Multiple non-cell-autonomous defects underlie neocortical callosal dysgenesis in Nfib-deficient mice

Abstract

BackgroundAgenesis of the corpus callosum is associated with many human developmental syndromes. Key mechanisms regulating callosal formation include the guidance of axons arising from pioneering neurons in the cingulate cortex and the development of cortical midline glial populations, but their molecular regulation remains poorly characterised. Recent data have shown that mice lacking the transcription factor Nfib exhibit callosal agenesis, yet neocortical callosal neurons express only low levels of Nfib. Therefore, we investigate here how Nfib functions to regulate non-cell-autonomous mechanisms of callosal formation.ResultsOur investigations confirmed a reduction in glial cells at the midline in Nfib-/- mice. To determine how this occurs, we examined radial progenitors at the cortical midline and found that they were specified correctly in Nfib mutant mice, but did not differentiate into mature glia. Cellular proliferation and apoptosis occurred normally at the midline of Nfib mutant mice, indicating that the decrease in midline glia observed was due to deficits in differentiation rather than proliferation or apoptosis. Next we investigated the development of callosal pioneering axons in Nfib-/- mice. Using retrograde tracer labelling, we found that Nfib is expressed in cingulate neurons and hence may regulate their development. In Nfib-/- mice, neuropilin 1-positive axons fail to cross the midline and expression of neuropilin 1 is diminished. Tract tracing and immunohistochemistry further revealed that, in late gestation, a minor population of neocortical axons does cross the midline in Nfib mutants on a C57Bl/6J background, forming a rudimentary corpus callosum. Finally, the development of other forebrain commissures in Nfib-deficient mice is also aberrant.ConclusionThe formation of the corpus callosum is severely delayed in the absence of Nfib, despite Nfib not being highly expressed in neocortical callosal neurons. Our results indicate that in addition to regulating the development of midline glial populations, Nfib also regulates the expression of neuropilin 1 within the cingulate cortex. Collectively, these data indicate that defects in midline glia and cingulate cortex neurons are associated with the callosal dysgenesis seen in Nfib-deficient mice, and provide insight into how the development of these cellular populations is controlled at a molecular level.

Combined allelic dosage of Nfia and Nfib regulates cortical development

“…To exclude the possibility that knockout of Nfia and Nfib affected different subpopulations of radial glia, we analysed their co-expression at a cellular level in the developing cerebrum. The Nfib locus carries a knock-in β-galactosidase reporter gene substituted into the deleted exon of the Nfib allele as a marker for NFIB expression (Betancourt et al, 2014;Piper et al, 2009;Steele-Perkins et al, 2005). For each stage examined, we validated β-galactosidase and NFIB co-expression (Figure 1) to ensure that we were able to analyse co-expression of NFIA and NFIB using β-galactosidase protein expression as a reporter with fidelity (Figure 2).…”

“…In the context of the brain, NFIA and NFIB share many common transcriptional downstream targets, including Gfap, Hes1, Hes5, Sox3, Fgf9, Gli3, Id4, Cntn2, Efnb1 and Cdh2 (Barry et al, 2008;Betancourt et al, 2014;Brun et al, 2009;Cebolla and Vallejo, 2006;Harris et al, 2016;Piper et al, 2009Piper et al, , 2010Piper et al, , 2014Wang et al, 2007Wang et al, , 2010. In vitro experiments for targets such as Gfap and Cntn2 have validated their direct regulation by both NFIA and NFIB.…”

“…Analyses of knockout mouse models suggest that the general underlying cause of these phenotypes is a cellular differentiation defect which affects radial glia, the embryonic neural progenitor cells of the cerebral cortex, which give rise to mature neurons and glia in the cortex (Barry et al, 2008;Betancourt et al, 2014;Gobius et al, 2016;Piper et al, 2009Piper et al, , 2010Piper et al, , 2014. Knockout of either Nfia or Nfib causes the radial glia to remain proliferative and undifferentiated for an extended period of time.…”

Nuclear factor one B regulates neural stem cell differentiation and axonal projection of corticofugal neurons

“…One genetic candidate in neural progenitor cell maintenance is NFIB, a member of the Nuclear Factor One (NFI) family of transcription factors that is essential for normal brain development. Mice with an NfiB gene mutation (NfiB −/− ) have enlarged lateral ventricles, agenesis of the corpus callosum, defects in the formation of the hippocampus, basilar pons and midline structures and die at birth due to lung defects (Plachez et al, 2008;Kumbasar et al, 2009;Piper et al, 2009). NFIB regulates the migration and axonal projection of cerebellar granule neurons (Wang et al, 2007;Mason et al, 2009;Heng et al, 2012).…”

NFIA Controls Telencephalic Progenitor Cell Differentiation through Repression of the Notch Effector Hes1

“…After completion of the PCR amplification, the data were analyzed with the Rotor-Gene software as described previously (Piper et al, 2009b). When quantifying the mRNA expression levels, the housekeeping gene HPRT was used as a relative standard.…”