4831 DEVELOPMENT AND STEM CELLS RESEARCH ARTICLE INTRODUCTION In mammals, severe injury to the adult brain has catastrophic effects and significant regeneration does not occur. By contrast, adult non- mammalian vertebrates can regenerate considerable regions of their central nervous system (CNS) (Endo et al., 2007; Font et al., 2001; Kaslin et al., 2008; Kizil et al., 2011; Tanaka and Ferretti, 2009). Although different means of cellular regeneration, such as transdifferentiation, dedifferentiation and activation of endogenous progenitors have been proposed, the mechanisms are currently not known (Echeverri and Tanaka, 2002; Kikuchi et al., 2010; Kirsche, 1965; Kragl et al., 2009; Yoshii et al., 2007). Work in frogs, lizards, salamanders and fish suggests that neural stem/progenitor cells at the ventricle react to CNS injury by increasing proliferation and neurogenesis, and might be the origin of regenerated neurons (Endo et al., 2007; Font et al., 2001; Kaslin et al., 2008; Kirsche and Kirsche, 1961; Parish et al., 2007; Tanaka and Ferretti, 2009; Zupanc and Clint, 2003). However, the experimental evidence for this notion is indirect: it relies on the observation of upregulated proliferation and neurogenesis after lesion in the ventricular zone and enhanced migration of newborn neurons to the lesion site (Endo et al., 2007; Font et al., 2001; Kaslin et al., 2008; Kirsche, 1965; Romero-Aleman et al., 2004; Zupanc and Ott, 1999). In addition, removal of the constitutive proliferation zones in the carp optic tectum prevents restoration of the tissue architecture (Kirsche and Kirsche, 1961). Short-term lineage-tracing analyses of ventricular radial glia (RG) in the brain and spinal cord suggested that RG could give rise to new neurons after injury. Because these studies relied on transient protein persistence, and did not permit long-term cell fate analysis (Berg et al., 2010; Echeverri and Tanaka, 2002; Reimer et al., 2008), the cellular basis of neuronal regeneration in the CNS remained unresolved. To study the origin of newly generated neurons in regenerating brains, we developed a novel traumatic stab lesion model. We find that the adult zebrafish brain efficiently regenerates and restores the tissue architecture completely. Using conditional CreER T2 -loxP lineage-tracing (Hans et al., 2009), we show that ventricular RG expressing her4.1 (an orthologue of mammalian hes5) serve as neuronal progenitors that respond to the lesion. Upon injury, these cells increase proliferation, upregulate neuronal-fate determining gene transcription, and give rise to neuroblasts that migrate into the periventricular zone and deeper into the parenchyma to the site of injury, where they differentiate into mature neurons. MATERIALS AND METHODS All animal experiments were conducted according to the guidelines and under supervision of the Regierungspräsidium Dresden (permit AZ 24D- 9168.11-1/2008-1 and AZ 24D-9168.11-1/2008-4). All efforts were made to minimize animal suffering and the number of animals used. Fish maintenance Fish were kept under standard conditions as described previously (Brand et al., 2002). Wild-type fish were from the gol-b1 line in the AB genetic background. Stab lesion assay Adult fish were 6-10 months old and had a body length of 25-32 mm. Fish were anaesthetized using Tricaine (Sigma). A canula (30 gauge, outer diameter 300 m) was pushed through a nostril ~6-8 mm deep along the rostrocaudal body axis, through the olfactory bulb until reaching the caudal part of the telencephalon. The nostrils helped to guide the canula into the dorsal telencephalon (Fig. 1A). Stab-lesioned fish survived well (>90%) with only minor bleeding, and recovered in fresh fishwater. Sham-operated animals were treated equally, except that the canula was inserted into the nostril and not further into the brain. Plasmid construction and germline transformation To create the pTol her4.1:mCherryT2ACreER T2 plasmid, the her4.1 promoter (Yeo et al., 2007) was PCR amplified (her4-for, 5'-agtaGGG- CCCctgtgtgagcagtcatgtt; her4-rev, 5'-cattGGCCGGCCgtcaggatcagatctg- agct) flanked by ApaI and FseI restriction sites that allowed substitution of the hsp70l promoter of the pTol her4.1:mCherryT2ACreER T2 plasmid (Hans et al., 2011). To generate pTol hsp70l:DsRed2(floxed)EGFP Development 138, 4831-4841 (2011) doi:10.1242/dev.072587 © 2011. Published by The Company of Biologists Ltd Biotechnology Center and DFG-Research Center for Regenerative Therapies Dresden (CRTD), Technische Universität Dresden, Tatzberg 47/49, 01307 Dresden, Germany. *Author for correspondence (michael.brand@biotec.tu-dresden.de) Accepted 6 September 2011 SUMMARY Severe traumatic injury to the adult mammalian CNS leads to life-long loss of function. By contrast, several non-mammalian vertebrate species, including adult zebrafish, have a remarkable ability to regenerate injured organs, including the CNS. However, the cellular and molecular mechanisms that enable or prevent CNS regeneration are largely unknown. To study brain regeneration mechanisms in adult zebrafish, we developed a traumatic lesion assay, analyzed cellular reactions to injury and show that adult zebrafish can efficiently regenerate brain lesions and lack permanent glial scarring. Using Cre-loxP-based genetic lineage-tracing, we demonstrate that her4.1-positive ventricular radial glia progenitor cells react to injury, proliferate and generate neuroblasts that migrate to the lesion site. The newly generated neurons survive for more than 3 months, are decorated with synaptic contacts and express mature neuronal markers. Thus, regeneration after traumatic lesion of the adult zebrafish brain occurs efficiently from radial glia-type stem/progenitor cells. Key words: CNS, Adult neural stem cells, Brain injury, Genetic lineage-tracing, Neurogenesis, Teleost Regeneration of the adult zebrafish brain from neurogenic radial glia-type progenitors Volker Kroehne, Dorian Freudenreich, Stefan Hans, Jan Kaslin and Michael Brand* DEVELOPMENT