European Journal of Neuroscience European Journal of Neuroscience, pp. 1–12, 2011 doi:10.1111/j.1460-9568.2010.07570.x Emx1-expressing neural stem cells in the subventricular zone give rise to new interneurons in the ischemic injured striatum Bin Wei,* Yanzhen Nie,* Xiaosu Li, Congmin Wang, Tong Ma, Zengjin Huang, Miao Tian, Chifei Sun, Yuqun Cai, Yan You, Fang Liu and Zhengang Yang Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Fudan University, 138 Yi Xue Yuan Road, Shanghai, 200032 China Keywords: calretinin, hypoxia-ischemia, mouse, Pax6, Sp8, striatum Abstract Neural stem cells from different regions within the subventricular zone (SVZ) are able to produce several different subtypes of interneurons in the olfactory bulb throughout life. Previous studies have shown that ischemic stroke induces the production of new neurons in the damaged striatum from the SVZ. However, the origins and genetic profiles of these newborn neurons remain largely unknown as SVZ neural stem cells are heterogeneous. In the present study, using a mouse model of perinatal hypoxic-ischemic (H ⁄ I) brain injury combined with BrdU labeling methods, we found that, as in rat brains, virtually all newborn neuroblasts that migrate from the SVZ into the ischemic injured striatum exclusively express the transcription factor Sp8. Furthermore, although newborn neuroblasts are plentiful in the damaged striatum, only a few can differentiate into calretinin-expressing (CR+) interneurons that continuously express Sp8. Genetic fate mapping reveals that newly born CR+ interneurons are generated from Emx1-expressing neural stem cells in the dorsal–lateral SVZ. These results suggest that the fate of the Emx1-expressing lineage in the ischemic damaged striatum is restricted. However, when Sp8 was conditionally inactivated in the Emx1-lineage cells, Pax6 was ectopically expressed by a subpopulation of Emx1-derived CR+ cells in the normal and damaged striatum. Interestingly, these cells possessed large cell bodies and long processes. This work identifies the origin of the newly born CR+ interneurons in the damaged striatum after ischemic brain injury. Introduction The existence of neural stem cells and the persistence of neurogenesis Experimentally induced perinatal hypoxic-ischemic (H ⁄ I) brain in the postnatal subventricular zone (SVZ) (Ming & Song, 2005; Zhao injury in rodents, which primarily damages the ipsilateral hemisphere, et al., 2008; Kriegstein & Alvarez-Buylla, 2009) have raised hopes for is a widely used model system for studying neurodegeneration the development of effective methods for treating neurodegenerative (Vannucci& Vannucci, 1997). We have previously shown that induction diseases, stroke and traumatic brain injury. Indeed, numerous studies of H ⁄ I injury in the perinatal rat brain can result in the newly born have shown that ischemic stroke induces the production of new neuroblasts migrating from the SVZ into the damaged striatum and neurons in the damaged striatum from SVZ neural stem cells. differentiating into calretinin-expressing (CR+) interneurons (Yang However, the potential for plasticity of these cells after brain injury et al., 2008), a phenomenon similar to that observed in the adult brain remains a highly controversial issue (Kernie & Parent, 2010). after stroke (Liu et al., 2009). This response, usually referred to as Furthermore, little is known about the origins and genetic profiles of neurogenesis in the damaged striatum, is much more robust in the these newborn neurons in the damaged striatum after brain injury. immature brain than adult brain. Previous studies have shown that Thus, there has been a gap in our understanding of normal neural neural stem cells in the normal mouse brain comprise a heterogeneous development and ectopic neurogenesis after brain injury. In this study, population of progenitors that are restricted in their developmental we have taken advantage of genetic fate mapping to permanently label potential (Merkle et al., 2007). Neural stem cells in the perinatal SVZ, newly born neurons in the ischemic damaged striatum to address this however, are actually more diverse than those in the adult SVZ (Merkle issue. et al., 2007; Young et al., 2007; Batista-Brito et al., 2008; Yang, 2008). As more transgenic or knockout mice are now available, the perinatal mouse H ⁄ I model provides perhaps the best system for exploring the Correspondence: Dr Z. Yang, as above. potential for plasticity of neural stem cells in the pathological brain and E-mail: [email protected] the underlying molecular mechanisms. *B.W. and Y.N. contributed equally to this work. In the present study, using a genetic fate mapping approach, we Received 7 November 2010, revised 21 November 2010, accepted 23 November 2010 found that neuroblasts generated from Emx1-expressing neural stem ª 2011 The Authors. European Journal of Neuroscience ª 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd 2B.Weiet al. cells in the dorsal–lateral SVZ only give rise to CR+ interneurons in goat anti-CR (1 : 2000; Millipore), rabbit anti-CR (1 : 8000; Milli- the damaged striatum. As in rat brains, nearly all newborn young and pore), rabbit anti-DARPP-32 (1 : 200; Cell Signaling, Danvers, MA, mature neurons in the mouse striatum continuously express the USA), goat anti-Dcx (1 : 100; Santa Cruz Biotechnology, Santa transcription factor Sp8. By contrast, no newborn neuroblasts Cruz, CA, USA), rabbit anti-Dcx (1 : 500; Abcam, Cambridge, MA, differentiate into medium spiny neurons, the primary striatal projec- USA), rabbit anti-Foxp1 (1 : 500; Abcam), chicken anti-green tion neurons. Surprisingly, when Sp8 was conditionally inactivated in fluorescent protein (GFP, 1 : 2000; Aves Labs, Tigard, OR, USA), the Emx1-expressing lineage, a subpopulation of CR+ cells in the mouse anti-NeuN (1 : 500; Millipore), mouse anti-parvalbumin normal and damaged striatum ectopically expressed Pax6. Moreover, (1 : 1000; Millipore), rabbit anti-Pax6 (1 : 300; Covance, Princeton, these cells had large cell bodies and long processes. The present study MA, USA), rabbit anti-somatostatin (1 : 100; Santa Cruz) and goat identifies the origin of the newly born CR+ interneurons in the anti-Sp8 (1 : 500; Santa Cruz). Secondary antibodies against the damaged striatum after ischemic brain injury. This also indicates that appropriate species were incubated for 2 h at room temperature Emx1-expressing neural stem cells might be a valuable manipulation (from Jackson; 1 : 200). Fluorescently stained sections were then target for brain repair after injury. washed, counterstained with DAPI (Sigma; 200 ng ⁄ mL) for 5 min and coverslipped with Gel ⁄ Mount (Biomeda, Foster City, CA, USA). Double immunostaining of lateral wall whole mounts was Materials and methods performed as previously described (Mirzadeh et al., 2008). Omission Mice of primary antibodies eliminated staining. C57BL ⁄ 6 mice were obtained from Shanghai SLAC Laboratory Animal Co. Ltd (Shanghai, China). Z ⁄ EG (Novak et al., 2000) and Microscopy Emx1-Cre (Gorski et al., 2002) mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Sp8 conditional mutant mice Fluorescently immunolabeled sections were analysed on an Olympus were obtained from crossing double heterozygous males (Emx1-Cre; FV1000 confocal laser scanning microscope. Confocal Z sectioning Sp8Flox ⁄ +) with Sp8 homozygous flox (Sp8Flox ⁄ flox) females (Waclaw was performed at 1.0- or 0.5-lm intervals using a ·20 (NA = 0.75) or et al., 2006). All experiments using animals were carried out in ·60 (NA = 1.42) objective for single, double and triple labeling. accordance with institutional guidelines, and the study was approved Images were acquired and a Z-stack was reconstructed using the by the Fudan University Animal Care and Use Committee. All efforts FV10-ASW software, cropped, adjusted and optimized in Photoshop were made to minimize the number of animals used. 9.0. Images of fluorescently immunostained sections were also acquired using an Olympus BX 51 microscope. Induction of perinatal H ⁄ I injury Electron microscopy Perinatal H ⁄ I injury was produced in 8-day-old mice (day of birth was designated P0) by a permanent unilateral common carotid artery For Sp8 pre-embedding immunostaining, adult mice were perfused ligation followed by systemic hypoxia. Briefly, pups were lightly with 4% paraformaldehyde and 0.5% glutaraldehyde, and postfixed anesthetized with isoflurane (4% induction, 2% maintenance). Once overnight. Fifty-micron sections were cut on the vibratome and fully anesthetized, a midline neck incision was made and the right washed in TBS three times for 10 min. Sections were then blocked for common carotid artery was identified. The right common carotid 1 h in 10% donkey serum and incubated for 48 h at 4 °C with anti- artery was separated from the vagus nerve and then ligated with 4-0 Sp8. Secondary antibody (biotinylated donkey-anti goat) was incu- silk. Pups were returned to the dam for 2 h. Before being exposed to bated for 3 h at room temperature. Sections were then incubated with Streptavidin-coupled horseradish peroxidase for 1 h and revealed with hypoxia (8% O2 ⁄ 92% N2), the pups were prewarmed for 10 min in jars submerged in a 37 °C water bath. The pups were then exposed to diaminobenzidine (DAB). After immunostaining the sections were hypoxia for 50 min. After hypoxia the pups were returned to the dam post-fixed in 2% osmium for 2 h, rinsed, dehydrated and embedded in and maintained under standard conditions. Araldite. To recognize the SVZ, 1-lm semithin sections were stained with 1% toluidine blue. To further identify individual cells in the SVZ, we cut 60-nm ultrathin sections with a diamond knife. The sections were stained with lead citrate and uranyl acetate, and examined under BrdU injections a Philips CM120 electron microscope. BrdU (50 mg ⁄ kg; Sigma, St. Louis, MO, USA) was injected intraperitoneally (i.p.) twice daily (8-h intervals) during days 3–6 after H ⁄ I injury (in total, 4 days eight times). Animals were perfused Cell quantification 3, 5 or 13 weeks after H ⁄ I injury.