NFIX Regulates Proliferation and Migration Within the Murine SVZ Neurogenic Niche

Cerebral Cortex, October 2015;25: 3758–3778 doi: 10.1093/cercor/bhu253 Advance Access Publication Date: 19 October 2014 ORIGINAL ARTICLE ORIGINAL ARTICLE NFIX Regulates Proliferation and Migration Within Yee Hsieh Evelyn Heng1, Bo Zhou5, Lachlan Harris1, Tracey Harvey1, Aaron Smith1, Elise Horne1, Ben Martynoga4, Jimena Andersen4, Angeliki Achimastou4, Kathleen Cato1, Linda J. Richards1,2, Richard M. Gronostajski5, Giles S. Yeo6, François Guillemot4, Timothy L. Bailey3, and Michael Piper1,2 1 The School of Biomedical Sciences, 2Queensland Brain Institute, 3Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia, 4Division of Molecular Neurobiology, MRC-National Institute for Medical Research, London NW7 1AA, UK, 5Department of Biochemistry, Programs in Neuroscience and Genetics, Genomics & Bioinformatics, Developmental Genomics Group, New York State Center of Excellence in Bioinformatics and Life Sciences, State University of New York at Buffalo, Buffalo, NY 14203, USA, and 6MRC Metabolic Diseases Unit, University of Cambridge Metabolic Research Laboratories, Wellcome Trust-MRC Institute of Metabolic Science, Addenbrooke’s Hospital, Cambridge CB2 0QQ, UK Address correspondence to Michael Piper, The School of Biomedical Sciences and the Queensland Brain Institute, The University of Queensland, Brisbane 4072, Australia. Email: Abstract Transcription factors of the nuclear factor one (NFI) family play a pivotal role in the development of the nervous system. One member, NFIX, regulates the development of the neocortex, hippocampus, and cerebellum. Postnatal Nfix−/− mice also display abnormalities within the subventricular zone (SVZ) lining the lateral ventricles, a region of the brain comprising a neurogenic niche that provides ongoing neurogenesis throughout life. Specifically, Nfix−/− mice exhibit more PAX6-expressing progenitor cells within the SVZ. However, the mechanism underlying the development of this phenotype remains undefined. Here, we reveal that NFIX contributes to multiple facets of SVZ development. Postnatal Nfix−/− mice exhibit increased levels of proliferation within the SVZ, both in vivo and in vitro as assessed by a neurosphere assay. Furthermore, we show that the migration of SVZ-derived neuroblasts to the olfactory bulb is impaired, and that the olfactory bulbs of postnatal Nfix−/− mice are smaller. We also demonstrate that gliogenesis within the rostral migratory stream is delayed in the absence of Nfix, and reveal that Gdnf (glial-derived neurotrophic factor), a known attractant for SVZ-derived neuroblasts, is a target for transcriptional activation by NFIX. Collectively, these findings suggest that NFIX regulates both proliferation and migration during the development of the SVZ neurogenic niche. Key words: neuroblast, nuclear factor one X, olfactory bulb, rostral migratory stream, subventricular zone © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: the Murine SVZ Neurogenic Niche NFIX Regulates Proliferation and Migration Within the SVZ Introduction | 3759 exhibit increased numbers of neural progenitor cells within the SVZ, a finding supported by the increased numbers of spheres formed by Nfix−/− SVZ tissue in vitro in a neurosphere assay. Despite the increased levels of SVZ proliferation, the olfactory bulbs of Nfix−/− mice are smaller, with reduced numbers of interneurons expressing PAX6, calbindin and calretinin. Birthdating experiments further reveal deficits in the migration of SVZderived neuroblasts to the olfactory bulb. Finally, we demonstrate that gliogenesis within the RMS is delayed and identify Gdnf as a target for transcriptional activation by NFIX. Thus, NFIX regulates both proliferation and migration within the postnatal mouse SVZ/RMS. Materials and Methods Mouse Strains Wild-type and Nfix−/− littermate mice were used in this study. These mice were maintained on a C57Bl/6J background. Timedpregnant females were obtained by placing Nfix+/− male and Nfix+/− female mice together overnight. The following day was designated as embryonic day (E) 0 if the female had a vaginal plug. Mice were genotyped by polymerase chain reaction (PCR; Campbell et al. 2008). Transgenic mice expressing green fluorescent protein (GFP) under control of the glutamic acid decarboxylase 67 (Gad67) promoter were also used (Tamamaki et al. 2003), as were mice expressing GFP under the control of the Dcx promoter (Walker et al. 2007). The former mice have GFP knocked into the Gad67 locus, and expression of GFP has previously been shown to colocalize with GAD67 expression (Tamamaki et al. 2003). The latter strain (Dcx-GFP/bacterial artificial chromosome [BAC]) was originally obtained from the Mutant Mouse Regional Resource Center and the Gene Expression Nervous System Atlas BAC transgenic project. The pattern of GFP expression in these animals matches previously reported expression of DCX (Gleeson et al. 1999). Finally, we used another BAC transgenic line expressing GFP under the control of the Hes5 promoter. These mice have been shown previously to express GFP in neural stem cells within the adult brain (Jhaveri et al. 2010). All animals were bred at The University of Queensland under approval from the Institutional Animal Ethics Committee, and were performed according to the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Hematoxylin Staining Brains from wild-type or Nfix −/− mice were dissected from the skull, blocked in 3% noble agar (Difco), and sectioned coronally at 50 µm on a vibratome (Leica). Sections were mounted and stained with Mayer’s hematoxylin using standard protocols. Immunohistochemistry Embryos, postnatal pups and adult mice were transcardially perfused with 0.9% saline, followed by 4% paraformaldehyde, and postfixed in 4% paraformaldehyde at 4 °C. Brains were removed and sectioned at 50 μm using a vibratome. Immunohistochemistry (IHC) using the chromogen 3,30 -diaminobenzidine (DAB) was performed as described previously (Piper et al. 2009). Biotinconjugated goat anti-rabbit IgG (BA-1000, Vector Laboratories) and biotin-conjugated donkey anti-mouse IgG (715-065-150, Jackson Immunoresearch) secondary antibodies were used at 1/1000. To perform co-immunofluorescence (IF) labeling, sections were incubated overnight with the primary antibodies at 4 °C. The subventricular zone (SVZ) is one of the 2 neurogenic niches in the rodent brain that continuously generates neurons throughout adult life (Kriegstein and Alvarez-Buylla 2009). Within the SVZ, the division of neural stem cells produces transitamplifying cells, and ultimately neuroblasts. These SVZ-derived neuroblasts migrate away from the SVZ anteriorly along a distinct pathway known as the rostral migratory stream (RMS) to the olfactory bulb. Here, they differentiate into interneurons that migrate radially within the olfactory bulb to either the granule cell layer (...truncated)