Development 127, 2515-2522 (2000) 2515 Printed in Great Britain © The Company of Biologists Limited 2000 DEV4317 Glial cell fate specification modulated by the bHLH gene Hes5 in mouse retina Masato Hojo1,2, Toshiyuki Ohtsuka1, Nobuo Hashimoto2, Gérald Gradwohl3, François Guillemot3 and Ryoichiro Kageyama1,* 1Institute for Virus Research, Kyoto University and 2Department of Neurosurgery, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto 606-8507, Japan 3Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/Université Louis Pasteur/Collège de France, 67404 Illkirch, CU de Strasbourg, France *Author for correspondence (e-mail: [email protected]) Accepted 29 March; published on WWW 23 May 2000 SUMMARY Neurons and glial cells differentiate from common retrovirus significantly increased the population of glial precursors. Whereas the gene glial cells missing (gcm) cells at the expense of neurons. Conversely, Hes5-deficient determines the glial fate in Drosophila, current data about retina showed 30-40% decrease of Müller glial cell number the expression patterns suggest that, in mammals, gcm without affecting cell survival. These results indicate that homologues are unlikely to regulate gliogenesis. Here, we Hes5 modulates glial cell fate specification in mouse retina. found that, in mouse retina, the bHLH gene Hes5 was specifically expressed by differentiating Müller glial cells Key words: bHLH, Glial specification, Hes5, Müller glia, Retina, and that misexpression of Hes5 with recombinant Mouse, Cell fate INTRODUCTION homologues have been characterized (Gcm1/Gcma and Gcm2/Gcmb) (Akiyama et al., 1996; Kim et al., 1998; Retina provides a powerful model system to investigate the Kanemura et al., 1999; Reifegerste et al., 1999) and it was mechanisms of cell fate decision in mammalian central nervous shown that one of them, Gcm1, can substitute functionally for system. From common retinal precursors, six types of neurons Drosophila gcm by transforming neurons into glia in and one type of glial cells (Müller glia) differentiate in the Drosophila embryos (Kim et al., 1998; Reifegerste et al., 1999), overlapping but precise time course (Cepko et al., 1996). These suggesting that mammalian gcm homologues may have a seven types of cells constitute three nuclear layers in the retina: conserved gliogenic function, at least, in Drosophila embryos. the outer nuclear layer (ONL), which contains rod and cone However, neither of the mammalian gcm homologues show the photoreceptors, the inner nuclear layer (INL), which contains expression pattern that may account for generation of glial cells bipolar, horizontal and amacrine interneurons as well as Müller in mammalian nervous system and therefore these genes are glia, and the ganglion cell layer, which contains ganglion cells unlikely to regulate glial fate decision in mammals (Kim et al., (projection neurons). Although the kinetic pattern of generation 1998). of these cell types has been well characterized, the molecular Here, we found that the bHLH gene Hes5 (Akazawa et al., mechanism for glial cell fate determination is not well 1992), one of Notch effectors (Kageyama and Nakanishi, 1997; understood. It was previously shown that activation of EGF Ohtsuka et al., 1999), is specifically expressed by differentiat- receptor increases the population of Müller glial cells (Lillien, ing glial cells in mouse retina and that misexpression of Hes5 1995), but the intracellular mechanism for gliogenesis still directs neural precursor cells toward the glial fate at the expense remains to be determined. Recent data demonstrated that of the neuronal fate. Furthermore, Müller glial cell number was transcription factors of the bHLH and homeobox classes reduced in Hes5-deficient retina. These results demonstrate positively or negatively regulate differentiation of many retinal that, in mouse retina, Hes5 modulates the glial cell fate cells, but so far no genes that determine the glial fate are known specification. in the retina (Tomita et al., 1996a,b; Burmeister et al., 1996; Chen et al., 1997; Freund et al., 1997; Furukawa et al., 1997a,b; Kanekar et al., 1997; Mathers et al., 1997; Brown et al., 1998; MATERIALS AND METHODS Jean et al., 1998; Morrow et al., 1999). In situ hybridization In Drosophila, the gene glial cells missing (gcm) is known to In situ hybridization analysis of Hes5 was performed as previously determine the glial fate: in loss-of-function gcm mutants, nearly described (Ishibashi et al., 1995; Ishii et al., 1997). Digoxigenin- all glial cells fail to differentiate while ectopic gcm expression labeled antisense RNA probe was synthesized in vitro, as previously converts presumptive neurons into glia (Hosoya et al., 1995; described (Akazawa et al., 1992). The probe was hybridized to 12 µm Jones et al., 1995; Vincent et al., 1996). Two mammalian gcm cryostat sections of mouse retinae. 2516 M. Hojo and others Dissociation of retinal cells Amersham), mouse anti-HPC1 (1:200; Sigma), mouse anti-calbindin To count the cell numbers, retinae were dissociated, as previously (1:200; Sigma) and rabbit rhodopsin antibodies (1:2000; LSL). described (Morrow et al., 1998). Briefly, neural retinae were incubated Affinity-purified rabbit anti-Hes5 antibody was raised against the with 0.1% trypsin in HBSS lacking Ca2+/Mg2+ for 10 minutes at room peptide, AGPKSLHQDYSEGYSW, which is common to mouse and temperature and then soybean trypsin inhibitor was added (a final rat Hes5. To detect these antibodies, FITC-conjugated goat anti-mouse concentration of 2 mg/ml). The cells were pelleted, resuspended in IgG (1:200; Vector), FITC-conjugated goat anti-rabbit IgG (1:200; HBSS containing 100 µg/ml DNaseI, triturated and plated on poly-D- Vector), biotinylated anti-rabbit IgG (1:200; Vector), biotinylated anti- lysine-coated eight-well glass slides (Nalge Nunc). After incubation mouse IgG (1:200; Vector), FITC-avidin (1:2000; Vector) and Texas for 90 minutes at 37°C in 5% CO2, the cells were fixed with 4% red avidin (1:2000; Vector) were used. For BrdU staining, BrdU was paraformaldehyde for 10 minutes at room temperature and subjected to applied (2 µM) to the retinal explants 2 days after viral infection and immunostaining (see below). Cells were counted manually with the explants were cultured in the continued presence of BrdU. Sections masking the genotypes. were then made and stained with anti-BrdU antibody (1:5; Becton Dickinson). TUNEL assay was performed with a detection kit Retinal explant culture (Boehringer-Mannheim). Pictures were taken by confocal microscopy Retinal explant culture was performed, as previously described (Caffé (Carl Zeiss). et al., 1989; Sparrow et al., 1990; Stoppini et al., 1991; Tomita et al., 1996a). Eyes were isolated from mouse embryos and neonates and transferred to PBS solution. The neural retina without pigment RESULTS epithelium was placed onto a Millicell chamber filter (Millipore: diameter 30 mm, pore size 0.4 µm) with the ganglion cell layer upward. The chamber was placed in a 6-well culture plate, which Hes5 is specifically expressed by differentiating contained 0.8 ml of 50% MEM with Hepes, 25% Hank’s solution, 25% Müller glial cells heat-inactivated horse serum, 200 µM L-glutamine, and 6.75 mg/ml In situ hybridization analysis showed that, at postnatal day 1 glucose (Stoppini et al., 1991). Explants were incubated at 34°C in 5% (P1), Hes5 was expressed in the ventricular zone of the CO2. developing mouse retina, which contains common precursors Construction and infection of retrovirus for neurons and glial cells (Fig. 1A). During the postnatal period Hes5 expression was gradually restricted to the inner CLIG was constructed as follows. The SphI-SacI fragment of pIRES- EGFP (Clontech) was isolated, and the SphI and SacI sites were nuclear layer (INL) and, by P6, Hes5 was expressed only in the converted to EcoRI and the blunt end, respectively. This fragment was INL (Fig. 1B). After P7, Hes5 expression in the INL decreased ligated to EcoRI- and HpaI-digested pCLNCX v.2 (kindly provided by and became undetectable by P14 (Fig. 1C-E). At P6-8, Müller I. Verma) to replace the neo and the CMV promoter with the IRES- glial cells (vimentin+) were differentiating in the INL, where EGFP. The resultant vector was pCLIG. Hes5 cDNA was cloned into Hes5 expression was observed (Fig. 1G-I). Expression of the EcoRI site of pCLIG. The retroviral DNAs were transfected into the ecotropic packaging cells ψ2mp34 (Yoshimatsu et al., 1998), and 3 days later the medium was recovered and concentrated, as previously described (Tsuda et al., 1998). Virus solution was applied to the retinal explants, and after 2 weeks of culture, the explants were fixed with 4% paraformaldehyde, dehydrated in 25% sucrose, embedded in OCT compound (Miles) and sectioned. Immunostaining and TUNEL Sections and dissociated cells on slides were preincubated in PBS containing 5% goat serum and 0.1% Triton X-100 for 30 minutes and incubated in 1% goat serum and 0.1% Triton X-100 with the following primary antibodies (dilution rate): mouse anti-myc (1:500; Invitrogen), mouse anti- Fig. 1. Hes5 expression and Müller glial differentiation in the developing retina. (A-E) In situ hybridization vimentin (1:1; Histofine), of Hes5. Hes5 was initially expressed in the ventricular zone at P1 and later the expression was restricted to mouse anti-glutamine the INL. By P14, the expression disappears. (F-J) Expression of the Müller glial marker vimentin (Vim). synthetase (1:200; Chemicon), Müller glial cells differentiated in the INL, where Hes5 expression is observed. (K-O) Expression of the anti-GFP (1:500; Clontech), Müller glial marker glutamine synthetase (GS). By the time when GS was well expressed (P14), Hes5 mouse anti-PKC (1: 200; expression became undetectable. VZ, the ventricular zone. Scale bar, 50 µm. Glial cell fate specification by Hes5 2517 2D). In contrast, none of Hes5+ cells (green in Fig. 2F,H) expressed the retinal neuronal markers such as protein kinase C (PKC) (red in Fig.