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NFIX Regulates Neural Progenitor Cell Differentiation During Hippocampal Morphogenesis

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NFIX Regulates Neural Progenitor Cell Differentiation During Hippocampal Morphogenesis Cerebral Cortex Advance Access published October 4, 2012 Cerebral Cortex doi:10.1093/cercor/bhs307 NFIX Regulates Neural Progenitor Cell Differentiation During Hippocampal Morphogenesis Yee Hsieh Evelyn Heng1, Robert C. McLeay3, Tracey J. Harvey1, Aaron G. Smith1, Guy Barry2, Kathleen Cato1, Céline Plachez4, Erica Little2, Sharon Mason2, Chantelle Dixon1, Richard M. Gronostajski5, Timothy L. Bailey3, Linda J. Richards1,2 and Michael Piper1,2 1The School of Biomedical Sciences, 2Queensland Brain Institute, 3Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia, 4School of Medicine, The University of Maryland, Baltimore, MD, USA and 5Department of Biochemistry and the Program in Neuroscience, Developmental Genomics Group, New York State Center of Excellence in Bioinformatics and Life Sciences, State University of New York at Buffalo, Buffalo, NY, USA Address correspondence to M. Piper, The School of Biomedical Sciences and the Queensland Brain Institute, The University of Queensland, Brisbane 4072, Queensland, Australia. Email: [email protected] Neural progenitor cells have the ability to give rise to neurons and coordinated during development. Examples include the glia in the embryonic, postnatal and adult brain. During development, Notch (Shimojo et al. 2008; Imayoshi et al. 2010), fibroblast the program regulating whether these cells divide and self-renew or growth factor (Sahara and O’Leary 2009; Rash et al. 2011), Downloaded from exit the cell cycle and differentiate is tightly controlled, and imbal- and Sonic hedgehog (SHH; Komada et al. 2008) signaling ances to the normal trajectory of this process can lead to severe pathways, all of which have been implicated in regulating pro- functional consequences. However, our understanding of the mol- genitor cell identity during development of the cortex. Tran- ecular regulation of these fundamental events remains limited. scription factors of the Sry-related HMG box (SOX) family Moreover, processes underpinning development of the postnatal have also been shown to play a role in the maintenance of neurogenic niches within the cortex remain poorly defined. Here, we progenitor cell identity (Stolt and Wegner 2010). For instance, http://cercor.oxfordjournals.org/ demonstrate that Nuclear factor one X (NFIX) is expressed by neural both SOX2 and SOX3 are expressed by neural progenitor progenitor cells within the embryonic hippocampus, and that pro- cells within the developing and adult forebrain (Avilion et al. genitor cell differentiation is delayed within Nfix−/− mice. Moreover, 2003; Wang et al. 2006), and SOX2 has been implicated in we reveal that the morphology of the dentate gyrus in postnatal maintaining progenitor cell identity within the developing Nfix−/− mice is abnormal, with fewer subgranular zone neural pro- neocortex (Bani-Yaghoub et al. 2006) and the adult hippo- genitor cells being generated in the absence of this transcription campus (Suh et al. 2007). Another suite of molecules known factor. Mechanistically, we demonstrate that the progenitor cell to play a role in regulating the differentiation of neural pro- maintenance factor Sry-related HMG box 9 (SOX9) is upregulated in genitor cells are the transcription factors of the Nuclear factor Nfix−/− the hippocampus of mice and demonstrate that NFIX can one (NFI) family (Piper et al. 2007; Mason et al. 2009), which at Buffalo State on August 30, 2013 repress Sox9 promoter-driven transcription. Collectively, our findings in vertebrates comprises 4 members; Nfia, Nfib, Nfic, and Nfix demonstrate that NFIX plays a central role in hippocampal morpho- (Rupp et al. 1990; Kruse et al. 1991). Mice lacking either Nfia genesis, regulating the formation of neuronal and glial populations or Nfib display neurological phenotypes including dysgenesis within this structure. of the corpus callosum (Shu, Butz, et al. 2003; Shu, Puche, et al. 2003; Piper, Moldrich, et al. 2009; Piper, Plachez, et al. Keywords: glia, glial fibrillary acidic protein, neural progenitor cell, nuclear 2009), hippocampal malformation (Barry et al. 2008; Piper factor one X, SOX9 et al. 2010), and delays in cerebellar development (Steele- Perkins et al. 2005; Wang et al. 2007). Mechanistically, Nfi genes have been implicated in regulating glial development Introduction via promoting the expression of astrocyte-specific genes fi fi During nervous system formation, one of the most important (Gopalan et al. 2006; Brun et al. 2009), and N a and N b developmental events to occur is the differentiation of neural were recently shown to promote progenitor cell differen- progenitor cells into neurons and glia. The embryonic fore- tiation in a complementary fashion within the developing brain provides a cogent example of this, with neural progeni- telencephalon through the repression of the Notch signaling tor cells within the proliferative ventricular zone, region pathway (Piper et al. 2010). fi −/− executing a program of proliferation, then differentiation, to N x mice also display severe neurological phenotypes generate the postmitotic cells of the cortex and hippocampus (Driller et al. 2007; Campbell et al. 2008), and NFIX has pre- (Sauvageot and Stiles 2002). The abnormal proliferation or viously been implicated in driving the expression of astrocytic differentiation of cortical neural progenitor cells during devel- genes during neural development (Gopalan et al. 2006; Piper opment can lead to severe functional consequences, such as et al. 2011). However, the mechanism by which NFIX regu- lissencephaly and microcephaly, both of which can cause lates morphogenesis of the nervous system in vivo remains fi fi −/− mental retardation (Manzini and Walsh 2011). As such, under- unde ned. Here, using the developing hippocampus of N x standing the regulatory processes controlling whether neural mice as a model, we reveal that NFIX regulates the differen- progenitor cells either divide and self-renew or exit the cell tiation of neural progenitor cells through the transcriptional fi cycle and differentiate is critical to our understanding of both regulation of progenitor-speci c pathways. Our data demon- fi −/− normal and pathological cortical development. strate that N x mice display delayed progenitor cell differ- fi A number of recent studies have begun to elucidate some entiation, which culminates in de cits in both neuronal and of the key molecules and signaling pathways that control how glial formation. Moreover, the formation of progenitor cells fi −/− neural progenitor cell proliferation and differentiation is within the postnatal dentate gyrus is abnormal in N x © The Author 2012. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: [email protected] mice. Finally, we show that SOX9, a central mediator of pro- Switzerland); anti-calretinin (rabbit polyclonal, 1/50 000, SWANT); genitor cell self-renewal that acts downstream of SHH signal- anti-reelin (mouse monoclonal, 1/100 000, a gift from Dr Andre Goffi- ing during corticogenesis (Scott et al. 2010), is a target for net, University of Louvain Medical School, Brussels, Belgium); anti-SOX2 (rabbit polyclonal, 1/1000; Cell Signaling Technology, transcriptional repression by NFIX. Taken together, these data Danvers, MA, United States of America); anti-cleaved caspase-3 (rabbit reveal a central role for NFIX in orchestrating the timely differ- polyclonal, 1/5000, Cell Signaling Technology); anti-nestin (mouse entiation of neural progenitor cells within the embryonic hip- monoclonal, 1/1500, Developmental Studies Hybridoma Bank); and pocampus and for regulating the development of neural anti-tenascin C (rabbit polyclonal, 1/5000, Millipore Bioscience Re- progenitor cells within the subgranular zone of the postnatal search Reagents). Primary antibodies used for immunohistochemistry fi dentate gyrus. on paraf n sections were anti-PAX6 (rabbit polyclonal, 1:1000; Milli- pore Bioscience Research Reagents), anti-SOX2 (1:1000), anti-SOX9 (rabbit polyclonal, 1:1000; a gift from Dr Peter Koopman, Institute for Materials and Methods Molecular Bioscience, University of Queensland, Brisbane, Australia), and anti-phosphohistone H3 (rabbit polyclonal, 1:1000; Millipore Bio- science Research Reagents). Mouse Strains − − Nfix+/+ and Nfix / littermate mice were used for the majority of this study. These mice were maintained on a C57Bl/6J background. These Quantification of Ventricular Zone Width/Hippocampal animals were bred at the University of Queensland under approval Cell Counts from the institutional animal ethics committee. Timed-pregnant – − − To measure the ventricular zone width in E14 18 wild-type and +/ +/ − − fi fi Downloaded from females were obtained by placing N x male and N x female Nfix / brains, sections were hematoxylin-stained and imaged with mice together overnight. The following day was designated as em- an upright microscope coupled to AxioVision software (Zeiss). The bryonic day (E)0 if the female had a vaginal plug. Embryos were gen- width of the ventricular zone was measured at 3 points along the hip- otyped by polymerase chain reaction (PCR; Campbell et al. 2008). For pocampus for each section. Data for both wild-type and knockout the in utero electroporation experiments, wild-type CD-1 mice were hippocampi at each age were then pooled for the comparison of ven- used. tricular zone width. For phosphohistone H3-, PAX6-, SOX2-, TBR2-, and SOX9-expressing cell counts, the total number of immunopositive http://cercor.oxfordjournals.org/ cells per 100
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