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Ebf1 Involvement in Striatum Development

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Ebf1 Involvement in Striatum Development Development 126, 5285-5294 (1999) 5285 Printed in Great Britain © The Company of Biologists Limited 1999 DEV1475 Ebf1 controls early cell differentiation in the embryonic striatum Sonia Garel1, Faustino Marín1, Rudolf Grosschedl2,* and Patrick Charnay1,‡ 1Unité 368 de l’Institut National de la Santé et de la Recherche Médicale, Ecole Normale Supérieure, 46 rue d’Ulm, 75230 Paris Cedex 05, France 2Howard Hughes Medical Institute, Department of Microbiology and Immunology, University of California, San Francisco, California 94143, USA *Present address: Institute for Biochemistry and Gene Centre, University of Munich, Feodor-Lynen Str. 25, 81377 München, Germany ‡Author for correspondence (e-mail: [email protected]) Accepted 22 September; published on WWW 9 November 1999 SUMMARY Ebf1/Olf-1 belongs to a small multigene family encoding adhesion/guidance molecules. These early defects in the closely related helix-loop-helix transcription factors, SVZ/mantle transition are followed by an increase in cell which have been proposed to play a role in neuronal death, a dramatic reduction in size of the postnatal differentiation. Here we show that Ebf1 controls cell striatum and defects in navigation and fasciculation of differentiation in the murine embryonic striatum, where it thalamocortical fibres travelling through the striatum. Our is the only gene of the family to be expressed. Ebf1 targeted data therefore show that Ebf1 plays an essential role in the disruption affects postmitotic cells that leave the acquisition of mantle cell molecular identity in the subventricular zone (SVZ) en route to the mantle: they developing striatum and provide information on the genetic appear to be unable to downregulate genes normally hierarchies that govern neuronal differentiation in the restricted to the SVZ or to activate some mantle-specific ventral telencephalon. genes. These downstream genes encode a variety of regulatory proteins including transcription factors and Key words: Ebf, Olf, Striatum, Neurogenesis, Apoptosis, Axonal proteins involved in retinoid signalling as well as navigation INTRODUCTION non-basic HLH dimerisation domain (Hagman et al., 1995). Two closely related mouse genes were subsequently identified, The vertebrate central nervous system (CNS) is a highly Ebf2 (also known as O/E-3 or Mmot1) and Ebf3 (also known complex structure whose normal activity relies on the as O/E-2) (Garel et al., 1997; Malgaretti et al., 1997; Wang et appropriate differentiation and interconnection of a variety of al., 1997). In the embryonic CNS, the three genes show neuronal subtypes. The control of neuronal differentiation overlapping expression patterns with two salient features: (1) therefore constitutes a key aspect of CNS development. In the they are transiently expressed in differentiating neurons, Ebf2 last few years a number of vertebrate genes involved in this being expressed in early postmitotic neurons and Ebf1 and process have been identified on the basis of homology with Ebf3 in more differentiated cells; (2) they are expressed along Drosophila genes. Hence, genes encoding members of the basic the entire rostrocaudal axis from the midbrain to the spinal helix-loop-helix (bHLH) family of transcription factors like cord, whereas in the forebrain they are regionally restricted Mash-1 and the neurogenins have been implicated in various (Garel et al., 1997). These results suggested a role for Ebf steps in the determination and differentiation of the CNS and genes in neuronal differentiation and specification of forebrain of the peripheral nervous system (PNS) (Guillemot et al., 1993; subpopulations, which is supported by experiments in Xenopus Cau et al., 1997; Fode et al., 1998; Hirsch et al., 1998; Ma et and C. elegans embryos: alteration of the Ebf2 ortholog al., 1998; Casarosa et al., 1999). Nevertheless our understanding (XCoe2) activity in Xenopus embryos revealed a role for this of the genetic networks governing neuronal differentiation is factor in early steps of primary neuron differentiation (Dubois still very fragmentary. In the present paper, we provide direct et al., 1998), and mutations in the C. elegans Ebf ortholog, unc- evidence of the involvement of another gene family, the Ebf/Olf 3, affect motor neuron differentiation and ventral nerve cord family (also called O/E, Coe), in this process. fasciculation (Prasad et al., 1998). However, the role of Ebf The founding member of the family, Ebf1/Olf-1, was genes in mouse CNS development remains to be elucidated. originally cloned independently in mouse and rat on the basis Ebf1-deficient mice have been generated and show an early of its putative role in B-cell and olfactory neurons block in the differentiation of the B-cell lineage at the pro-B differentiation, respectively (Hagman et al., 1991, 1993; Wang stage (Lin and Grosschedl, 1995). In contrast, no abnormality and Reed, 1993). Ebf1 encodes a transcription factor in the olfactory epithelium and no global defect in the containing an atypical zinc finger DNA binding domain and a CNS were observed in these mice, presumably because of 5286 S. Garel and others complementation or redundancy between the different Ebf fixed by immersion or perfused through the heart with 4% genes. However, since Ebf1 is the only member of the family paraformaldehyde (PFA) in PBS. Brains were cryoprotected in 30% expressed in the developing striatum (Garel et al., 1997), this sucrose in PBS and cut into 20-30 µm sections on a cryotome or area of the telencephalon deserved further analysis. embedded in paraffin and cut into 10 µm sections on a microtome. The striatum is a large structure located in the ventral Sections were preincubated in 5% foetal calf serum, 0.1% Tween-20 in telencephalon. During embryogenesis, the telencephalic vesicles PBS for 1 hour at room temperature (RT) and incubated overnight at 4°C with: mouse anti-TH, 1:200 dilution (Boehringer Mannheim); differentiate dorsally into the cortex and ventrally into two bulges, mouse anti-DARPP-32, 1:5000 (a kind gift of J.A. Girault); rabbit anti- the lateral (LGE) and medial (MGE) ganglionic eminences GAD 67 kDa, 1:2000 (Chemicon); rabbit anti-CaBP 28 kDa, 1:3000 (Smart and Sturrock, 1979). The LGE will give rise to the (Swant); goat anti-ChAT, 1:100 (Chemicon). Sections were washed in striatum and the MGE will form the pallidum (Smart and PBS-0.1% Tween-20 and incubated with horseradish peroxidase Sturrock, 1979; Deacon et al., 1994), two structures that are part (HRP)-conjugated secondary antibodies for 2 hours at RT: HRP-goat of the basal ganglia and are involved in motion control. In both anti-mouse antibodies, 1:100 (Sigma); HRP-goat anti-rabbit antibodies, the cortex and the ganglionic eminences, two distinct proliferative 1:200 (Sigma), biotin-conjugated donkey anti-goat antibodies, 1:100 zones contribute to the generation of neuronal precursors: a (Sigma). For biotin-conjugated antibodies, the sections were incubated ventricular zone (VZ) organised as a pseudostratified with streptavidin biotinylated-HRP complex, 1:100 (Amersham) for 1 neuroepithelium and a VZ-derived subventricular zone (SVZ), hour at RT. HRP activity was detected with diaminobenzidine (DAB) or with DAB and nickel ammonium sulphate. NADPH histochemistry where dividing cells are scattered and do not form an epithelial and MAP2 immunohistochemistry were performed as described structure (Smart, 1976; Halliday and Cepko, 1992; Bhide, 1996). (Vincent et al., 1983; Anderson et al., 1997a). After their exit of the cell cycle in the VZ or in the SVZ, cells migrate laterally into the mantle where they undergo terminal In situ hybridisation differentiation. Two histochemical compartments designated In situ hybridisation was performed on 70-80 µm vibratome sections as patch (striosome) and matrix can be distinguished in the postnatal described previously (Garel et al., 1997) with the following probes: striatum. The patch and matrix compartments differ in their Ebf1, Ebf2 and Ebf3 (Garel et al., 1997); Dlx-1 (Smith-Fernandez et al., expression of neurotransmitters, neuropeptides and receptors as 1998); Dll1 (Bettenhausen et al., 1995); Dll3 (Dunwoodie et al., 1997); α β γ well as in their input from the cortex and output to the pallidum RAR , RAR , and CRABP I (Ruberte et al., 1993); RXR (Dollé et al., and substantia nigra (reviewed in Gerfen, 1992). In addition, the 1994); EphA4 (Gilardi-Hebenstreit et al., 1992); SCIP/Oct-6 (Monuki et al., 1990); Dlx-5 and Dlx-6 (Anderson et al., 1997a); cadherin-8 peak of generation of patch neurons occurs earlier than that of (Korematsu and Redies, 1997a); netrin-1 (Serafini et al., 1996). the matrix neurons (van der Kooy and Fishell, 1987). In this paper, we establish that Ebf1 plays an essential role Bromodeoxyuridine labelling and codetection in the development of the striatum. Ebf1 targeted inactivation Pregnant mice were injected intraperitoneally with 40 mg/kg of specifically affects cell differentiation at the SVZ/mantle bromodeoxyuridine (BrdU, Sigma) in PBS and killed 30 minutes or transition in the embryonic LGE, resulting in the expression of several days later. Embryo heads were fixed for 2 hours in Carnoy’s an aberrant combination of regulatory genes in the mantle. fixative, embedded in paraffin and cut in 10 µm sections. Anti-BrdU These early differentiation defects may be responsible for an immunohistochemistry was performed as described (Garel et al., 1997). increase in cell death and a dramatic atrophy observed in the For quantification of BrdU-positive cells, the scoring was performed on perinatal striatum. Interestingly, we show that defective striatal two wild-type and two homozygous mutant embryos. For each embryo, four transverse sections defined as follows were analysed: (1) the first cell differentiation is also accompanied by fasciculation and section containing the corpus callosum; (2) the first section containing navigation defects in fibre tracts passing through the striatum. the posterior branch of the anterior commissure; (3) two equidistant sections in between the previous ones. The scoring area was delimited MATERIALS AND METHODS by tangental lines drawn at the sulci between the cortex and the LGE, and between the LGE and the MGE.
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