Professor Richard M. Gronostajski, USA : “NFI genes in CNS development”
Speaker:
Professor Richard M. Gronostajski
Department of Biochemistry, State University of New York at Buffalo
Title: “NFI genes in CNS development”
Abstract: Understanding postnatal neural stem/progenitor cell (pNSPC) self-renewal and lineage specification is key to future stem cell therapies. Here we assess the effects of loss of single or multiple Nfi genes on murine pNSPC self-renewal and differentiation in vitro. Germline loss of Nfia or Nfib reduces astrogenesis in cortex and spinal cord and results in prenatal dysgenesis of the corpus callosum. Conversely, germline loss of Nfix has minor effects on astrogenesis but promotes oligodendrogenesis. We generated floxed alleles of Nfia, Nfib and Nfix. Mice homozygous for these alleles and carrying R26CreERT2 are viable and pNSPCs were cultured from the subventricular zone (SVZ) of such mice.
pNSPCs of P10-20 mice were cultured in the presence of EGF and bFGF (proliferation (prolif.) conditions) then placed into medium lacking these growth factors (differentiation (differen.) conditions). Transcript levels of markers of self-renewal and differentiation were assessed by qPCR from RNA of cells cultured without (WT) or with 4HT (NFI-deleted) during both prolif. and differen. conditions. Treatment with 4HT during prolif. efficiently deleted all floxed alleles with >99% loss of transcripts within 3 days.
Deletion of Nfix resulted in little or no changes in prolif. or neuronal or astrocytic differen., but a bias towards the oligodendrocyte lineage, consistent with our previous studies on germline loss of Nfix. Deletion of Nfib also resulted in no obvious changes in self-renewal or neuronal differentiation but reduced the expression of astrocyte markers, consistent with our previous studies on loss of Nfib in vivo. Surprisingly, simultaneous deletion of Nfia & Nfib resulted in a major reduction in self-renewal as seen by reduced PCNA and Nestin expression and the loss of colony forming ability. Upon differentiation there were increases in the neuroblast marker DCX and reduced expression of astrocyte and oligodendrocyte markers. This loss of self renewal appears specific for the combined loss of Nfia & Nfib as the combined loss of Nfib & Nfix does not result in this phenotype.
We are currently assessing the molecular mechanisms that influence these changes in self-renewal and lineage-specification by RNA-seq analysis of prolif. WT and NFI-deleted pNSPCs and quantification of the cell types formed upon differentiation. We will also present evidence for a combined roll of Nfia and Nfib in gliogenesis in the mouse retina.
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Neuroscience seminars at the QBI play a major role in the advancement of neuroscience in the Asia-Pacific region. The primary goal of these seminars is to promote excellence in neuroscience through the exchange of ideas, establishing new collaborations and augmenting partnerships already in place.
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