Inactivation of the gene for the nuclear receptor tailless in the brain preserving its function in the eye Thorsten Belz, 1 Hai-Kun Liu, 1 Dagmar Bock, 1, * Andrea Takacs, 1, * Miriam Vogt, 2 Tim Wintermantel, 1,3 Christiane Brandwein, 2 Peter Gass, 2 Erich Greiner 1,4 and Gu ¨ nther Schu ¨tz 1 1 Division of Molecular Biology of the Cell I, German Cancer Research Center, D-69120 Heidelberg, Germany 2 Central Institute of Mental Health Mannheim, University of Heidelberg, D-68159 Mannheim, Germany 3 CRBA Gynaecology & Andrology, SCHERING AG, D-13342 Berlin, Germany 4 Evotec AG, D-22525 Hamburg, Germany Keywords: aggressiveness, blindness, conditional mutant, learning and memory, mouse Abstract During embryogenesis, tailless, an orphan member of the nuclear receptor family, is expressed in the germinal zones of the brain and the developing retina, and is involved in regulating the cell cycle of progenitor cells. Consequently, a deletion of the tailless gene leads to decreased cell number with associated anatomical defects in the limbic system, the cortex and the eye. These structural abnormalities are associated with blindness, increased aggressiveness, poor performance in learning paradigms and reduced anxiousness. In order to assess the contribution of blindness to the behavioural changes, we established tailless mutant mice with intact visual abilities. We generated a mouse line in which the second exon of the tailless gene is flanked by loxP sites and crossed these animals with a transgenic line expressing the Cre recombinase in the neurogenic area of the developing brain, but not in the eye. The resulting animals have anatomically indistinguishable brains compared with tailless germline mutants, but are not blind. They are less anxious and much more aggressive than controls, like tailless germline mutants. In contrast to germline mutants, the conditional mutants are not impaired in fear conditioning. Furthermore, they show good performance in the Morris water-maze despite severely reduced hippocampal structures. Thus, the pathological aggressiveness and reduced anxiety found in tailless germline mutants are due to malformations caused by inactivation of the tailless gene in the brain, but the poor performance of tailless null mice in learning and memory paradigms is dependent on the associated blindness. Introduction The exact regulation of proliferation and differentiation is essential for correct brain development. One factor involved in this process is the nuclear receptor tailless (tlx; Monaghan et al., 1997; Land & Monaghan, 2003; Roy et al., 2004; Shi et al., 2004). Strong tlx expression is detected in forebrain structures including the optic stalk, neural retina and the embryonic germinal zones during development (Monaghan et al., 1995). Previously we showed that mice with a targeted tlx mutation have a reduction of limbic and rhinencephalic structures, including the entorhinal cortex, amygdala and dentate gyrus (Monaghan et al., 1997). The loss of tlx causes a reduction of the thickness of the cerebral cortex, the striatum and other structures due to impaired proliferation of neuronal progenitor cells (Roy et al., 2002; Stenman et al., 2003). Tlx-deficient mice are abnormally aggressive, less anxious and show poor cognition (Monaghan et al., 1997; Roy et al., 2002; Young et al., 2002; Abrahams et al., 2005). This is in agreement with studies showing that hippocampal and amygdala lesions cause learning and memory deficits (Morris et al., 1982; Phillips & LeDoux, 1992), and that the limbic system is involved in anxiety and aggression (Davis, 1992; Guillot et al., 1994). Because tlx is involved in ocular development (Hollemann et al., 1998), tlx knockout mice additionally have a dramatic reduction in retina thickness and optic nerve diameter with associated blindness (Yu et al., 2000; Young et al., 2002; Miyawaki et al., 2004; Zhang et al., 2006). Comprehensive behavioural studies showed that impaired visual abilities severely influence the performance of rodents in the Morris water-maze and contextual fear-conditioning paradigm (Buhot et al., 2001; Garcia et al., 2004; Clapcote et al., 2005), and also have an impact on anxiety (Cook et al., 2001; Garcia et al., 2004). We therefore asked whether the set of behavioural abnormalities found in tlx mutants are caused by the brain defects per se or if they are a reflection of the mutation-associated blindness. To circumvent the disadvantage of the tlx germline deletion leading to blindness, we analysed tlx mutants with intact visual abilities. Therefore, we engineered a floxed tlx allele for conditional inactiva- tion of the tlx gene. Because we have observed expression of the calmodulin-dependent protein kinase II alpha (CaMKIIa) gene during development in the brain, but not in the eye, we investigated if CaMKIIa gene-driven expression of the Cre recombinase (CaMKCre; Casanova et al., 2001) would allow targeting of tlx in the developing brain, sparing the eye. Indeed, mutants obtained by crossing tlx flox ⁄ flox and CaMKCre mice show similar brain defects to tlx germline mutants, but show no abnormalities in the retina and are not blind. By comparing results from several behavioural tests of the conditional tlx mutants with tlx null mice, we conclude that the poor performance of tlx germline mutants in fear conditioning (Roy et al., 2002) results from a deficit at the level of vision. In contrast, the increased Correspondence: Professor Dr G. Schu ¨tz, as above. E-mail: g.schuetz@dkfz.de *D.B. and A.T. contributed equally to this work. Received 16 March 2007, revised 20 August 2007, accepted 21 August 2007 European Journal of Neuroscience, Vol. 26, pp. 2222–2227, 2007 doi:10.1111/j.1460-9568.2007.05841.x ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd