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Conditional Gene Expression and Lineage Tracing of Tuba1a Expressing Cells During Zebrafish Development and Retina Regeneration

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Conditional Gene Expression and Lineage Tracing of Tuba1a Expressing Cells During Zebrafish Development and Retina Regeneration RESEARCH ARTICLE Conditional Gene Expression and Lineage Tracing of tuba1a Expressing Cells During Zebrafish Development and Retina Regeneration Rajesh Ramachandran,1 Aaron Reifler,1,2 Jack M. Parent,2,3 and Daniel Goldman1,2,4* 1Molecular and Behavioral Neuroscience Institute, University of Michigan, Ann Arbor, Michigan 48109 2Neuroscience Program, University of Michigan, Ann Arbor, Michigan 48109 3Department of Neurology, University of Michigan, Ann Arbor, Michigan 48109 4Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109 ABSTRACT During development, recombination revealed transient The tuba1a gene encodes a neural-specific a-tubulin tuba1a expression in not only neural progenitors but isoform whose expression is restricted to the develop- also cells that contribute to skeletal muscle, heart, and ing and regenerating nervous system. By using zebra- intestine. In the adult, recombination revealed tuba1a fish as a model system for studying CNS regeneration, expression in brain, olfactory neurons, and sensory cells we recently showed that retinal injury induces tuba1a of the lateral line, but not in the retina. After retinal gene expression in Mu¨ller glia that reentered the cell injury, recombination showed tuba1a expression in cycle. However, because of the transient nature of Mu¨ller glia that had reentered the cell cycle, and line- tuba1a gene expression during development and regen- age tracing indicated that these cells are responsible eration, it was not possible to trace the lineage of the for regenerating retinal neurons and glia. These results tuba1a-expressing cells with a reporter directly under suggest that tuba1a-expressing progenitors contribute the control of the tuba1a promoter. To overcome this to multiple cell lineages during development and that limitation, we generated tuba1a:CreERT2 and b-actin2: tuba1a-expressing Mu¨ller glia are retinal progenitors in loxP-mCherrry-loxP-GFP double transgenic fish that the adult. J. Comp. Neurol. 518:4196–4212, 2010. allowed us to label tuba1a-expressing cells conditionally and permanently via ligand-induced recombination. VC 2010 Wiley-Liss, Inc. INDEXING TERMS: Cre; recombination; neural development; regeneration; retina; Mu¨ller glia Unlike mammals, adult teleost fish such as zebrafish layer cell proliferation precedes retina regeneration sug- are able to regenerate a damaged central nervous system gested that retinal stem cells residing in the inner nuclear and therefore provide a good model for investigating layer are the predominant source of progenitors for regener- mechanisms underlying neuronal repair. The retina is a ation. Mu¨ller glia whose cell bodies reside in the inner nu- readily accessible portion of the central nervous system clear layer were observed to reenter the cell cycle follow- that has served as a model for studying both axonal and ing retinal damage (Braisted et al., 1994; Cameron, 2000; neuronal regeneration in fish (Hitchcock et al., 2004; Becker and Becker, 2007, 2008). The robust regenerative power of the teleost retina is demonstrated by the obser- Additional Supporting Information may be found in the online version of this article. vation that chemically induced whole-retina destruction Grant sponsor: National Eye Institute; Grant number: RO1 EY018132 (to results in retinal regeneration that is accompanied by res- D.G.); Grant sponsor: National Institute of Neurological Disorders and Stroke; Grant number: 1 R21 NS061293 (to J.P. and D.G.); Grant sponsor: toration of visual function (Sherpa et al., 2007). The cells National Institute of Mental Health; Grant number: 5T32MH014279 (to that contribute to retinal regeneration appear to be resi- A.R.); Grant sponsor: National Institute of Child Health and Human dent in the inner nuclear layer, where clusters or columns Development; Grant number: T32HD007507 (to R.R.). *CORRESPONDENCE TO: Daniel Goldman, University of Michigan, 5045 of proliferating cells are observed (Raymond and BSRB, 109 Zina Pitcher Place, Ann Arbor, MI 48109. Hitchock, 2000; Vihtelic and Hyde, 2000; Wu et al., 2001; E-mail: [email protected] Faillace et al., 2002). The observation that inner nuclear Received August 26, 2009; Revised December 18, 2009; Accepted June 1, 2010 DOI 10.1002/cne.22448 Published online June 17, 2010 in Wiley Online Library (wileyonlinelibrary. VC 2010 Wiley-Liss, Inc. com) 4196 The Journal of Comparative Neurology | Research in Systems Neuroscience 518:4196–4212 (2010) Conditional gene expression and lineage tracing in zebrafish Wu et al., 2001). These observations, combined with moters (Boniface et al., 2009; Hans et al., 2009). One reports that Mu¨ller glia in postnatal chicks and rodents study, with fish embryos that harbor the pax2a:CreERT2 have a limited capacity to regenerate neurons following transgene and a recombination reporter driven by the retinal injury (Fischer and Reh, 2001; Ooto et al., 2004), EF1a promoter, found ligand-dependent CreERT2 activa- implicated Mu¨ller glia as a source of retinal progenitors tion and recombination in developing embryos (Hans that participate in repair of damaged retinas. et al., 2009). In contrast, another study, using the her4.1 Support for Mu¨ller glia as a source of retinal progeni- promoter to drive CreERT2 expression, reported ligand-in- tors comes from studies with transgenic fish, in which the dependent CreERT2 activation that could be prevented by fate of green fluorescent protein (GFP)-expressing Mu¨ller appending an additional ER ligand binding domain to the glia can be followed for short periods of time following CreERT2 fusion (Boniface et al., 2009). These studies retinal injury (Fausett and Goldman, 2006; Bernardos were restricted to the evaluation of conditional gene et al., 2007; Fimbel et al., 2007; Thummel et al., 2008). expression in early developing embryos by bathing fish By using transgenic fish harboring a 1,016-bp fragment of embryos in water containing 4-OHT. Although these stud- the tuba1a promoter driving GFP expression (1016 ies suggest that the CreERT2/LoxP system will be useful tuba1a:GFP), we found that tuba1a-expressing Mu¨ller glia for conditional gene expression during development, its respond to retinal injury by dedifferentiating and prolifer- suitability for conditional gene expression in adults and ating (Fausett and Goldman, 2006). However, because its use for lineage tracing in developing and adult animals the tuba1a promoter was only transiently expressed in remained untested. these dedifferentiated Mu¨ller glia, it was not possible to Motivated by the need for a conditional gene expres- follow their fate over long periods of time and confirm sion system that would allow gene recombination at any they were stably integrated into the retinal architecture. stage of development, including adulthood, and that was To follow the fate of these tuba1a-expressing progenitors amenable for lineage tracing of Mu¨ller glia-derived pro- in the adult retina, we developed a conditional expression genitors in the injured retina, we developed the following system that allowed for permanent labeling of tuba1a- transgenic fish: 1) 1016 tuba1a:CreERT2, in which a expressing Mu¨ller glia. Based on previous success in 1,016-bp fragment of the tuba1a promoter directs mice, we chose to use the CreERT2/LoxP recombination CreERT2 expression to the developing and regenerating system for this purpose (Branda and Dymecki, 2004). CNS; 2) b-actin2:LCLG, in which the ubiquitously Cre-mediated recombination offers many advantages expressed b-actin2 promoter drives mCherry (C) expres- for studying gene function by stimulating targeted dele- sion that is flanked by loxP (L) sites and followed by an tion, insertion, inversion, and exchange of chromosomal out-of-frame EGFP (G) sequence; and 3) 1016 DNA (Branda and Dymecki, 2004). Recently, the Cre/ tuba1a:CreERT2;b-actin2:LCLG double transgenic fish, in LoxP system has been used for conditional gene expres- which the latter transgene serves as a recombination re- sion in developing zebrafish in which either a tissue-spe- porter and allows one to label permanently cells that ei- cific or a ubiquitously expressed heat shock-regulated ther constitutively or transiently express CreERT2 driven promoter was used to drive Cre recombinase expression by the 1016 tuba1a promoter. With these fish, we show (Feng et al., 2007; Le et al., 2007; Liu et al., 2008; Wang that transgenic lines expressing CreERT2 at low levels do et al., 2008; Hans et al., 2009; Collins et al., 2010). How- not exhibit basal ligand-independent CreERT2 activity. ever, these promoters have limitations insofar as they These low-expressing lines allowed us to map the fate of lack temporal control (tissue-specific promoters) or may cells expressing the 1016 tuba1a promoter during devel- exhibit leaky expression (heat shock promoters). In addi- opment and in the adult injured retina. We found that this tion, stressing fish by a heat shock might have unin- recombination system revealed very low and transient tended consequences on cell function. For the mouse, 1016 tuba1a promoter activity that could not be observed these limitations have been surmounted by taking advant- with traditional 1016 tuba1a:GFP transgenes. This age of a ligand-dependent chimeric Cre recombinase improved sensitivity allowed us to identify descendents of where Cre is fused to the mutant ligand-binding domain tuba1a-expressing cells early in development that include of the human estrogen receptor (CreERT and CreERT2; neural and
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