cortical midline
Nfixexpression during hippocampal development
These data indicate that Nfix, like Nfia and Nfib, plays a major role in brain development. In addition, the poor weight gain and delayed eye and ear opening (Fig. 2) indicate that Nfix may play an important role in the development of the gut and possibly the epithelial components of other organs. While this manuscript was in preparation another group generated and characterized mice containing a non-conditional lacZ-tagged Nfix disrupted allele . Since there are both similarities and differences between the phenotypes of these two distinct mouse mutants we will discuss these in detail below.
The delayed eye and ear opening, poor weight gain, and lethality by ~P22 of Nfix-/- animals (Figs. 2A, C–D) are the most striking and earliest visible phenotypes due to loss of Nfix. We observed that supplementing the diets of Nfix-/- animals with a soft dough supplement helped alleviate the runting of the animals and allowed survival of most animals past P28 (Fig. 2B). Such supplementation had previously allowed us to rescue runting due to tooth defects in Nfic-/- mice . However, we have seen no tooth defects in Nfix-/- mice. Thus, this partial rescue of the lethality and runting with dietary supplement points strongly towards either a neurological defect that affects eating or digestion, or a defect or delay in gut development in the Nfix-/- animals. Our preliminary data (not shown) indicate some changes in the timing of gene expression patterns in the small intestine. While the diet-supplemented Nfix-/- animals still have lower weights than their heterozygous or WT littermates (Fig. 2B), the weight reduction is far less severe than is seen in non-supplemented animals and ~65% of diet-supplemented Nfix-/- mice survive past P40 and ~10% past P90. In one case, an Nfix-/- male has sired multiple litters, showing that Nfix is not essential for male fertility. A single adult Nfix-/- female has given birth to a small litter but failed to nurture it, resulting in death of the pups. Thus it appears that Nfix is not essential for reproduction per se, but may affect rearing behavior. The leg clasping phenotype seen previously in Nfix targeted mice  was also seen in our Nfix-/- animals maintained on soft dough, indicating that the neuroanatomical defects described below likely have behavioral consequences independent of the nutritional status of the animals. It will be of great interest to determine whether the observed failure to thrive is due to direct defects or delays in intestine development or function, or whether the neuroanatomical defects discussed below could influence food sensing or eating behavior. To distinguish between these possibilities it will be necessary to perform detailed analysis of intestine development and function, careful behavioral studies on the mutant mice, and to use the conditional allele described here to delete Nfix solely from brain or intestine cells and determine which region is responsible for the phenotype.
Brain developmental changes in Nfix-/-mice
Previous studies showed that both Nfia and Nfib are essential for normal brain development [12, 13, 16]. Loss of either gene results in agenesis of the corpus callosum although it is unclear whether the mechanisms leading to this loss are identical in the two mutants. Loss of Nfia also results in postnatal hydrocephalus in mice, while the P0 lethality seen in Nfib-/- mice precludes analysis of postnatal phenotypes in these animals. The changes seen in Nfix-/- brains are quite distinct from those seen with loss of either Nfia or Nfib. For example, callosal axons cross the midline in Nfix-/- animals (Figs. 3B–C, 4B &4E, 5B &5F). This differs markedly from the complete callosal agenesis seen in homozygous Nfia and Nfib knockouts. In a recently published study of a non-conditional Nfix disruption the authors conclude that loss of Nfix results in both hydrocephalus and partial callosal agenesis . One possible explanation for this partial agenesis of the corpus callosum is the observation that virtually all 129 strain mice contain a recessive gene that results in callosal agenesis in ~25% of offspring and a reduced callosum size in 50% of progeny . To allow robust analysis of callosal defects, all mouse knockouts derived from 129 strain ES cells should be backcrossed at least 10 generations onto a strain that does not carry this trait (e.g. C57BL/6). Thus it will be important to determine whether callosal defects are evident when the Nfix-/- mice are backcrossed to C57BL/6 for 10 or more generations.
Unlike the previous report, we have not detected hydrocephalus in our Nfix-/- mice, but instead find unusual Pax6- and DCX-positive cells within the lateral ventricles of the mice (Figs. 3C–E). These aberrant cells are present from at least P0 until past P69 although their abundance appears to decrease somewhat with age. These cells have been detected in every Nfix-/- brain sectioned (N = 10) and in no WT brains. While the nature and source of these cells is as yet unknown, it is intriguing that at P12–16 they express Pax6, a marker for neural progenitor cells [19, 20]. The normal expression of Nfix in the ventricular zone of postnatal animals at this age (Figs. 7F &7G, 8F–H), together with the knowledge that ventricular zone progenitor cells express Pax6, strongly suggests that these cells are aberrant ventricular zone cells and may represent a hyperproliferation of these potential neural progenitor cells. Of possible relevance to this change in ventricular cell number, Nfix had previously been implicated in the etiology of glioblastoma formation in a mouse model system [25, 26]. Nfix is a recurrent integration target in glioblastomas generated by intraventricular injection of retroviruses carrying the PDGF-B chain . This system is used to identify oncogenes or tumor suppressor genes that act in concert with PDGF-B chain to generate glioblastomas. Interestingly, Nfia, Nfib and Nfic were also shown to be integration targets in this system . Thus it will be of great interest to determine whether Nfix-/- mice are more prone to glioblastoma formation induced by PDGF-B retroviruses and other agents, and whether double mutants of Nfix with Nfia, Nfib or Nfic have additional neurological or oncological phenotypes. It is unclear why the previous study did not note these aberrant ventricular zone cells, since they have been seen in 100% of the Nfix-/- brains from P0–P69 (N = 10). Given that the phenotype is seen in 100% of the homozygous Cre-deleted animals, but not in any heterozygous or homozygous conditional allele-containing animals, this phenotype cannot be due to the presence of a linked or unlinked secondary mutation.
It is of interest that a fraction of these aberrant ventricular cells also express DCX, a marker of migratory neurons . The presence of this marker could indicate the differentiation of aberrant ventricular zone cells into migratory neurons. However, an alternative explanation is that these aberrant ventricular cells represent migratory neurons destined normally for the rostral migratory stream (RMS), which fail to follow their normal migratory route to the olfactory bulb and instead populate the ventricle. In this case, these cells would be exhibiting a cell migration defect that could be related to the axonal guidance defects that cause callosal agenesis in Nfia and Nfib KO mice. Were the phenotype due to a failure of ventricular zone cells to enter the RMS, one might expect a decrease in the size of the olfactory bulb in the mutants as has been seen with some other mutations affecting cell migration or survival [27–29]. While gross examination and preliminary data from sections of the olfactory bulb do not show major changes of structure as have been seen with some mutations affecting RMS migration , careful birthdating, cell marking, and migration studies must be performed to determine whether these aberrant ventricular cells are indeed migratory neurons. Should defects in olfactory bulb structure or function be detected, they could contribute to feeding defects in the animals and the observed failure to thrive.
Our current working hypothesis is that the expanded cortex (Figs. 3, 4, 5), and the distortion of the hippocampus (Fig. 5), may be directly related to the process that produces these aberrant ventricular zone cells. Both the cortical expansion and aberrant ventricular cells have been seen in 100% of Nfix-/- animals (N = 10) in multiple sections along the anterior-posterior axis. Similar or even more dramatic expansions of the cortical surface area have previously been detected due to increased β-catenin signaling  or by deletion of the pro-apoptotic caspase, Casp9 . While the cortical expansion seen here is less than that seen with these other mutations, it will be of interest to determine whether either of these pathways may be affected by loss of Nfix. In particular, it will be important to determine whether the cortical expansion is consistent with recent models of mechanisms regulating cortex development . If the aberrant ventricular cells detected here indeed represent an increase in the neural stem cell population, it is possible that continued slow proliferation and differentiation of such stem cells could both expand the cortex and cause distortions of the hippocampus due to excessive cell production. We will test this hypothesis with cell-marking and birthdating studies and assessing neural stem cell numbers in WT and Nfix-/- animals.
Possible differences in phenotype compared with previous studies
As noted above, previous investigators found postnatal lethality, partial callosal agenesis, severe hydrocephalus and skeletal defects in Nfix-/- mice which were either not seen or were significantly less severe in this study. Both studies deleted exon 2 of Nfix which as discussed above should generate a similar null allele. Some possible reasons for these apparent phenotype differences include mouse strain differences, nutritional differences, and subtle differences in the disrupted allele, each of which is discussed below. Different ES cells lines were used in the two studies and the mice analyzed were at differences stages of back-crossing to the C57BL/6 background. In the current study, animals were backcrossed from 3–5 generations to C57BL/6 and thus should contain predominantly C57BL/6 genetic alleles. As discussed above, callosal agenesis is a known phenotype of 129 strain mice and it is necessary to backcross to a strain with normal callosum formation (e.g. C57BL/6) before assessing callosum formation. Hydrocephalus is also sometimes associated with callosal agenesis and thus these two apparent phenotypic differences may be linked. Any differences in these phenotypes between the two KO strains should diminish with further backcrossing if strain differences are significant.
In the current study, we were able to rescue substantially the postnatal lethality of the Nfix-/- mice by supplementation with a soft dough diet. Without this supplementation, all Nfix-/- animals lost over half their body weight from P10–P22 and died, apparently of starvation. The previous study examined animals that were in the process of severe malnutrition and it is possible that such malnutrition could contribute to both hydrocephalus and skeletal defects. Given the normal cranial bones seen in our Nfix-/- animals (not shown), we feel it is unlikely that bone defects contribute in any way to the brain malformations seen here. It will be necessary to rear the two KO strains under identical nutritional conditions to assess whether this is a true difference in phenotype. Finally, the two strains differ in the structure of the final Nfix KO allele. The earlier KO allele, after Cre deletion of the neomycin-resistance marker, contained a fusion of a lacZ coding region to the first 4 coding residues of the 2nd exon, a deletion of the remainder of the 2nd exon and 32 residues of the adjacent intron, and addition of a loxP site 3' to the lacZ coding region. The current study generated a conditional allele with an insertion of a floxed 2nd exon and a neomycin-resistance gene within the adjacent 3' intron. This conditional allele was then bred to a ZP3-Cre transgenic strain resulting in deletion of the 2nd exon, ~1 kb of intron sequence 5' and 3' to exon 2 and the neomycin resistance marker, and retention of only a single loxP site. Thus the previous allele deleted all but 4 residues of exon 2 and some adjacent intron sequence and contains a lacZ coding region and loxP site, while the current allele deletes all of exon 2 and some adjacent intron sequence and contains a single loxP site. Thus it is possible that either the addition of the lacZ coding region or differences in the amount of intron sequence deleted between the two KO alleles could affect either the expression of adjacent genes or possible expression of downstream exons of either Nfix KO allele. The presence of termination codons at the 3' end of the lacZ sequence of the previous allele and early within the out-of-frame spliced exon1-exon 3 transcript of the current KO allele significantly reduces the probability of any translation of downstream exons from either locus. However it is possible that aberrant internal initiation of translation from exons downstream of the deleted exon 2 could occur with either allele. Since such unusual downstream internal initiation could occur theoretically at any initiation codon downstream of exon 2, to formerly test this hypothesis it would be necessary to generate antibodies to all of the potential in- and out-of-frame coding regions downstream of exon 2 to test for the presence of such peptides. This would be particularly difficult given the known alternative splicing of Nfix transcripts downstream of exon 2. Since all previous studies indicate that exon 2 codes for the DNA-binding and dimerization domains of NFI proteins, the current evidence makes it unlikely but not impossible that such aberrant downstream peptides could mediate some of the apparent differences in phenotype seen between the current and previous KO strains. We would reiterate that the lack of phenotype seen in the current conditional Nfix allele, combined with the highly penetrant phenotype of the Cre-deleted KO allele, strongly indicates that there are no unlinked or linked mutations that contribute to the observed phenotype in the current KO allele. Additional studies may be needed to reconcile the apparent differences seen between the previous and current Nfix KO alleles.
It has been noted that the temporal expression pattern seen here for NFIX protein in developing cortex may differ somewhat from the pattern seen previously for Nfix mRNA . In previous studies Nfix mRNA was clearly detected in the dorsal VZ/SVZ region by E11.5 while here only weak expression of NFIX protein is seen in these regions at E12 and E13, with robust expression being seen at E17. Such differences may be due to differential translation of Nfix mRNA at these times, or to differences in the sensitivity and cellular resolution of in situ mRNA detection versus immunostaining. We are currently examining in more detail both the Nfix mRNA and protein expression patterns from E12–E17 to resolve these possible discrepancies.
Given the high degree of sequence homology among NFI family members and the overlapping patterns of expression of the 4 mouse NFI genes, it is perhaps surprising that each of the mouse genes has been shown to have distinct roles in development. However, we are now beginning to see some similarities and possibly shared functions among the 4 mouse NFI genes. Loss of Nfia results in hydrocephalus, agenesis of the corpus callosum, defects in midline structures and changes in gene expression associated with a delay in oligodendrocyte maturation [12, 13, 33]. Many of these defects could be related to altered glial cell development in the brain. Similarly, loss of Nfib is also associated with midline glial cell defects and agenesis of the corpus callosum . The recent observation that either Nfia or Nfib can promote gliogenesis in the developing spinal cord also points to potential sharing of function during development . We are currently testing for direct evidence of overlapping or shared functions between the 4 NFI genes by generating double-mutant mice in specific pairs of NFI genes.
Together with previous studies, these data indicate that 3 of the 4 NFI genes (Nfia, Nfib and Nfix) play important roles in brain development. In addition to their functions in brain development, at least 3 of the NFI genes also have essential roles in other organ systems including lung (Nfib) , tooth (Nfic)  and intestine (Nfix). Since products of all 4 NFI genes appear to bind to the same DNA sequences with similar affinities, it will be of great interest to determine whether there are shared or distinct target genes for the different NFI isoforms in tissues affected by loss of individual NFI genes.
Gene targeting and mouse strains
Genomic clones encompassing Nfix exon 2 were isolated from a mouse 129/Sv phage library as described previously [12, 17]. A targeting vector was constructed with a 4.2 kb 5' homology arm containing all of exon 2 and 633 bp of intron 2, with a loxP site inserted 406 bp 5' to the start of exon 2. The 5' arm was followed by an FRT-flanked PGK-neo expression cassette in the opposite transcriptional orientation, a 3' loxP site, the contiguous 2.7 kb 3' homology arm and a PGK-diphtheria toxin A chain cassette in the opposite transcriptional orientation. Cre-mediated recombination deletes all of exon 2, 406 bp of intron 1 and 633 bp of intron 2. The vector was electroporated into J1 ES cells (129S4, ), G418-resistant colonies were picked, expanded and banked until PCR screening was completed. DNA from ES cell clones was isolated and subjected to nested PCR using pairs of primers with one primer located outside of the 5' and 3' ends of the targeting vector and the other primer located within the 5' and 3' lox P sites (see Fig. 1 for details). 4 of 361 colonies showed correct targeting at the 5' and 3' ends and clone 38 was injected into C57Bl/6 blastocysts to generate chimeric animals. Chimeras were bred with C57Bl/6 females and agouti progeny were screened for the presence of the targeted conditional KO allele by PCR with primers flanking the 5' loxP site. To generate the Cre-deleted knockout allele (KO), males containing one or two Conditional Alleles (CA) were bred with females expressing Cre recombinase in their oocytes from a ZP3-Cre transgene . Progeny were screened for Cre-mediated loss of exon 2 by PCR with a primer 5' to the 5' loxP site and 3' to the 3' loxP site of the Conditional Allele. Male mice heterozygous for the Conditional Allele or the Knockout Allele were backcrossed to C57Bl/6 mice and to heterozygous females to generate homozygous progeny. All experiments were performed on a mixed 129S4/C57Bl/6 background and mice backcrossed for 2–5 generations to C57Bl/6 have indistinguishable phenotypes.
Analysis of animal and brain weights and gross morphology
Animals were weighed from postnatal day 6 (P6). Animals were observed visually daily from postnatal day 1 (P1) and the days at which the eyelids and ear canals opened were noted. The times shown are for complete eye and ear canal opening. Brain weights were assessed on brains fixed in 4% PFA at the ages indicated.
PCR and QPCR transcript analysis
Tissues were extracted immediately following dissection with TRIzol (Invitrogen), total RNA was isolated and cDNA was generated from 5 μg RNA with Superscript II reverse transcriptase (Invitrogen) and random primers as recommended by the manufacturer. Nfix transcripts were analyzed by PCR using an exon 1-based sense primer and an exon 3-based anti-sense primer and products were resolved on 2.5% agarose gels. Transcript levels of all genes analyzed were quantified by QPCR with a Bio-Rad iCycler real-time PCR machine using gene-specific primers that span multiple exons as described previously . Sequences of the primers are available upon request.
Embryonic mouse brains were collected from embryonic day (E) E12 to P0. Prior to E15, brains were drop-fixed in 4% paraformaldehyde in PBS. From E15 onwards mice were perfused transcardially with saline followed by 4% paraformaldehyde in PBS. For Figures 6, 7, 8, 9, brains were sectioned at 45–50 μm using a vibratome (Leica, Deerfield, IL). For experiments shown in Figures 3, 4, 5, mice were sacrificed by decapitation under Nembutal anesthesia and the brains of postnatal day 7 (P7) to adult Nfix-/- and Nfix+/+ mice were excised, fixed, embedded, sectioned and stained using standard procedures. Brains were fixed overnight in 10% buffered-formalin or 4% paraformaldehyde in PBS and then cryoprotected in fixative with 15% then 30% sucrose and stored at 4°C. Some brains were embedded in 15% gelatin/5% albumin matrix and equilibrated in 30% sucrose in fixative. 40 μm frozen sections were cut on a sliding microtome or a cryostat. Sections were stored in fixative at 4°C or in a cryoprotection solution of glycerol/ethylene glycol at -20°C. Sections were mounted on gelatin coated slides and stained with cresyl violet. Some animals were subjected to deep anesthesia and transcardial perfusion with saline followed by 4% paraformaldehyde in PBS. The brains were removed and postfixed in 4% paraformaldehyde for 1–3 days, cryoprotected in sucrose in PBS and sectioned and stained as shown above.
NFIX expression in brain sections was analyzed using a rabbit polyclonal anti-NFIX serum raised against amino acids 277–291 of human NFIX made by Geneka Biotechnology Inc (cat# 16021118, Montreal, Canada) and now available from Active Motif (cat# 39072, Carlsbad, CA, USA). Other antibodies used were as follows: Pax6 (Chemicon, rabbit), doublecortin (DCX, Santa Cruz sc-8606, goat) and phospho-histone H3 (pH3, Upstate, rabbit). Sections were washed with 1X phosphate buffered saline (PBS) and incubated for 2 hours with a blocking buffer containing 2% goat serum (v/v, S-1000, Vector Laboratories, Burlingame, CA) and 0.2% Triton X-100 (v/v; Sigma, St. Louis, MO) in 1X PBS. Anti-NFIX was used at 1/20,000 for embryonic tissues and 1/12,500 for postnatal tissues, anti-Pax6 was used at 1/25,000, anti-DCX was used at 1/500 and anti-pH3 was used at 1/2000. Sections were incubated overnight at room temperature then washed several times in PBS and incubated with a biotinylated secondary antibody (Vector Laboratories, Burlingame, CA) for 1 to 2 hours at room temperature, followed by processing with a VECTASTAIN ABC kit for 1 hour at room temperature (A used at 1/500, B used at 1/500, PK6100, Vector Laboratories). Sections were processed for color reaction using nickel- 3,3'-Diaminobenzidine (DAB, D5905, Sigma) solution (2.5% nickel sulfate and 0.02% DAB in 0.175 M sodium acetate) activated with 0.01% (v/v) hydrogen peroxide or the glucose-oxidase modification thereof , washed with 1X PBS multiple times, mounted on 3% gelatin-coated slides and coverslipped with DPX mounting medium (Electron Microscopy Sciences, PA). Immunofluorescent staining was performed using a Cy3-labeled goat anti-rabbit secondary antibody and DAPI (300 nM) staining of nuclei. Images were acquired on a PowerPhase digital camera (PhaseOne, Coppenhagen, Denmark), Zeiss AxioCam HRc (Zeiss, Germany) or a Zeiss LSM 510 Meta confocal microscope and were processed using Adobe Photoshop.
Western Blot analysis
Extracts of JEG3 choriocarcinoma cells transfected with vectors expressing N-terminal HA-tagged versions of NFIA, NFIB, NFIC and NFIX were subjected to SDS-polyacrylamide gel electrophoresis and Western blot analysis as described previously . Duplicate gels containing the samples indicated were blotted to PVDF membranes, probed with antibodies against the HA-tag (Roche, C125A) or NFIX (Active Motif, described above) and subject to chemiluminescent detection (ECL, Amersham).
List of abbreviations used
Nuclear Factor I
- Nfia :
Nuclear Factor I A gene
- Nfib :
Nuclear Factor I B gene
- Nfic :
Nuclear Factor I C gene
- Nfix :
Nuclear Factor I X gene
Nuclear Factor I X protein
phosphate buffered saline.
We thank Aimee Stablewski and the Roswell Park Transgenic/Knockout facility for the electroporation and isolation of ES cells. We want to acknowledge Linette Philip for her initial studies on Nfix transcripts and for Fig. 2E and Raquel Lima for histological studies. This work was supported in part by an IRDF grant from the University at Buffalo (RMG), NASA grant NNJ06HA59G (RMG) and NIH/NIHLB grant HL080624 (RMG). Work in LJR's laboratory was supported by grant #401616 from the National Health and Medical Research Council, Australia, and that in EDL's laboratory by NIH/NINDS grant NS050515. LJR is supported by a Senior Research Fellowship and MJP is supported by a Howard Florey Centenary Fellowship, both from the National Health and Medical Research Council, Australia.
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