Nfix expression critically modulates early B lymphopoiesis and myelopoiesis

The commitment of stem and progenitor cells toward specific hematopoietic lineages is tightly controlled by a number of transcription factors that regulate differentiation programs via the expression of lineage restricting genes. Nuclear factor one (NFI) transcription factors are important in regulating hematopoiesis and here we report an important physiological role of NFIX in B- and myeloid lineage commitment and differentiation. We demonstrate that NFIX acts as a regulator of lineage specification in the haematopoietic system and the expression of Nfix was transcriptionally downregulated as B cells commit and differentiate, whilst maintained in myeloid progenitor cells. Ectopic Nfix expression in vivo blocked early B cell development stage, coincident with the stage of its downregulation. Furthermore, loss of Nfix resulted in the perturbation of myeloid and lymphoid cell differentiation, and a skewing of gene expression involved in lineage fate determination. Nfix was able to promote myeloid differentiation of total bone marrow cells under B cell specific culture conditions but not when expressed in the hematopoietic stem cell (HSPC), consistent with its role in HSPC survival. The lineage choice determined by Nfix correlated with transcriptional changes in a number of genes, such as E2A, C/EBP, and Id genes. These data highlight a novel and critical role for NFIX transcription factor in hematopoiesis and in lineage specification

The transcription factor Nfix is essential for normal brain development

Background: The Nuclear Factor I (NFI) multi-gene family encodes site-specific transcription factors essential for the development of a number of organ systems. We showed previously that Nfia-deficient mice exhibit agenesis of the corpus callosum and other forebrain defects; Nfib-deficient mice have defects in lung maturation and show callosal agenesis and forebrain defects resembling those seen in Nfia-deficient animals, while Nficdeficient mice have defects in tooth root formation. Recently the Nfix gene has been disrupted and these studies indicated that there were largely uncharacterized defects in brain and skeletal development in Nfix-deficient mice. Results: Here we show that disruption of Nfix by Cre-recombinase mediated excision of the 2nd exon results in defects in brain development that differ from those seen in Nfia and Nfib KO mice. In particular, complete callosal agenesis is not seen in Nfix-/- mice but rather there appears to be an overabundance of aberrant 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. Conclusion: These data show that Nfix is essential for normal brain development and may be required for neural stem cell homeostasis. The delays seen in eye and ear opening and the brain morphology defects appear independent of the nutritional deprivation, as rescue of perinatal lethality with soft dough does not eliminate these defects

Loss of NFIX transcription factor biases postnatal stem/progenitor cells towards oligodendrogenesis

Murine postnatal neural stem cells (NSCs) give rise to neurons, astrocytes, or oligodendrocytes (OLs); however, our knowledge of the genes that control this lineage specification is incomplete. In this study, we show that nuclear factor I X (NFIX), a transcription factor known to regulate NSC quiescence, also suppresses oligodendrogenesis (ODG) from NSCs. Immunostaining reveals little or no expression of NFIX in OL lineage cells both in vivo and in vitro. Loss of NFIX from subventricular zone (SVZ) NSCs results in enhanced ODG both in vivo and in vitro, while forced expression of NFIX blocks NSC differentiation into OLs in vitro. RNA-seq analysis shows that genes previously shown to be differentially expressed in OL progenitors are significantly enriched in RNA from Nfix(-/-) versus wild-type NSCs. These data indicate that NFIX influences the lineage specification of postnatal SVZ NSCs, specifically suppressing ODG

Nfix expression in stem and progenitor populations.

<p><b>(A)</b> Analysis of <i>Nfix</i> expression during murine B cell differentiation using the B-cell lineage microarray dataset (GSE11110)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120102#pone.0120102.ref041" target="_blank">41</a>]. Error bars represent max and min <i>Nfix</i> expression values. <b>(B)</b> Quantitative RT-PCR analysis of <i>Nfix</i> expression in murine B cell populations fractionated by flow cytometry. Data are representative of three independent experiments, performed in duplicate. Error bars denote ±SD. <b>(C)</b> Analysis of <i>NFIX</i> expression in human B cell development from the human hematopoiesis microarray dataset (GSE24759) (21). Plots represent raw values, error bars represent max and min values. <b>(D)</b> Analysis of <i>Nfix</i> expression by qRT-PCR in murine stem and progenitor cells isolated from bone marrow. <b>(E)</b> Graph of percentage expression of CD43⁺, B220⁺, and B220^loCD43⁺ populations in BM cells from chimeras established with MigR1 or <i>Nfix</i> progenitors 8–10 wks previously. Error bars denote +/- SD of 2 independent experiments (n = 3). <b>(F)</b> Representative flow cytometric analysis, with gates and percentages showing B220^loCD43⁺ populations (early B cells) in GFP^- and GFP⁺ populations.</p

<i>Nfix</i> blocks B cell differentiation in vitro in favour of myelopoiesis.

<p><b>(A)</b> Total FL was transduced with control MigR1 or <i>Nfix</i>, plated on OP9 cells and analyzed by flow cytometry on day 16. FACS plots of GFP⁺ B cells (B220⁺CD19⁺, top panels) and GFP⁺ myeloid cells (CD11b⁺Gr-1⁺, lower panels) representative of 3 replicates from 2 independent experiments. <b>(B)</b> Graph of average percentage of B220⁺CD19⁺ (left) and CD11b⁺Gr-1⁺ (right) from (A). Error bars denote +/- SD of 2 independent experiments (n = 3). Graph shows a statistically significant decrease in <i>Nfix</i> derived B220⁺CD19⁺ cells (p = 0.02) and a significant increase in <i>Nfix</i> derived CD11b⁺Gr-1⁺ cells (p = 0.01) when compared with MigR1 controls. <b>(C)</b> E14.5 FL HSPCs (Lin^-Sca1⁺cKit⁺Flt3^-) transduced with control MigR1 or <i>Nfix</i> retrovirus were plated on OP9 cells (Day 0) and analysed by flow cytometry on day 12 with FACs plots of GFP expression (left histograms) and GFP^- and GFP⁺ B220⁺CD19⁺ cells (right dot plots) representative of 2 independent experiments. <b>(D)</b> Graph of average percentage of B220⁺CD19⁺ cells from (C), error bars denote +/- SD of 2 independent experiments. Experiment shows significant decrease in <i>Nfix</i> expressing B cells compared with control MigR1 (p = 0.01) <b>(E)</b> Total BM cells were sorted for MigR1 and <i>Nfix</i> expression 24 hr post transduction and qRT-PCR was performed. Graphical presentation of relative mRNA expression of <i>Nfix</i>, E2A, ID2 and ID3 genes and presented relative to control MigR1 cells. Error bars denote +/- SD of 3 technical replicates. Data representative of 2 biological replicates.</p

Decreased B cells and increased myeloid cells in <i>Nfix</i> expressing cells in the periphery.

<p>C57Bl/6 mice were reconstituted with BM cells transduced with control MigR1 or <i>Nfix</i> vectors. <b>(A)</b> Graph of percentage number of GFP⁺ B cells (B220⁺, CD19⁺), myeloid cells (Gr-1⁺CD11b⁺), and <b>(C)</b> T cells (CD4⁺, CD8⁺) in the PB of MigR1 and <i>Nfix</i> chimeric animals 6 weeks post-transplant. <b>(B)</b> Representative flow cytometric analysis of PB cells in MigR1 and <i>Nfix</i> chimeric animals, 10 wk post-transplant, showing engraftment of GFP (left panel), B220⁺CD19⁺ B cells in the GFP^- and GFP⁺ fractions (middle panel, percentages given) and the Gr-1⁺CD11b⁺ myeloid cells in the GFP^- and GFP⁺ fractions (right panels, percentages given). Results are representative of 2 independent BMT experiments.</p

Combined allelic dosage of Nfia and Nfib regulates cortical development

Nuclear factor I family members nuclear factor I A and nuclear factor I B play important roles during cerebral cortical development. Nuclear factor I A and nuclear factor I B regulate similar biological processes, as their expression patterns, regulation of target genes and individual knockout phenotypes overlap. We hypothesised that the combined allelic loss of and would culminate in more severe defects in the cerebral cortex than loss of a single member.We combined immunofluorescence, co-immunoprecipitation, gene expression analysis and immunohistochemistry on knockout mouse models to investigate whether nuclear factor I A and nuclear factor I B function similarly and whether increasing allelic loss of and caused a more severe phenotype.We determined that the biological functions of nuclear factor I A and nuclear factor I B overlap during early cortical development. These proteins are co-expressed and can form heterodimers . Differentially regulated genes that are shared between and knockout mice are highly enriched for nuclear factor I binding sites in their promoters and are associated with neurodevelopment. We found that compound heterozygous deletion of both genes resulted in a cortical phenotype similar to that of single homozygous or knockout embryos. This was characterised by retention of the interhemispheric fissure, dysgenesis of the corpus callosum and a malformed dentate gyrus. Double homozygous knockout of and resulted in a more severe phenotype, with increased ventricular enlargement and decreased numbers of differentiated glia and neurons.In the developing cerebral cortex, nuclear factor I A and nuclear factor I B share similar biological functions and function additively, as the combined allelic loss of these genes directly correlates with the severity of the developmental brain phenotype

Expansion of the lateral ventricles and ependymal deficits underlie the hydrocephalus evident in mice lacking the transcription factor NFIX

Nuclear factor one X (NFIX) has been shown to play a pivotal role during the development of many regions of the brain, including the neocortex, the hippocampus and the cerebellum. Mechanistically, NFIX has been shown to promote neural stem cell differentiation through the activation of astrocyte-specific genes and via the repression of genes central to progenitor cell self-renewal. Interestingly, mice lacking Nfix also exhibit other phenotypes with respect to development of the central nervous system, and whose underlying causes have yet to be determined. Here we examine one of the phenotypes displayed by Nfix -/- mice, namely hydrocephalus. Through the examination of embryonic and postnatal Nfix -/- mice we reveal that hydrocephalus is first seen at around postnatal day (P) 10 in mice lacking Nfix, and is fully penetrant by P20. Furthermore, we examined the subcommissural organ (SCO), the Sylvian aqueduct and the ependymal layer of the lateral ventricles, regions that when malformed and functionally perturbed have previously been implicated in the development of hydrocephalus. SOX3 is a factor known to regulate SCO development. Although we revealed that NFIX could repress Sox3-promoter-driven transcriptional activity in vitro, SOX3 expression within the SCO was normal within Nfix -/- mice, and Nfix mutant mice showed no abnormalities in the structure or function of the SCO. Moreover, these mutant mice exhibited no overt blockage of the Sylvian aqueduct. However, the ependymal layer of the lateral ventricles was frequently absent in Nfix -/- mice, suggesting that this phenotype may underlie the development of hydrocephalus within these knockout mice

Opposing, spatially-determined epigenetic forces impose restrictions on stochastic olfactory receptor choice

Olfactory receptor (OR) choice represents an example of genetically hardwired stochasticity, where every olfactory neuron expresses one out of ~2000 OR alleles in the mouse genome in a probabilistic, yet stereotypic fashion. Here, we propose that topographic restrictions in OR expression are established in neuronal progenitors by two opposing forces: polygenic transcription and genomic silencing, both of which are influenced by dorsoventral gradients of transcription factors NFIA, B, and X. Polygenic transcription of OR genes may define spatially constrained OR repertoires, among which one OR allele is selected for singular expression later in development. Heterochromatin assembly and genomic compartmentalization of OR alleles also vary across the axes of the olfactory epithelium and may preferentially eliminate ectopically expressed ORs with more dorsal expression destinations from this ‘privileged’ repertoire. Our experiments identify early transcription as a potential ‘epigenetic’ contributor to future developmental patterning and reveal how two spatially responsive probabilistic processes may act in concert to establish deterministic, precise, and reproducible territories of stochastic gene expression