Search tips
Search criteria 


Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
Mol Cell Biol. 2005 January; 25(2): 685–698.
PMCID: PMC543431

The Transcription Factor Gene Nfib Is Essential for both Lung Maturation and Brain Development


The phylogenetically conserved nuclear factor I (NFI) 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, whereas Nfic-deficient mice have agenesis of molar tooth roots and severe incisor defects. Here we show that Nfib-deficient mice possess unique defects in lung maturation and exhibit callosal agenesis and forebrain defects that are similar to, but more severe than, those seen in Nfia-deficient animals. In addition, loss of Nfib results in defects in basilar pons formation and hippocampus development that are not seen in Nfia-deficient mice. Heterozygous Nfib-deficient animals also exhibit callosal agenesis and delayed lung maturation, indicating haploinsufficiency at the Nfib locus. The similarity in brain defects in Nfia- and Nfib-deficient animals suggests that these two genes may cooperate in late fetal forebrain development, while Nfib is essential for late fetal lung maturation and development of the pons.

Nuclear factor I (NFI) transcription and replication proteins function both in adenoviral DNA replication (12, 43, 44) and in the regulation of transcription throughout development (21). There are four NFI genes in mammals (Nfia, Nfib, Nfic, and Nfix) and single NFI genes in Drosophila melanogaster, Caenorhabditis elegans, Anopheles spp., and other simple animals (21, 30, 50). No NFI genes have been found in plants, bacteria, or single-cell eukaryotes. In mammals, NFI proteins function as homo- or heterodimers and are expressed in complex, overlapping patterns during embryogenesis (6, 31). NFI proteins bind to a dyad-symmetric binding site (TTGGCN5GCCAA) with high affinity (20, 40), and NFI proteins have been shown to either activate or repress gene expression depending on the promoter and cellular context (21, 42). The presence of four NFI genes in mammals with possibly overlapping functions makes it a challenge to identify in vivo targets of individual NFI proteins and the roles of NFI genes in development.

We showed previously that disruption of Nfia causes late gestation neuroanatomical defects, including agenesis of the corpus callosum, size reductions in other forebrain commissures, and loss of specific midline glial populations (11, 56). In contrast, disruption of Nfic results in early postnatal defects in tooth formation, including the loss of molar roots and aberrant incisor development (61). In a previous study, insertion of a lacZ reporter gene into the Nfib locus resulted in defects in lung maturation but no apparent defects in brain development (22). Here we report the replacement of the essential exon 2 of the Nfib gene with a lacZ reporter gene and show that mice homozygous for our replacement mutation have major neuroanatomical defects similar to, but more severe than, those of Nfia/ mice. These defects include callosal agenesis, aberrant hippocampus and pons formation, and loss of specific midline glial populations. In addition, as was seen in the previously described Nfib insertion mutant, these Nfib-deficient animals show defects in lung development and die at birth with immature lungs. The loss of one copy of Nfib results in callosal agenesis in some animals and reduced lung maturation in all animals, demonstrating haploinsufficiency at the Nfib locus. Together, these defects indicate that Nfia and Nfib may function cooperatively during mouse forebrain development and that Nfib alone is essential for late gestational lung maturation and hippocampus and pons development.


Gene targeting and mouse strains.

Genomic clones containing Nfib exons 1 and 2 were isolated from a mouse 129/Sv phage library as described previously (11, 61). We modified lacZ to encode the simian virus 40 T antigen nuclear localization signal (NLS) at the β-galactosidase (β-Gal) N terminus and to include a phosphoglycerate kinase (PGK) gene poly(A) signal at its 3′ end (64), generating NLS-β-Gal-pA. Using a convenient EcoRI site at the 5′ end of exon 2 we cloned NLS-β-Gal-pA in frame with a 5.65-kb Nfib fragment ending at base 9 of exon 2 to form a translational fusion protein with the product of exon 2 of Nfib. A PGK-Neo-bGHpA cassette (60) was cloned downstream of the lacZ fusion cassette, followed by ~3 kb of intron 2. The NLS-β-Gal-pA and Neo cassettes replace 700 bp of Nfib, including all but 9 bp of exon 2. A diphtheria toxin A chain-expressing negative selection cassette (PGK-DTA-bGHpA) was assembled and cloned downstream of the 3′ homologous region in the opposite transcriptional orientation (67). E14-1 mouse embryonic stem (ES) cells (5 × 106) were electroporated with 5 μg of gel-purified 15.2-kb XhoI targeting vector fragment and then selected in 0.2-mg/ml G418 (GIBCO/BRL) for 6 days. Colonies were picked and expanded twice in 96-well plates. Two plates were frozen, and two were used for DNA isolation and Southern blot analyses.

Correctly targeted ES cells were injected into C57BL/6 blastocysts, and stable transmission of the targeted allele was assessed by PCR genotyping of the progeny of founder male chimeras. The targeted (knockout [KO]) allele was crossed into Black Swiss (Taconic), C57BL/6 (Taconic and Jackson Laboratories), and FVB/N (Jackson Laboratories) strains for analysis. Mice derived from two independent targeted ES cell clones were analyzed and had indistinguishable phenotypes. Since some 129 strains of mice have a recessive callosal agenesis phenotype (35, 36) and since the ES cells used were derived from 129S6 mice, all brain sections were prepared from mice backcrossed into the Black Swiss (one generation) or C57BL/6 (more than five generations) strain to avoid the as yet uncharacterized 129 recessive acallosal-susceptibility locus. The lung morphology phenotypes of Nfib mutant animals of the Black Swiss, C57BL/6, and FVB/N strains were indistinguishable. Each figure legend contains the strain used in the figure.

PCR genotyping.

Postnatal day 10 (P10) to P14 0.5-cm tail tip biopsy samples were shaken at 55°C overnight in 0.5 ml of tail lysis buffer (100 mM Tris-Cl [pH 8.5], 5 mM EDTA, 200 mM NaCl, 0.2% sodium dodecyl sulfate) including 0.1 mg of proteinase K/ml. One microliter of the lysate diluted 1:10 in H2O was analyzed by multiplex PCR (0.5 U of Platinum Taq DNA polymerase [Invitrogen], 1.5 mM MgCl2, 0.2 mM [each] deoxynucleoside triphosphate, 1× Rediload [Research Genetics], 1× PCR buffer [Invitrogen; 10 mM Tris-Cl {pH 8.3}, 50 mM KCl, 0.02 mg of bovine serum albumin/ml], five primers at 0.2 μM each). Primers were Nfib specific (a and b), lacZ specific (c), or mouse Y chromosome specific (SRY-1 and -2); sequences are as follows: a, 5′-GCTGAGTTGGGAGATTGTGTC-3′; b, 5′-TTCTGCTTGATTTCGGGCTTC-3′; c, 5′-CATCGTAACCGTGCATCTGCC-3′; SRY-1, 5′-AACAACTGGGCTTTGCACATTG-3′; SRY-2, 5′-GTTTATCAGGGTTTCTCTCTAGC-3′). PCR products were resolved on 2.0% agarose gels and visualized with ethidium bromide.

PCR and QPCR transcript analysis.

Total RNA was isolated with TRIzol reagent (Invitrogen), and cDNA was generated from 2 to 5 μg of RNA with Superscript II reverse transcriptase (Invitrogen) and random primers as recommended by the manufacturer. Nfib transcripts were analyzed by PCR with an exon 1-based sense primer (5′-GATCGGCCTCACGGGCCGATGATGTATTCTCCCATCTG-3′) and an exon 4-based antisense primer (5′-CTCTGATACATTGAAGACTCCG-3′), and products were resolved on a 2.5% agarose gel. Transcript levels of all genes shown were quantified by quantitative PCR (QPCR) with a Bio-Rad iCycler real-time PCR machine and gene-specific primers that span multiple exons as described previously (61). Primer sequences for the genes quantified are available upon request.

Tissue preparation and lacZ detection.

Embryos were harvested at various times after detection of a coital plug. For total lung DNA analysis, either the dissected left lobe (embryonic day 18.5 [E18.5]) or the whole lung (E15.5) was harvested into 1 ml of 2 M NaCl-2 mM EDTA in phosphate-buffered saline (PBS) and sonicated on ice, and DNA content was assayed fluorimetrically as described previously (32) with a 96-well plate fluorimeter and purified calf thymus DNA as a standard. Whole thoraces were immersed in cold 4% paraformaldehyde (PFA) overnight, washed in PBS, dehydrated in ethanol, embedded in paraffin, and transversely sectioned at 5 μm for hematoxylin and eosin (H&E) staining and immunohistochemistry.

β-Gal staining was performed on cryostat sections of frozen fixed tissues. Lungs were fixed in PBS containing 0.2% glutaraldehyde, 5 mM EGTA (pH 7.3), and 0.1 M MgCl2 for 3 h on ice and rinsed three times for 15 min with cold PBS. Fixed tissues were cryoprotected overnight in cold 30% sucrose in 1× PBS and then embedded in OCT embedding medium and snap-frozen on dry ice. Cryostat sections of frozen tissue (10 μm) were loaded onto polylysine-coated slides and allowed to dry at room temperature for 2 h and incubated in β-Gal solution (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 1 mg of X-Gal [5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside]) in buffer (100 mM sodium phosphate [pH 7.3], 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% NP-40) at room temperature for 6 h. Stained sections were washed three times in PBS and then postfixed in distilled water containing 2% glutaraldehyde, 2% PFA, and 0.1 M sodium cacodylate for 1 h at 4°C. The slides were rinsed in three changes of cold PBS for 10 min each, cleared in distilled water for 5 min, and then counterstained with 0.1% nuclear fast red in 5% aluminum sulfate for 2 min, washed in running water, dehydrated, and topped with coverslips for analysis.

For whole-mount staining fixed lungs were washed and immediately incubated in β-Gal solution at room temperature overnight without cryoprotection. Stained lung lobes were washed three times with PBS and postfixed as described above, and washed left lobes were photographed through a dissecting microscope.

Brain sectioning and staining.

Brains were perfused with 4% PFA, fixed overnight in 4% PFA, and stored in PBS until use. Brains were blocked in 3% agar and cut at 40 to 50 μm on a vibratome (Leica, Deerfield, Ill.). The sections were counterstained with 2% Mayer's hematoxylin (Sigma Chemical Co.) for 6 min, or alternatively with 1% thionin, rinsed, dehydrated, and mounted for analysis.


All procedures were performed at room temperature. Brain sections were washed three times in PBS and blocked in a solution of 2% (vol/vol) serum and 0.2% (vol/vol) Triton X-100 (Sigma) in PBS for 2 h. Normal goat serum (S-1000; Vector Laboratories, Burlingame, Calif.) or normal donkey serum (017-000-121; Jackson ImmunoResearch Laboratories, West Grove, Pa.) was used as the blocking agent. The sections were incubated with the primary antibody, rabbit anti-glial fibrillary acidic protein (GFAP; 1:30,000; Z0334; Dako, Glostrup, Denmark) or rat anti-L1 (1:5,000; MAB5272; Chemicon, Temecula, Calif.), overnight. Sections were then washed three times in PBS and incubated with the biotinylated secondary antibody (biotinylated goat anti-rabbit secondary antibody [1:500; Vector Laboratories] or biotinylated donkey anti-rat secondary antibody [1:500; Jackson ImmunoResearch Laboratories]) for 2 h. After three washes in PBS, sections were incubated in avidin-biotin solution (1:500; Vector Laboratories) for 1 h, followed by three washes in PBS. Sections were then immersed in a nickel-3,3′-diaminobenzidine (DAB; D-5905; Sigma)-chromogen solution (2.5% nickel sulfate and 0.02% DAB in 0.175 M sodium acetate) activated with 0.01% (vol/vol) H2O2 until a dark purple-black precipitate formed. Sections were washed in PBS, mounted, and placed on coverslips in DPX mounting medium (Electron Microscopy Services). Labeling was analyzed with an upright light microscope (Leica). Images were made with a PowerPhase digital camera (PhaseOne, Copenhagen, Denmark) directly into Adobe Photoshop software.

Immunohistochemistry for α smooth muscle actin (αSMA) was performed essentially as described previously (41). The smooth muscle differentiation marker αSMA was detected with mouse monoclonal anti-αSMA 1A4 (1:20,000; Sigma). Staining was performed with the Mouse on Mouse kit (Vector Laboratories). Biotinylated secondary antibodies were used with a nickel-enhanced DAB protocol essentially as described for the brain except that slides were counterstained with nuclear fast red.


Nfib gene targeting and confirmation of Nfib null allele.

We constructed a replacement-type targeting vector by replacing the most 3′ 523 bp of exon 2 and the first 177 bp of intron 2 with a translational-fusion β-Gal reporter gene and a neomycin resistance gene (lacZ and Neo, respectively, in Fig. Fig.1A).1A). Since exon 2 encodes both the DNA-binding and dimerization activities of NFI, homologous recombination of the native gene and the targeting vector should generate a null allele. SacI digests of targeted ES cell genomic DNA analyzed with a DNA probe that maps external to the targeted region generated 8.2-kb targeted-allele and 22.7-kb native-allele fragments (Fig. (Fig.1A)1A) in heterozygous targeted ES cells. A Southern blot screen of 80 G418-resistant ES clones identified 6 clones harboring a single targeted mutation, and a composite of the results of the screen is shown (Fig. (Fig.1B).1B). Further Southern blot analyses using Neo- and lacZ-specific probes showed that Nfib-targeted clones harbored no other vector integrations. PCR analyses confirmed homologous recombination of the 3′ flanking region (data not shown).

FIG. 1.
Disruption of Nfib gene and generation of null allele. (A) Nfib targeting vector (top) generated by replacement of most of exon 2 with the coding sequence for an NLS-β-Gal translational fusion protein and a PGK-neo selection cassette. Correct ...

We injected two ES clones heterozygous for the targeted Nfib mutation (Nfib/+) into C57BL/6 mouse blastocysts to generate chimeras. Male chimeras of one ES clone were crossed to Black Swiss, 129S6, FVB/N, and C57BL/6 strains, and genomic DNA from tail biopsy samples of the progeny was screened by PCR for the mutated allele. Both ES clones transmitted the mutant allele into the germ line, and the phenotypes of mice derived from the two ES clones were indistinguishable. To confirm that the targeted Nfib allele was null, we analyzed brain, liver, and lung Nfib transcripts isolated from E17.5 littermates of each Nfib genotype. Total RNA was reverse transcribed, and the cDNA then used as the template in PCRs with Nfib exon 1-specific (forward) and exon 4-specific (reverse) primers (Fig. (Fig.1C).1C). The major product generated from wild-type (WT; Nfib+/+) and heterozygote (Nfib/+) brain, liver, and lung cDNA corresponds to a predicted 684-bp product that includes exon 2 (532 bp), exon 3 (54 bp), and parts of exons 1 and 4 (Fig. (Fig.1C,1C, lanes +/+ and −/+). However, no such product was generated from Nfib homozygous mutant (Nfib/) brain, liver, and lung cDNA (Fig. (Fig.1C,1C, lanes −/−). A much less abundant product having a migration corresponding to that of a predicted 152-bp amplicon was generated only from Nfib/+ and Nfib/ brain cDNA. This product likely represents an Nfib transcript containing exon 1 spliced directly to exon 3, therefore lacking exon 2. Because exon 1 ends at codon base 3 and exon 3 begins at codon base 2, such an aberrantly spliced Nfib transcript would encode a 35-amino-acid missense polypeptide that terminates at a TAG codon in exon 4. These data indicate that replacement of all but 3 residues encoded by exon 2 with the β-Gal fusion protein generates a null allele of Nfib.

Lung maturation defects in Nfib-deficient mice.

A previous study showed that mice with an insertion of a lacZ translational fusion gene into the same EcoRI site in exon 2 of Nfib targeted here exhibited defects in lung maturation (22). We therefore examined lung development in our Nfib-null mice. Mouse lung maturation occurs relatively late in gestation, with final prenatal maturation (sacculation) occurring from E16.5 to P3 (10). Histological examination of lungs in E17.5 Nfib-deficient mice showed that lungs from Nfib/ mice were severely defective in maturation, while those from Nfib/+ animals had a clear developmental delay in maturation. WT lungs at E17.5 were well sacculated (Fig. (Fig.2A),2A), while lungs from Nfib/+ mice had smaller saccules (Fig. (Fig.2B)2B) and lungs of Nfib/ mice completely lacked saccules (Fig. (Fig.2C).2C). The morphology of the E17.5 Nfib/ lungs resembled that of less-mature WT E15.5 lungs (Fig. (Fig.2,2, compare panels C and D). To assess the approximate time in development at which maturation was arrested, we examined the morphology of E15.5 WT, Nfib/+, and Nfib/ lungs (Fig. 2D to F, respectively). Unlike the case at E17.5, at E15.5 the WT and mutant lungs had similar morphologies with dense mesenchyme and no saccules. In addition, levels of smooth muscle actin expression around the bronchioles (Fig. 2D to F) and vessels (Fig. 2D and F) in WT and mutant mice were similar. These data indicate that lung development in Nfib/ mice appears arrested between E15.5 and E16.5 at the late-pseudoglandular-to-early-canalicular stage of development and that Nfib/+ mice have a less severe phenotype of delayed lung maturation.

FIG. 2.
Decreased lung maturation in Nfib/+ and Nfib/ mice. Transverse sections of E17.5 lungs (A to C) were stained with H&E, and E15.5 lungs (D to F) were stained with a monoclonal antibody against a smooth muscle ...

Perinatal lethality in Nfib-deficient mice.

The difference in severity of the lung maturation defects between Nfib/ and Nfib/+ animals was confirmed by the finding that no Nfib/ animals survived for 24 h after birth (Table (Table1;1; 10 litters, 0 of 23 Nfib/ animals alive at day 1), while ~60% of the predicted number of Nfib/+ animals survived (34 of 58 predicted Nfib/+ animals). The lethality appears to be perinatal since the genotypes of E17.5 to E18.5 embryos were present at nearly the expected Mendelian frequency of 1:2:1 (66 Nfib+/+ mice, 115 Nfib/+ mice, and 70 Nfib/ mice; 31 litters). Compared to Nfib+/+ animals in the same litters, only ~55% of Nfib/+ animals in the C57BL/6 strain survived to genotyping, suggesting that the delay in lung maturation in Nfib/+ animals reduces early postnatal survival in this strain (Table (Table2;2; 70 Nfib/+ mice survived to day 14 versus 124 Nfib+/+ mice; 26 litters). Surprisingly, little or no loss of heterozygotes was seen during backcrossing to the FVB/N strain, indicating that this reduction in heterozygote survival is strain specific (Table (Table2;2; 83 Nfib/+ mice versus 75 Nfib+/+ mice survived to day 14; 17 litters). Histological evaluation of heterozygote E18.5 lungs showed that delays in sacculation were indistinguishable among the strains. Whether this difference in survival of Nfib-deficient heterozygotes among strains is dependent on modifier genes and/or maternal rearing differences remains to be determined. These data strongly support a model for haploinsufficiency at the Nfib locus.

Lethality in Nfib-deficient animalsa
Loss of Nfib−/+ animals during backcrossing of heterozygous Nfib−/+ animals to the C57BL/6 but not the FVB/N straina

Changes in gross morphology, DNA content, and epithelial differentiation markers in lungs of Nfib/ mice.

A previous report on the insertion of a lacZ transgene into exon 2 of Nfib described the lungs of the resulting mice as hypoplastic (22). We dissected lungs from our E15.5 and E18.5 WT and Nfib-deficient embryos to assess their gross morphology. The gross morphologies of WT and Nfib-deficient lungs were almost indistinguishable at E15.5 (Fig. (Fig.3A;3A; left lobes are shown for comparison). Upon close inspection, slight indentations were detected on the surfaces of Nfib/ lungs (Fig. (Fig.3A).3A). At later times in gestation these indentations became more pronounced and obvious surface clefts or crevices were seen in all Nfib/ lungs examined (Fig. 3B and C). Despite the presence of these defects in the pleural surface, Nfib/ lungs were not obviously hypoplastic.

FIG. 3.
Gross morphological changes in Nfib/ lungs. Dissected left lobes are shown unstained at E15.5 (A), whole mount stained for β-Gal at E18.5 (B; βgal), and H&E stained in transverse section at E18.5 (B; H&E). ...

To determine whether there was overall cell loss (hypoplasia) in the Nfib/ lungs, we assessed the left lung DNA contents and total body weights of E18.5 embryos (Table (Table3).3). While the total body weights of WT, Nfib/+, and Nfib/ animals were similar, there was a substantial increase in the total DNA in the lungs of Nfib/ animals, resulting in an increased ratio of DNA content to total body weight for these animals (Table (Table3;3; Nfib/ versus Nfib+/+). Thus while differentiation of type I and II epithelial cells appears defective in the Nfib/ lungs (see below), there is an apparent increase in cell number in the Nfib/ lungs. These data indicate that, rather than hypoplasia or cell loss in the Nfib/ lungs, there is instead an increase in DNA content, suggesting a defect in either the turning off of cell proliferation or in apoptosis during lung maturation (62).

Total body weights and DNA contents of the left lunga

We also measured proliferating cell nuclear antigen (PCNA) levels in WT and Nfib mutant lungs to assess overall levels of cell proliferation. PCNA transcripts were increased approximately fourfold at E18.5 in Nfib/ versus Nfib+/+ lungs, suggesting that higher levels of proliferation occurred in the mutant lungs (Table (Table4).4). This apparent increase in proliferation is consistent with the thickened mesenchyme and increased DNA content in Nfib/ lungs and may reflect a failure of the normal reduction in mesenchymal proliferation seen during lung maturation (62). As an initial screen for signaling molecules that may influence cell proliferation during lung maturation, we used QPCR to assess the levels of Fgf-10, Fgf-7, and Midkine transcripts in WT and Nfib/ lungs. Fibroblast growth factor signaling is essential for early lung development, and loss of Fgf-10 results in a lack of branching morphogenesis (53). While there was an ~2-fold increase in Fgf-10 levels and no change in Fgf-7 levels in E18.5 Nfib/ lungs relative to Nfib+/+ lungs, there was an ~11-fold increase in Midkine expression in the mutant lungs (Table (Table4).4). Since increased Midkine levels have been associated with the decreased lung maturation seen in glucocorticoid receptor-deficient mice (28) and with lung vascular remodeling after hypoxia (48), it will be important to assess Midkine's role in the apparent increase in mesenchymal cell density in Nfib/ lungs.

Transcript levels in Nfib−/+ and Nfib−/− lungs relative to those in Nfib+/+ lungsa

To determine if morphological immaturity corresponded with reduced expression of maturation-associated genes, we examined the transcript levels at E18.5 of a number of genes whose expression is known to increase from E16.5 to E18.5 in normal mouse lung development. Lung maturation from E16.5 to E18.5 is characterized by large increases in markers of type II epithelial and type I squamous epithelial cell differentiation (65). Using QPCR we showed that a number of type II cell markers, including surfactant protein A (SP-A), SP-B, SP-C, and SP-D, are greatly reduced in Nfib/ lungs and partially reduced in Nfib/+ lungs relative to WT lungs at E18.5 (Table (Table4).4). Also, type I cell markers Aquaporin-1 (Aqp-1) and the epithelial sodium channel gene (ENaC) were strongly reduced in Nfib/ lungs and partially reduced in Nfib/+ lungs (Table (Table4).4). These data indicate that loss of Nfib results in a significant reduction in expression of terminal differentiation markers of type II and type I cells, suggesting that Nfib is essential for the normal differentiation of these cells.

Nfib::lacZ expression during lung development.

Lung development requires many reciprocal epithelial-mesenchymal inductions (see references 25 and 54 for reviews). While our previous in situ data showed that Nfib is highly expressed in the developing lung (6), the cellular expression pattern was not determined. To identify the specific cell compartments where Nfib is expressed during lung development, we stained lungs of mice heterozygous for the Nfib::lacZ allele for β-Gal activity. Nfib::lacZ was uniformly expressed at high levels throughout the mesenchyme at E14.5, whereas weaker expression was detected in a few scattered epithelial cells at this time (Fig. (Fig.4A).4A). Nfib::lacZ was expressed at similar levels in most epithelial and mesenchymal cells at E16.5, with increased expression in smooth muscle (Fig. (Fig.4B).4B). At E18.5, Nfib::lacZ was expressed in a subset of both proximal and distal epithelial cells, while mesenchymal expression was decreased but still present in the smooth muscle (Fig. 4C and D). LacZ staining was not detected in WT E18.5 lung (Fig. (Fig.4E).4E). In adult lung Nfib::lacZ was expressed primarily in the bronchiolar epithelium and type II cells (Fig. (Fig.4F).4F). Preliminary immunohistochemistry with Nfib-specific antibodies supports this pattern for endogenous Nfib in WT mice (C. Bachurski, unpublished data). These data indicate that Nfib could play essential roles in either the lung mesenchyme or epithelium during late prenatal lung maturation.

FIG. 4.
Expression of β-Gal from the Nfib-lacZ allele in Nfib/+ lungs. Frozen sections of lungs of E14.5 (A), E16.5 (B), E18.5 (C and D), and adult Nfib/+ (F) or E18.5 Nfib+/+ (E) mice were stained for ...

Nfib−/− mice display agenesis of the corpus callosum, enlargement of the lateral ventricles, and malformation of midline glial populations.

We showed previously that loss of Nfia resulted in major neurological defects, including agenesis of the corpus callosum and defects in other forebrain commissures (11, 56). Although the previous study of a lacZ insertion into the Nfib locus reported no effects on brain development (22), we found multiple profound defects in brain development in our Nfib/ mice. As seen previously in our Nfia/ mice (56), Nfib/ mice exhibit agenesis of the corpus callosum on both mixed Black Swiss/129S6 (Fig. 5A to C) (n = 4) and C57BL/6 (Fig. 5D to I) (n = 3) backgrounds. This was evident with hematoxylin staining (Fig. 5A to F) and was confirmed by L1 cell adhesion molecule (CAM) immunohistochemistry, which labels axonal tracts (Fig. 5G to I). On the mixed Black Swiss background, most heterozygous Nfib/+ animals also displayed a callosal phenotype (n = 8 of 11), with the formation of Probst bundles at the midline (n = 11 of 11; Fig. Fig.5B).5B). However this phenotype in heterozygotes was less penetrant (n = 1 of 7) after backcrossing the allele onto a C57BL/6 background (Fig. (Fig.5E5E).

FIG. 5.
Agenesis of the corpus callosum in Nfib/ mice. Coronal 50-μm vibratome sections from E17.5 Nfib brains on either a Black Swiss (A to C) or C57BL/6 (D to F) background were mounted and stained with hematoxylin (A to F) or stained ...

Surviving postnatal Nfia/ mice on a mixed Black Swiss-129S6-C57BL/6 background exhibit severe hydrocephalus (11). Although Nfib/ mice die at birth, precluding analysis of postnatal hydrocephalus, embryonic Nfib/ mice have larger ventricles (Fig. 5C and F) than their WT and heterozygote littermates on both mixed Black Swiss and C57BL/6 backgrounds. These data suggest that both Nfib and Nfia may influence cerebrospinal fluid transport and/or production in the brain.

Midline glial populations are important for the formation of the corpus callosum, as they secrete guidance factors, such as Slit2, that guide callosal axons both before and after they cross the midline (58, 59). Figure Figure66 shows an immunohistochemical analysis of GFAP expression. Three midline glial populations are evident in WT animals: the glial wedge (GW), glia within the indusium griseum (IGG), and the midline zipper glia (MZG; Fig. Fig.6D)6D) (57). Nfib/ mice showed a reduction in GFAP expression at the midline, which likely reflects a loss or reduction in these glial populations. This phenotype is similar to that observed in Nfia/ mice (56). Nfib/ mice display a complete absence of GFAP labeling in the regions of both the IGG and the MZG (Fig. 6F and L). Furthermore, although a few glial cells were present, the GW was greatly reduced in the Nfib/ animals (Fig. 6F and L). Since there are NFI binding sites in the GFAP gene promoter, these results, together with our results for Nfia/ mice (56), suggest that NFI genes may regulate both GFAP expression and glial development. Further evidence for such regulation is that Nfib/+ mice on a C57BL/6 background display lower levels of expression of GFAP than their Nfib+/+ littermates (compare Fig. 6K and J). Although all three midline glial populations were present in Nfib/+animals on both the Black Swiss and C57BL/6 background, the IGG appeared disorganized on the Black Swiss background, probably due to the formation of Probst bundles at the midline (Fig. (Fig.6E6E).

FIG. 6.
Midline glial defects in Nfib/ mice. Coronal sections were labeled with an antibody against GFAP to label the midline glial populations. IGG (D, E, J, and K; arrowheads) and MZG (D, E, J, and K; thick arrows) were absent in Nfib ...

We showed previously that GFAP expression is decreased in Nfia/ brains relative to WT brains (11). Since we observed a decrease in GFAP-positive midline glial cells in Nfib/ brains, we examined GFAP transcript levels in Nfib/ whole brains. Although GFAP transcript levels were reduced ~5-fold in Nfib/ brains relative to WT, this decrease is less than the ~10-fold decrease seen in Nfia/ brains (Table (Table5).5). While examining the expression of genes proposed to influence forebrain development in Nfia KO mice, we noted that Tbr2 transcript levels were increased 1.9-fold in Nfia/ brains relative to Nfia+/+ brains (Table (Table5).5). However, Nfib/ brains had no higher levels of Tbr2 transcripts than did Nfib+/+ brains (Table (Table5).5). Thus, while the neuroanatomical defects in the Nfib/ brains appear more severe than those of Nfia/ brains, the changes in GFAP and Tbr2 expression in Nfib/ brains are less than those observed in Nfia/ animals. In addition, there were no apparent changes in the levels of transcripts for some other genes that have been proposed to play a role in callosum formation and forebrain development, including Slit2, Robo1 to -3, DCC, Netrin, and others (Table (Table5).5). Thus the reduction in GFAP transcripts is the only consistent change in gene expression so far detected in Nfib/ brains and is likely due at least in part to the observed loss of GFAP expression from specific midline glial cells.

Transcript levels in Nfib−/− and Nfia−/− brains relative to those in WT brainsa

Nfib regulates development of the hippocampus and the basilar pons.

Nfib/ mice also displayed severe disruptions in the formation of the hippocampus (Fig. (Fig.7).7). At E17.5 the dentate gyrus was absent (Fig. 7A, C, and F) and the entire CA3 region (Fig. (Fig.7A)7A) appeared to be absent or severely reduced (Fig. 7C and F). The fimbria, which contains the efferent projections from the hippocampus, was also greatly reduced (compare Fig. Fig.7C7C with A and B and F with D and E). This phenotype was evident on both the Black Swiss background and the C57BL/6 background, but the losses appeared to be more severe on the C57BL/6 background. To ask whether the defects were due to degeneration of these areas or, conversely, to a delay in development, we examined the hippocampal primordium at E15.5 (Fig. 7G to I). Even at E15.5 defects in hippocampal development were present. The entire hippocampal primordium, including the fimbria, was reduced in size. These data suggest that Nfib may regulate hippocampal cell proliferation and, since the CA1 region was less affected, possibly the regionalization of the hippocampus into different subfields.

FIG. 7.
Development of the hippocampus is severely disrupted in Nfib/ mice. Coronal 50-μm vibratome sections from E17.5 (A to F) and E15.5 (G to I) embryos were mounted and stained with hematoxylin to reveal the structural morphology ...

Previous studies showed that Nfib, Nfia, and Nfix are highly expressed in the basilar pons (6, 16), a hindbrain nucleus that provides significant mossy fiber input to the cerebellum. To investigate the role of Nfib in the development of the basilar pons, we examined Nfib/ hindbrains at E16.5 to E18.5 on C57BL/6 and Black Swiss backgrounds. The basilar pons is well formed at E18.5 in WT (n = 3) and heterozygote (n = 5) animals (Fig. 8A and B). However, the basilar pons was virtually absent in Nfib/ brains (n = 4), with only a small cap of cells remaining (Fig. (Fig.8C).8C). The pontine phenotype was evident from E16.5 to E18.5 in both Black Swiss (Nfib+/+, n = 3; Nfib/+, n = 5; Nfib/, n = 4) and C57BL/6 backgrounds. The rest of the hindbrain, including another precerebellar nucleus, the inferior olive, appears normal (Fig. 8B and C). In addition, the cerebellum is present and the normal lamination can be identified in the mutant. However, although foliation begins at this age in WT and heterozygous mice, the cerebellum appears unfoliated in Nfib/ mice (Fig. 8C versus A). It will be of interest to determine whether this apparent decreased foliation reflects changes in cerebellar cell proliferation or differentiation.

FIG. 8.
Absence of the basilar pons in Nfib/ mice. Sagittal sections of E18.5 Nfib+/+ (A), Nfib/+ (B), and Nfib/ (C) hindbrains stained with thionin. Brains were from litters backcrossed for ...

Expression of the other three Nfi genes in Nfib-deficient animals.

To determine if loss of Nfib affected the expression levels of the other three Nfi genes, we assessed Nfi transcript levels in the lungs and brains of E18.5 WT and Nfib/ animals. There were ~5.1, 3.3, and 2.8-fold increases in Nfia, Nfic, and Nfix expression, respectively, in Nfib/ lungs relative to WT lungs (Table (Table6).6). Thus, of the three NFI genes remaining in Nfib/ mice, the Nfia gene has the largest increase in expression in lung. In E18.5 whole brain, there was a 2.2-fold increase in Nfia expression but no significant change in Nfic or Nfix expression (Table (Table6).6). These data indicate that there were greater changes in the expression of the remaining Nfi genes in the lungs of Nfib-deficient animals than in the brains of these animals and that Nfia exhibited the greatest changes in both tissues. The significance of these changes and their relationship to the phenotypes seen in the tissues are being examined.

Levels of the other three Nfi genes in Nfib−/− lungs and brainsa


By replacing exon 2 of Nfib with a lacZ translational fusion construct (Fig. (Fig.1)1) we have shown that Nfib is essential for perinatal survival (Tables (Tables11 and and2),2), late fetal lung maturation (Fig. (Fig.22 and and3;3; Tables Tables33 and and4),4), and brain development (Fig. (Fig.55 to to88 and Table Table5).5). Loss of Nfib results in apparent arrest of fetal lung maturation at the late-pseudoglandular or early-canalicular stage, as indicated by both morphology (Fig. (Fig.2)2) and reduced expression of several late lung differentiation markers (Table (Table4).4). In addition, severe defects in development of the corpus callosum (Fig. (Fig.5),5), specific midline glial populations (Fig. (Fig.6),6), the hippocampus (Fig. (Fig.7),7), and the pons (Fig. (Fig.8)8) show that Nfib is essential for the proper development of both forebrain and hindbrain structures. Since our previous studies indicated that loss of Nfia resulted in callosal agenesis and loss of midline glial populations (11, 56), these data suggest that Nfia and Nfib may cooperate in the development of these brain regions.

Lung maturation defects in Nfib−/− animals.

Lung maturation defects that are morphologically similar to those seen in our Nfib/ animals were described previously for an insertion mutation in the Nfib locus (22). While that study described the defect as lung hypoplasia, our analysis shows that there is an apparent increase in the amount of DNA in Nfib/ lungs (Table (Table3),3), suggesting aberrant cell proliferation and/or apoptosis during lung maturation. Increased PCNA expression at E18.5 in Nfib/ versus WT lungs is also consistent with increased cell proliferation (Table (Table4).4). Prenatal lung maturation proceeds through a series of morphologically and biochemically defined stages (10) from budding (approximately E9.5), initial branching morphogenesis and expansion (pseudoglandular, approximately E9.5 to E16.5), continued branching of the distal epithelium and mesenchyme (canalicular; approximately E16.5 to E17.5), and formation of terminal saccules with terminal differentiation of type I and type II epithelial cells (sacculation; approximately E17.5 to P5). Final maturation of the terminal sacs into alveolar ducts and sacs (alveolization) occurs postnatally from approximately P5 to P30 (47, 65). The reduction of specific epithelial markers seen here (Table (Table4),4), together with the lack of saccule formation, indicates that the lungs in Nfib/ mice are arrested at the late-pseudoglandular or early-canalicular stage of maturation. Since the morbidity and mortality of premature infants are strongly associated with a failure of lung maturation (45), these mice may provide a useful model to investigate the later stages of prenatal lung maturation.

Failure of late fetal lung maturation has also been seen in mice containing targeted disruptions of the genes encoding glucocorticoid receptor (9), transforming growth factor β3 (55), endothelial nitric oxide synthase (24), and Sp3 (5) and other genes (66, 68, 69). The pathways linking this diverse set of molecules to the developmental events essential for lung maturation have not yet been determined. Given the possible increase in cell number seen in the Nfib/ lungs (Table (Table3),3), it is of interest that the combined loss of p21CIP and p57KIP2, two Cdk inhibitors that mediate cell cycle arrest, results in a maturation arrest that appears morphologically similar to that seen with loss of Nfib (69). Thus it is possible that Nfib is involved in either mesenchymal or epithelial cell exit from the cell cycle and that this cell cycle arrest is essential for normal maturation of the lung. Consistent with this model are the observations that overexpression of NFIB and other NFI proteins can suppress the oncogenic transformation of chicken embryo fibroblasts mediated by a number of nuclear oncogenes (52) and that rearrangement or altered expression or both of NFIB have been associated with the overgrowth syndromes of pleomorphic adenoma (17) and polycythemia vera (29). To test this model, it will be useful in future studies to determine whether the apparent increase in cell number in the Nfib/ lungs is due to changes in cell proliferation, apoptosis, or both.

Since Nfib/ mice have defects in both lung maturation and commissural tract formation (Fig. (Fig.22 to to8),8), it is noteworthy that loss of the known axon guidance receptor gene Robo1 also causes perinatal lethality attributed to a failure of lung maturation (66). Robo1 is a mouse ortholog of Roundabout, which is essential for normal midline axonal crossing in Drosophila (26). The Robo gene family encodes receptors with homology to the neural CAM family of cell surface adhesion molecules and appears to regulate axon guidance in both Drosophila and mice (49). This gene family has been implicated in regulating axon guidance in mice and humans (27, 37, 63), but its role in lung development is unknown (1). The expression patterns for Robo and Slit family members during lung maturation are consistent with a role for Robo signaling in epithelium-mesenchyme interactions (19). Although we observed no decrease in overall Robo1 expression in Nfib/ brains (Table (Table4),4), analysis of the expression patterns of Robo1 and other signaling molecules by in situ hybridization in Nfib/ animals might reveal regional changes in expression related to the observed phenotypes in brain and lung.

It will be important to determine the specific gene expression changes in Nfib/ lungs that affect maturation. However, given the failure of lung maturation in the Nfib/ mice, it will be essential to distinguish between changes that directly affect the maturation process and those that simply reflect the immature state of the Nfib/ lungs. For example, while we've seen decreases in the expression of a number of type II and type I epithelial markers in E18.5 Nfib/ lungs (Table (Table4),4), it is possible these changes merely reflect lack of maturity of the Nfib/ versus Nfib+/+ lungs. Since the expression levels of several of these genes normally increase during lung maturation, it is not surprising that their expression levels are reduced in the less mature Nfib/ lungs. Thus it will be important to determine the earliest time in development at which differences in gene expression between Nfib/ and Nfib+/+ lungs occur and which of the genes affected at this time are essential for maturation.

One known target for NFI genes in the developing lung epithelium is SP-C. Previous studies showed that the SP-C gene promoter is activated by NFI proteins, including NFI-B2, and that a transgene-encoded dominant repressor (NFI-engrailed) inhibits the expression of SP-C in mouse lung (2, 3). Thus SP-C is a good candidate for a direct target of Nfib in the developing lung. However, SP-C is highly expressed only late in lung maturation and SP-C mutant mice do not exhibit a failure in lung maturation (18). Given the expression pattern of the Nfib::lacZ allele (Fig. (Fig.4),4), it is possible that Nfib regulates both mesenchymal target genes that are essential for the maturation process early in lung development and also epithelial genes that are expressed later in lung development and that are important for physiological functioning of the lung. An important future goal is to distinguish between these two potential classes of Nfib target genes.

Neurological defects in Nfib/ animals.

While the callosal agenesis (Fig. (Fig.55 and and6)6) and loss of midline GFAP staining (Fig. (Fig.6)6) seen in Nfib/ animals is reminiscent of the phenotype seen previously in Nfia/ mice (11, 56), there are clear morphological differences between the two mutants. For example, Nfib/ animals usually lack large Probst bundles, regions of axonal overlap and swirling near the midline that are often present in Nfia/ animals (56). Such differences imply that, while both genes are essential for callosal formation, they may have both shared and distinct functions in this developmental process. Both Nfia and Nfib are expressed in the cortex (6, 56) (data not shown), but the lack of markers specific for callosally projecting neurons makes it difficult to assess the significance of this cortical expression. The similar losses of midline glial populations in Nfib/ mice (Fig. (Fig.6)6) and Nfia/ mice (56) suggest that both genes may cooperate in the growth and differentiation of these midline glial cells. Given the relatively restricted set of glial cells affected by loss of either Nfia or Nfib, it will be of interest to determine whether simultaneous loss of both Nfi genes produces a more severe phenotype in glial cell development.

While both Nfia- and Nfib-deficient mice exhibit agenesis of the corpus callosum, the Nfib-deficient mice have additional neurological defects not reported for Nfia-deficient animals, including severe defects in hippocampus (Fig. (Fig.7)7) and pons (Fig. (Fig.8)8) formation. Nfib had been shown previously to be expressed in both the hippocampus and the pons (6, 16), and thus it is possible that the defects in these regions are due to cell-autonomous defects in cells that normally express Nfib. Nfib is one of a small class of genes known to affect hippocampus development, a class which includes the genes Lhx-5 (70), Beta2/NeuroD (34), Lef-1 (15), CXCR4 (38), LRP6 (71), and Emx-2 (46, 51) and the Reelin-Disabled-1-beta(1)-class integrin pathway (14). The mechanisms by which these genes affect the molecular pathways and cellular processes essential for hippocampal development are beginning to be understood. LRP6-deficient mice have generalized defects in the Wnt/beta catenin signaling pathway because of the crucial function of LRP6 as a Wnt signaling coreceptor (71). These mice have severe defects in the dentate gyrus due to the decreased production of dentate granule cells. They also have abnormalities of the radial glial scaffolding in the forming dentate gyrus. By examining the hippocampal primordium at early stages, the authors showed a reduction in the number of dentate granule cell progenitors in the dentate ventricular zone prior to the emigration of the earliest granule neurons and precursors to form the dentate anlage. It is possible that loss of Nfib-regulated gene transcription may lead to a similar alteration of the hippocampal primordium, with resultant severe hypoplasia of the dentate gyrus. A more detailed analysis of early hippocampus development in the Nfib/ mice should allow us to test this model for Nfib function in the dentate gyrus.

The molecular mechanisms leading to the loss of the basilar pons in Nfib/ mice are unknown. It may be that Nfib regulates one or more signaling molecules, such as DCC or Rig-1/Robo3, that control the migration of pontine neurons to the midline (39). Alternatively, Nfib may interact with other transcription factors that regulate pontine formation (4, 13, 33). Distinguishing between these and other possibilities awaits characterization of the fate of pontine neurons in Nfib/ mice. Since analysis of whole-brain RNA has failed to find changes in the expression of some molecules known to influence axon guidance and cell migration (Table (Table5),5), determining whether there are common Nfib target genes that mediate the defects in callosal formation, hippocampal development, and pons formation will require a detailed analysis of Nfib target genes in each of these regions of the brain.

Given the severity of the neurological phenotypes seen in the Nfib/ animals, it is perhaps surprising that a previous study of an insertion of a lacZ translational fusion gene into the Nfib locus found no neurological defects (22). This is despite the finding that the lung maturation defects seen in our Nfib KO animals appear morphologically similar to those seen in animals with the Nfib insertion mutation. There are several possible explanations for such differences. (i) With both our Nfia/ and Nfic/ mice we showed that deletion of exon 2 results in aberrant splicing from the first to the third exons of the genes (11, 61). We show a similar aberrant transcript from our disrupted Nfib locus (Fig. (Fig.1C).1C). These aberrant transcripts do not encode functional proteins as they are missing DNA binding and dimerization domains and as the exons are out of frame, yielding short missense peptides. It is possible that aberrant splicing into the still-present second exon of the insertion mutant gene could generate a hypomorphic, but not null, allele in these animals. In our Nfib mutant allele the second exon is deleted, precluding this possibility. (ii) We showed previously that Nfib has an alternative first exon, expressed specifically in the brain (21, 42). Splicing from this brain-specific exon into the still present second exon of the insertion mutant gene may be why no brain phenotype for the insertion mutant was seen. Also, the report for the insertion mutant failed to show any β-Gal activity from the targeted allele or to demonstrate loss of Nfib expression, making it difficult to compare to the present study. While further studies are needed to resolve these differences, it is clear that our Nfib−/−mice have severe lung and brain phenotypes, making them an important tool with which to study Nfib function.

NFI gene family in mouse development.

Three of the four mouse NFI genes have now been disrupted, and each mutant has unique defects in development. Disruption of Nfia results in callosal agenesis, perinatal lethality, hydrocephalus, reduction in the hippocampal commissure, and loss of specific midline glial populations (11, 56). Some of these defects are also present in the Nfib/ mice described here. In contrast, disruption of the Nfic gene results in postnatal defects in tooth formation, with no observable defects in neuroanatomy and no lethality if the diet is composed of soft food (61). Since the Nfib/ mice do not survive postnatally, it is not possible to tell if they have tooth defects similar to those seen in Nfic/ mice. Since the four NFI genes encode proteins with highly homologous DNA-binding domains with similar DNA-binding specificities (40), it is possible that there are common downstream targets for all four NFI genes. It is unclear whether the diverse defects seen upon loss of the individual NFI genes are due to differences in the expression patterns of the four genes, differences in downstream target specificity, differences in gene activation or repression by specific NFI isoforms, or other factors. One feature that is common in the mutants is that the defects are seen relatively late in fetal or postnatal development. In Nfia/ and Nfib/ mice the first morphological indication of differences is at E14 to E15, while for Nfic/ mice the first changes are seen at approximately P10. Thus at least three of the four NFI genes are essential for relatively late developmental processes in the mouse. Since the four NFI genes are expressed in an overlapping pattern during development (6) and since changes in NFI transcript levels are first detected at the four-cell stage in embryogenesis (23), it will be of interest to determine whether multiple NFI genes cooperate at earlier stages of mouse development.

It is difficult to connect the diverse defects seen in the Nfib/ mice, callosal agenesis, aberrant gliogenesis, hippocampus and pons development, and failure of lung maturation, into a simple model of Nfib function. It will be necessary to determine the earliest stages at which gene expression changes occur in the affected tissues in order to determine whether common or unique Nfib target genes are affected in each organ. Since different NFI proteins have been found to either activate or repress expression from the same promoter depending on the cellular context (7, 8; reviewed in references 21 and 42), it will be important to analyze mice with mutations in multiple NFI genes to determine whether they cooperate or antagonize each other at earlier developmental stages. The future identification of direct downstream targets of Nfib, together with analysis of the effects of the combined loss of Nfib together with other genes known to affect brain and lung formation, should allow us to determine the molecular mechanisms through which Nfib functions in lung and brain development.


We thank Valerie Stewart of the Cleveland Clinic Transgenic/Knockout facility for blastocyst injections and the generation of founder chimeras and Clemencia Colmenares (Cleveland Clinic) for advise and assistance in ES cell culture. We also thank Kimberly Valentino and Rebecca Pacifico (UMB) for excellent technical assistance and Christine E. Campbell (SUNY-Buffalo) for reading the manuscript and helpful discussions.

This work was supported in part by Public Health Service grants HD34901 from the National Institute of Child Health and Development and DK58401 and DK48796 from the National Institute of Diabetes and Digestive and Kidney Diseases to R.M.G. and HL60907 from the National Heart, Lung and Blood Institute to C.J.B.


1. Anselmo, M. A., S. Dalvin, P. Prodhan, K. Komatsuzaki, J. T. Aidlen, J. J. Schnitzer, J. Y. Wu, and T. B. Kinane. 2003. Slit and robo: expression patterns in lung development. Gene Expr. Patterns 3:13-19. [PubMed]
2. Bachurski, C. J., S. E. Kelly, S. W. Glasser, and T. A. Currier. 1997. Nuclear factor I family members regulate the transcription of surfactant protein-C. J. Biol. Chem. 272:32759-32766. [PubMed]
3. Bachurski, C. J., G. H. Yang, T. A. Currier, R. M. Gronostajski, and D. Hong. 2003. Nuclear factor I/thyroid transcription factor 1 interactions modulate surfactant protein C transcription. Mol. Cell. Biol. 23:9014-9024. [PMC free article] [PubMed]
4. Ben-Arie, N., B. A. Hassan, N. A. Bermingham, D. M. Malicki, D. Armstrong, M. Matzuk, H. J. Bellen, and H. Y. Zoghbi. 2000. Functional conservation of atonal and Math1 in the CNS and PNS. Development 127:1039-1048. [PubMed]
5. Bouwman, P., H. Gollner, H. P. Elsasser, G. Eckhoff, A. Karis, F. Grosveld, S. Philipsen, and G. Suske. 2000. Transcription factor Sp3 is essential for post-natal survival and late tooth development. EMBO J. 19:655-661. [PubMed]
6. Chaudhry, A. Z., G. E. Lyons, and R. M. Gronostajski. 1997. Expression patterns of the four nuclear factor I genes during mouse embryogenesis indicate a potential role in development. Dev. Dyn. 208:313-325. [PubMed]
7. Chaudhry, A. Z., A. D. Vitullo, and R. M. Gronostajski. 1998. Nuclear factor I (NFI) isoforms differentially activate simple versus complex NFI-responsive promoters. J. Biol. Chem. 273:18538-18546. [PubMed]
8. Chaudhry, A. Z., A. D. Vitullo, and R. M. Gronostajski. 1999. Nuclear factor I-mediated repression of the mouse mammary tumor virus promoter is abrogated by the coactivators p300/CBP and SRC-1. J. Biol. Chem. 274:7072-7081. [PubMed]
9. Cole, T. J., N. M. Solomon, R. Van Driel, J. A. Monk, D. Bird, S. J. Richardson, R. J. Dilley, and S. B. Hooper. 2004. Altered epithelial cell proportions in the fetal lung of glucocorticoid receptor null mice. Am. J. Respir. Cell Mol. Biol. 30:613-619. [PubMed]
10. Costa, R. H., V. V. Kalinichenko, and L. Lim. 2001. Transcription factors in mouse lung development and function. Am. J. Physiol. Lung Cell. Mol. Physiol. 280:L823-L838. [PubMed]
11. das Neves, L., C. Duchala, F. Godinho, M. Haxhiu, C. Colmenares, W. Macklin, C. Campbell, K. Butz, and R. Gronostajski. 1999. Disruption of the murine nuclear factor I-A gene (Nfia) results in perinatal lethality, hydrocephalus and agenesis of the corpus callosum. Proc. Natl. Acad. Sci. USA 96:11946-11951. [PubMed]
12. de Jong, R. N., and P. C. van der Vliet. 1999. Mechanism of DNA replication in eukaryotic cells: cellular host factors stimulating adenovirus DNA replication. Gene 236:1-12. [PubMed]
13. Engelkamp, D., P. Rashbass, A. Seawright, and V. van Heyningen. 1999. Role of Pax6 in development of the cerebellar system. Development 126:3585-3596. [PubMed]
14. Forster, E., A. Tielsch, B. Saum, K. H. Weiss, C. Johanssen, D. Graus-Porta, U. Muller, and M. Frotscher. 2002. Reelin, Disabled 1, and beta 1 integrins are required for the formation of the radial glial scaffold in the hippocampus. Proc. Natl. Acad. Sci. USA 99:13178-13183. [PubMed]
15. Galceran, J., E. M. Miyashita-Lin, E. Devaney, J. L. Rubenstein, and R. Grosschedl. 2000. Hippocampus development and generation of dentate gyrus granule cells is regulated by LEF1. Development 127:469-482. [PubMed]
16. Gesemann, M., E. D. Litwack, K. T. Yee, U. Christen, and D. D. O'Leary. 2001. Identification of candidate genes for controlling development of the basilar pons by differential display PCR. Mol. Cell. Neurosci. 18:1-12. [PubMed]
17. Geurts, J. M., E. F. Schoenmakers, E. Roijer, A. K. Astrom, G. Stenman, and W. J. van de Ven. 1998. Identification of NFIB as recurrent translocation partner gene of HMGIC in pleomorphic adenomas. Oncogene 16:865-872. [PubMed]
18. Glasser, S. W., M. S. Burhans, T. R. Korfhagen, C. L. Na, P. D. Sly, G. F. Ross, M. Ikegami, and J. A. Whitsett. 2001. Altered stability of pulmonary surfactant in SP-C-deficient mice. Proc. Natl. Acad. Sci. USA 98:6366-6371. [PubMed]
19. Greenberg, J. M., F. Y. Thompson, S. K. Brooks, J. M. Shannon, and A. L. Akeson. 2004. Slit and robo expression in the developing mouse lung. Dev. Dyn. 230:350-360. [PubMed]
20. Gronostajski, R. M. 1986. Analysis of nuclear factor I binding to DNA using degenerate oligonucleotides. Nucleic Acids Res. 14:9117-9132. [PMC free article] [PubMed]
21. Gronostajski, R. M. 2000. Roles of the NFI/CTF gene family in transcription and development. Gene 249:31-45. [PubMed]
22. Grunder, A., T. T. Ebel, M. Mallo, G. Schwarzkopf, T. Shimizu, A. E. Sippel, and H. Schrewe. 2002. Nuclear factor I-B (Nfib) deficient mice have severe lung hypoplasia. Mech. Dev. 112:69-77. [PubMed]
23. Hamatani, T., M. G. Carter, A. A. Sharov, and M. S. Ko. 2004. Dynamics of global gene expression changes during mouse preimplantation development. Dev. Cell 6:117-131. [PubMed]
24. Han, R. N., S. Babaei, M. Robb, T. Lee, R. Ridsdale, C. Ackerley, M. Post, and D. J. Stewart. 2004. Defective lung vascular development and fatal respiratory distress in endothelial NO synthase-deficient mice: a model of alveolar capillary dysplasia? Circ. Res. 94:1115-1123. [PubMed]
25. Hogan, B. L., and J. M. Yingling. 1998. Epithelial/mesenchymal interactions and branching morphogenesis of the lung. Curr. Opin. Genet. Dev. 8:481-486. [PubMed]
26. Hummel, T., K. Schimmelpfeng, and C. Klambt. 1999. Commissure formation in the embryonic CNS of Drosophila. Development 126:771-779. [PubMed]
27. Jen, J. C., W. M. Chan, T. M. Bosley, J. Wan, J. R. Carr, U. Rub, D. Shattuck, G. Salamon, L. C. Kudo, J. Ou, D. D. Lin, M. A. Salih, T. Kansu, H. Al Dhalaan, Z. Al Zayed, D. B. MacDonald, B. Stigsby, A. Plaitakis, E. K. Dretakis, I. Gottlob, C. Pieh, E. I. Traboulsi, Q. Wang, L. Wang, C. Andrews, K. Yamada, J. L. Demer, S. Karim, J. R. Alger, D. H. Geschwind, T. Deller, N. L. Sicotte, S. F. Nelson, R. W. Baloh, and E. C. Engle. 2004. Mutations in a human ROBO gene disrupt hindbrain axon pathway crossing and morphogenesis. Science 304:1509-1513. [PMC free article] [PubMed]
28. Kaplan, F., J. Comber, R. Sladek, T. J. Hudson, L. J. Muglia, T. Macrae, S. Gagnon, M. Asada, J. A. Brewer, and N. B. Sweezey. 2003. The growth factor midkine is modulated by both glucocorticoid and retinoid in fetal lung development. Am. J. Respir. Cell Mol. Biol. 28:33-41. [PubMed]
29. Kralovics, R., Y. Guan, and J. T. Prchal. 2002. Acquired uniparental disomy of chromosome 9p is a frequent stem cell defect in polycythemia vera. Exp. Hematol. 30:229-236. [PubMed]
30. Kruse, U., F. Qian, and A. E. Sippel. 1991. Identification of a fourth nuclear factor I gene in chicken by cDNA cloning: NFI-X. Nucleic Acids Res. 19:6641. [PMC free article] [PubMed]
31. Kruse, U., and A. E. Sippel. 1994. Transcription factor nuclear factor I proteins form stable homo- and heterodimers. FEBS Lett. 348:46-50. [PubMed]
32. Labarca, C., and K. Paigen. 1980. A simple, rapid, and sensitive DNA assay procedure. Anal. Biochem. 102:344-352. [PubMed]
33. Li, S., F. Qiu, A. Xu, S. M. Price, and M. Xiang. 2004. Barhl1 regulates migration and survival of cerebellar granule cells by controlling expression of the neurotrophin-3 gene. J. Neurosci. 24:3104-3114. [PubMed]
34. Liu, M., S. J. Pleasure, A. E. Collins, J. L. Noebels, F. J. Naya, M. J. Tsai, and D. H. Lowenstein. 2000. Loss of BETA2/NeuroD leads to malformation of the dentate gyrus and epilepsy. Proc. Natl. Acad. Sci. USA 97:865-870. [PubMed]
35. Livy, D. J., and D. Wahlsten. 1997. Retarded formation of the hippocampal commissure in embryos from mouse strains lacking a corpus callosum. Hippocampus 7:2-14. [PubMed]
36. Livy, D. J., and D. Wahlsten. 1991. Tests of genetic allelism between four inbred mouse strains with absent corpus callosum. J. Hered. 82:459-464. [PubMed]
37. Long, H., C. Sabatier, L. Ma, A. Plump, W. Yuan, D. M. Ornitz, A. Tamada, F. Murakami, C. S. Goodman, and M. Tessier-Lavigne. 2004. Conserved roles for Slit and Robo proteins in midline commissural axon guidance. Neuron 42:213-223. [PubMed]
38. Lu, M., E. A. Grove, and R. J. Miller. 2002. Abnormal development of the hippocampal dentate gyrus in mice lacking the CXCR4 chemokine receptor. Proc. Natl. Acad. Sci. USA 99:7090-7095. [PubMed]
39. Marillat, V., C. Sabatier, V. Failli, E. Matsunaga, C. Sotelo, M. Tessier-Lavigne, and A. Chedotal. 2004. The slit receptor Rig-1/Robo3 controls midline crossing by hindbrain precerebellar neurons and axons. Neuron 43:69-79. [PubMed]
40. Meisterernst, M., I. Gander, L. Rogge, and E. L. Winnacker. 1988. A quantitative analysis of nuclear factor I/DNA interactions. Nucleic Acids Res. 16:4419-4435. [PMC free article] [PubMed]
41. Mucenski, M. L., S. E. Wert, J. M. Nation, D. E. Loudy, J. Huelsken, W. Birchmeier, E. E. Morrisey, and J. A. Whitsett. 2003. β-Catenin is required for specification of proximal/distal cell fate during lung morphogenesis. J. Biol. Chem. 278:40231-40238. [PubMed]
42. Murtagh, J., F. Martin, and R. M. Gronostajski. 2003. The nuclear factor I (NFI) gene family in mammary gland development and function. J. Mammary Gland Biol. Neoplasia. 8:241-254. [PubMed]
43. Nagata, K., R. Guggenheimer, T. Enomoto, J. Lichy, and J. Hurwitz. 1982. Adenovirus DNA replication in vitro. Identification of a host factor that stimulates synthesis of the preterminal protein-dCMP complex. Proc. Natl. Acad. Sci. USA 79:6438-6442. [PubMed]
44. Nagata, K., R. A. Guggenheimer, and J. Hurwitz. 1983. Specific binding of a cellular DNA replication protein to the origin of replication of adenovirus DNA. Proc. Natl. Acad. Sci. USA 80:6177-6181. [PubMed]
45. National Institutes of Health. 1994. Report of the Consensus Development Conference on the Effect of Corticosteroids for Fetal Maturation on Perinatal Outcomes. National Institutes of Health, Bethesda Md.
46. Pellegrini, M., A. Mansouri, A. Simeone, E. Boncinelli, and P. Gruss. 1996. Dentate gyrus formation requires Emx2. Development 122:3893-3898. [PubMed]
47. Prodhan, P., and T. B. Kinane. 2002. Developmental paradigms in terminal lung development. Bioessays 24:1052-1059. [PubMed]
48. Reynolds, P. R., M. L. Mucenski, T. D. Le Cras, W. C. Nichols, and J. A. Whitsett. 2004. Midkine is regulated by hypoxia and causes pulmonary vascular remodeling. J. Biol. Chem. 279:37124-37132. [PubMed]
49. Richards, L. J. 2002. Surrounded by Slit-how forebrain commissural axons can be led astray. Neuron 33:153-155. [PubMed]
50. Rupp, R., U. Kruse, G. Multhaup, U. Gobel, K. Beyreuther, and A. Sippel. 1990. Chicken NFI/TGGCA proteins are encoded by at least three independent genes: NFI-A, NFI-B and NFI-C with homologues in mammalian genomes. Nucleic Acids Res. 18:2607-2616. [PMC free article] [PubMed]
51. Savaskan, N. E., G. Alvarez-Bolado, R. Glumm, R. Nitsch, T. Skutella, and B. Heimrich. 2002. Impaired postnatal development of hippocampal neurons and axon projections in the Emx2−/− mutants. J. Neurochem. 83:1196-1207. [PubMed]
52. Schuur, E. R., U. Kruse, J. S. Iacovoni, and P. K. Vogt. 1995. Nuclear factor I interferes with transformation induced by nuclear oncogenes. Cell Growth Differ. 6:219-227. [PubMed]
53. Sekine, K., H. Ohuchi, M. Fujiwara, M. Yamasaki, T. Yoshizawa, T. Sato, N. Yagishita, D. Matsui, Y. Koga, N. Itoh, and S. Kato. 1999. Fgf10 is essential for limb and lung formation. Nat. Genet. 21:138-141. [PubMed]
54. Shannon, J. M., and B. A. Hyatt. 2004. Epithelial-mesenchymal interactions in the developing lung. Annu. Rev. Physiol. 66:625-645. [PubMed]
55. Shi, W., N. Heisterkamp, J. Groffen, J. Zhao, D. Warburton, and V. Kaartinen. 1999. TGF-β3-null mutation does not abrogate fetal lung maturation in vivo by glucocorticoids. Am. J. Physiol. 277:L1205-L1213. [PubMed]
56. Shu, T., K. G. Butz, C. Plachez, R. M. Gronostajski, and L. J. Richards. 2003. Abnormal development of forebrain midline glia and commissural projections in Nfia knock-out mice. J. Neurosci. 23:203-212. [PubMed]
57. Shu, T., A. C. Puche, and L. J. Richards. 2003. Development of midline glial populations at the corticoseptal boundary. J. Neurobiol. 57:81-94. [PubMed]
58. Shu, T., and L. J. Richards. 2001. Cortical axon guidance by the glial wedge during the development of the corpus callosum. J. Neurosci. 21:2749-2758. [PubMed]
59. Shu, T., V. Sundaresan, M. M. McCarthy, and L. J. Richards. 2003. Slit2 guides both precrossing and postcrossing callosal axons at the midline in vivo. J. Neurosci. 23:8176-8184. [PubMed]
60. Soriano, P., C. Montgomery, R. Geske, and A. Bradley. 1991. Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell 64:693-702. [PubMed]
61. Steele-Perkins, G., K. G. Butz, G. E. Lyons, M. Zeichner-David, H.-J. Kim, M. I. Cho, and R. M. Gronostajski. 2003. Essential role for NFI-C/CTF transcription-replication factor in tooth root development. Mol. Cell. Biol. 23:1075-1084. [PMC free article] [PubMed]
62. Stiles, A. D., D. Chrysis, H. W. Jarvis, B. Brighton, and B. M. Moats-Staats. 2001. Programmed cell death in normal fetal rat lung development. Exp. Lung Res. 27:569-587. [PubMed]
63. Sundaresan, V., E. Mambetisaeva, W. Andrews, A. Annan, B. Knoll, G. Tear, and L. Bannister. 2004. Dynamic expression patterns of Robo (Robo1 and Robo2) in the developing murine central nervous system. J. Comp. Neurol. 468:467-481. [PubMed]
64. Tybulewicz, V. L., C. E. Crawford, P. K. Jackson, R. T. Bronson, and R. C. Mulligan. 1991. Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene. Cell 65:1153-1163. [PubMed]
65. Warburton, D., M. Schwarz, D. Tefft, G. Flores-Delgado, K. D. Anderson, and W. V. Cardoso. 2000. The molecular basis of lung morphogenesis. Mech. Dev. 92:55-81. [PubMed]
66. Xian, J., K. J. Clark, R. Fordham, R. Pannell, T. H. Rabbitts, and P. H. Rabbitts. 2001. Inadequate lung development and bronchial hyperplasia in mice with a targeted deletion in the Dutt1/Robo1 gene. Proc. Natl. Acad. Sci. USA 98:15062-15066. [PubMed]
67. Yagi, T., Y. Ikawa, K. Yoshida, Y. Shigetani, N. Takeda, I. Mabuchi, T. Yamamoto, and S. Aizawa. 1990. Homologous recombination at c-fyn locus of mouse embryonic stem cells with use of diphtheria toxin A-fragment gene in negative selection. Proc. Natl. Acad. Sci. USA 87:9918-9922. [PubMed]
68. Yu, H., A. Wessels, J. Chen, A. L. Phelps, J. Oatis, G. S. Tint, and S. B. Patel. 2 February 2004, posting date. Late gestational lung hypoplasia in a mouse model of the Smith-Lemli-Opitz syndrome. BMC Dev. Biol. 4:1. [Online.] [PMC free article] [PubMed]
69. Zhang, P., C. Wong, D. Liu, M. Finegold, J. W. Harper, and S. J. Elledge. 1999. p21(CIP1) and p57(KIP2) control muscle differentiation at the myogenin step. Genes Dev. 13:213-224. [PubMed]
70. Zhao, Y., H. Z. Sheng, R. Amini, A. Grinberg, E. Lee, S. Huang, M. Taira, and H. Westphal. 1999. Control of hippocampal morphogenesis and neuronal differentiation by the LIM homeobox gene Lhx5. Science 284:1155-1158. [PubMed]
71. Zhou, C. J., C. Zhao, and S. J. Pleasure. 2004. Wnt signaling mutants have decreased dentate granule cell production and radial glial scaffolding abnormalities. J. Neurosci. 24:121-126. [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)