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BEN in the developing mouse spinal cord and various epithelia
Mechanisms of Development 95 (2000) 221±224
www.elsevier.com/locate/modo
Gene expression pattern
Expression of DM-GRASP/BEN in the developing mouse spinal cord and various epithelia Sandrine Fraboulet a,*, Tessa Schmidt-Petri b, Danielle Dhouailly a, Olivier Pourquie b a
Biologie de la DiffeÂrenciation EpitheÂliale, UMR CNRS 5538 LEDAC, Institut Albert Bonniot, Universite Joseph Fourier, Grenoble, France b Laboratoire de GeÂneÂtique et de Physiologie du DeÂveloppement, CNRS-INSERM-Universite de la MeÂditerraneÂe-AP de Marseille, Developmental Biology Institute of Marseille (IBDM), Marseille, France Received 29 February 2000; received in revised form 27 March 2000; accepted 28 March 2000
Abstract The expression pattern of the immunoglobulin DM-GRASP/BEN gene was studied in the mouse embryo using in situ hybridization. DMGRASP/BEN is expressed in the spinal cord in a subset of motoneurons expressing Islet-1, and non homogeneously in the dorsal root ganglia (DRG). In contrast, it's expression is homogeneous in the vestibulo-cochlear and trigemminal ganglia. DM-GRASP/BEN is also expressed in various epithelia of ectodermal or endodermal origin like the nasal, buccopharyngal and lung epithelia. In upper lip, DM-GRASP/BEN transcripts are present in the epidermal cells of the developing hair vibrissa follicles. First detected in the hair placode, DM-GRASP/BEN expression is localized in the central cells of the epithelial hair peg and then in a thin layer of cells crushed against the outer root sheath by the outgrowth of the hair shaft. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Mouse embryo; BEN/SC1/DM-GRASP; Spinal cord; Motor neuron; Subpopulation; Floor plate; Dorsal root ganglia; Fasciculation; Epithelia; In situ hybridization; Vibrissae; Hair placode; Central cells
BEN/SC1/DM-GRASP is a member of the CAM immunoglobulin (Ig) superfamily and was originally identi®ed by different groups in the chick (SC1: Tanaka and Obata, 1984; Tanaka et al., 1991; BEN: Pourquie et al., 1990, 1992a; DM-GRASP: Burns et al., 1991). DM-GRASP/BEN belongs to a new subfamily of the Ig superfamily which are characterized by an extracellular domain carrying two V-type and three C2-type Ig-like motifs followed by a transmembrane domain and a short cytoplasmic tail. DMGRASP/BEN is transiently expressed during avian embryogenesis by cells of the nervous and hemopoietic systems and by epithelia of various embryonic origins (Pourquie et al., 1990, 1992a; Corbel et al., 1992a,b, 1996). DM-GRASP/ BEN expression in the nervous system suggests that it could play a role in the fasciculation of axons, and in the establishment of synapses between neurons and their targets (Burns et al., 1991; Tanaka et al., 1991; Pourquie et al., 1992b; Fournier-Thibault et al., 1999). Although homologues of this protein have been identi®ed in ®sh, mouse, rat and human, expression of the mouse homologue has been poorly * Corresponding author. BDE, UMR CNRS 5538, Institut Albert Bonniot, 38706 La Tronche Cedex, France. Tel.: 133-4-7654-9573; fax: 133-4-7654-9425. E-mail address: [email protected] (S. Fraboulet).
studied, particularly outside the nervous system. Here we report the expression of the DM-GRASP/BEN mRNA in the developing mouse spinal cord and in a number of non neuronal sites in the embryo. In the mouse embryo, DM-GRASP/BEN mRNA can be detected in the ¯oor plate as early as embyonic day 9.5 (E9.5) and this expression is maintained until E15.5 (Figs. 1 and 2A, and data not shown). As has been reported for the other vertebrate species studied so far, motoneurons are the only mouse neuronal population that express DM-GRASP/ BEN in the spinal cord from E10.5 to E15.5 (Figs. 1B±G and 2A). In contrast to the chick embryo but as documented for rat and human embryos, DM-GRASP/BEN expression occurs too late to play a role in motor and sensory axon pioneering, and is only observed once the peripheral nerves have already fasciculated (Prince et al., 1992; Chedotal et al., 1995; Karagogeos et al., 1997). This suggests that in mammals, DM-GRASP/BEN might play a role in later events such as axon-target recognition and synaptogenesis. Double in situ hybridization using DM-GRASP/BEN and the pan-motoneuronal marker Islet-1 (Ericson et al., 1992) revealed that, at de®ned rostro-caudal positions in the embryo, only subsets of motoneurons express DMGRASP/BEN. At the brachial and lumbar levels (Fig. 1E,F), only a subpopulation of the motoneuronal cell popu-
0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(00)00330-0
222
S. Fraboulet et al. / Mechanisms of Development 95 (2000) 221±224
Fig. 1. BEN expression in mouse spinal cord during development. At E9.5 (A), BEN is expressed in neural crest cells (nc) and can be detected in the ¯oor plate (fp), where it is strongly expressed from day E10.5 (B) to at least day E12.5 (D). At E10.5 (B), BEN expression can be detected in the motoneurons (mn), in which a strong expression is maintained during later development (C±D). At E12.5, Fluorescein-labeled Islet-1 probe in red (empty arrowheads) and digoxigenin-labeled BEN probe in blue (full arrowheads) (E±G), reveal that some motoneuronal subpopulations are devoid of BEN mRNA expression at brachial (Br) (E) and lumbar levels (Lb) (F), whereas both mRNAs are co-expressed in the motoneurons at cervical level (Cerv) (G). Bars: 100 mm.
Fig. 2. BEN expression in different mouse ganglia at E13.5. BEN is expressed in dorsal root ganglia (drg) (A). At higher magni®cation (B) it is clear that this expression involves only a subpopulation of cells (arrowhead), whereas it is homogeneous in trigeminal ganglia (C) as well as in vestibulo-cochlear ganglia (vcg). Note that BEN can also be detected in the periosteum (p) (A) and in the epithelium of the cochlear-duct (cd) (D). Bars: 100 mm (A,C); 50 mm (B,D).
S. Fraboulet et al. / Mechanisms of Development 95 (2000) 221±224
lation which express Islet-1 co-express DM-GRASP/BEN mRNA. At the cervical level, however, all the motoneurons co-express DM-GRASP/BEN and islet-1 mRNA (Fig. 1G). At E9±E10, when neural crest cells located on either side of the neural tube condense to form the DRG, they express DM-GRASP/BEN (Fig. 1A). These cells maintain DMGRASP/BEN mRNA expression from E11.5 to at least E15.5 (Fig. 2A). The `salt and pepper' type of expression pattern observed in these ganglia suggests that not all the cell population expresses DM-GRASP/BEN (Fig. 2B). In contrast, DM-GRASP/BEN appears homogeneously expressed in cranial ganglia, most notably the trigeminal (Fig. 2C) and the vestibulo-cochlear (Fig. 2D). DM-GRASP/BEN was initially isolated from chick bursa of Fabricius epithelium (Pourquie et al., 1990) and its expression pro®le was subsequently described in peritoneal and gut epithelia (Pourquie et al., 1992). Here we show that, in addi-
223
tion, DM-GRASP/BEN is expressed in various epithelia of ectodermal or endodermal origin during mouse development. Expression is detected in the nasal (Fig. 3A), buccopharyngal (Fig. 3B) and lung epithelia (Fig. 3C). When the skin and hair vibrissae follicles form on the mouse upper-lip, from E12.5 to E16.5, DM-GRASP/BEN mRNA is expressed in the epidermal cells of the developing vibrissae. Expression is ®rst homogeneous in the hair placode (stage 1, named from Hardy, 1983) (Fig. 3D). Then, from stage 2±3 (Fig. 3E) to stage 4 (Fig. 3F), DM-GRASP/BEN expression is restricted to the central cells of the epithelial hair peg. At stage 5±6, the outgrowth of the hair shaft and of the inner root sheath push back the central DM-GRASP/BEN expressing cells, which form a plug at the skin surface or are crushed against the outer root sheath (Fig. 3G,H). Here we show that DM-GRASP/BEN is likely to play several roles during mouse development, not only in the
Fig. 3. BEN expression in different epithelia. At E12.5, BEN is speci®cally expressed in the nasal (A), pharynx (B), and lung bronchioles (C) epithelia (e). From E12.5 (D) to E16.5 (H), BEN is expressed in the developing vibrissae, at stage 1, in the hair placodes (pl) (D), and at stages 2±4 in the central cells (cc) of the hair peg (E). At stage 6, a transverse section at two levels (G) and a longitudinal section (H) show that BEN expressing cells are present in the central upper part of the follicle (a) and form a plug (pg) at the skin surface or are crushed against the outer root sheath (ORS). Note that the hair matrix (hm) and some dermal cells (arrowhead), corresponding to the prospective venous cavity, are also stained. d, dermis; e, epidermis; dp, dermal papilla; hs, hair shaft; IRS, inner root sheath. Bars: 100 mm (A±C); 50 mm (D±H).
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S. Fraboulet et al. / Mechanisms of Development 95 (2000) 221±224
differentiation of motoneuron subpopulations but also during the differentiation of many different epithelial appendages such as the nasal pit, the gut epithelium, the lung bronchioles, and the hair follicles. The obtention of mutant mice (BEN2/2) will allow a better understanding of BEN functions. 1. Experimental procedures OF1 E9.5±E15.5 embryos were ®xed overnight at 48C in 4% paraformaldehyde (PFA), 0.12 M PO4 pH7.4, 0.12 mM CaCl2, 2% sucrose. After washing in 0.12 M PO4, 15% sucrose for at least 24 h at 48C the embryos were embedded in gelatin compound and frozen in nitrogen-chilled isopentane for 1 min at 2708C. Serial sections (20-mm) were stored at 2808C before being used. A mouse BEN cDNA fragment was ampli®ed by RTPCR with oligonucleotides from a previously published sequence (Kanki et al., 1994). cDNA prepared from E12.5 mouse total RNA, was used to amplify a 1 kb cDNA fragment. This fragment was cloned into the pGEMT vector and sequenced. The mouse Islet-1 probe was a gift from Dr C. Henderson. In situ hybridization on cryostat sections was performed as described by Myat et al. (1996). Digoxigenin-labeled BEN probe was detected with NBT/BCIP (Roche diagnostics). For double in situ hybridizations, the ®rst probe labeled with ¯uorescein was revealed with Fast-red tablets (Roche diagnostics), phosphatase reaction was stopped in 100 mM glycine, pH 2.2, 0.1% Tween-20. Slides were ®xed in 4% PFA for 10 min, and washed in PBS before revelation of the second Digoxigenin-labeled probe. Acknowledgements Financial support was provided by the Centre National de la Recherche Scienti®que (CNRS), the Association FrancËaise contre les myopathies (AFM) and a Biotech contract of the European Union attributed to OP. S.F. is a recipient of a fellowship from the Association FrancËaise contre les Myopathies. References Burns, F.R., Von Kannen, S., Guy, L., Raper, J.A., Kaholz, J., Chang, S.,
1991. DM-GRASP, a novel immunoglobulin superfamily axonal surface protein that supports neurite extension. Neuron 7, 209±220. Chedotal, A., PourquieÂ, O., Sotelo, C., 1995. Initial tract formation in the brain of the chick embryo: selective expression of the BEN/SC1/DMGRASP cell adhesion molecule. Eur. J. Neurosci. 7, 198±212. Corbel, C., Bluestein, H., PourquieÂ, O., Vaigot, P., Le Dourin, N.M., 1992a. An antigen expressed by avian neuronal cells is also expressed by activated T lymphocytes. Cell. Immunol. 141, 99±110. Corbel, C., Cormier, F., PourquieÂ, O., Bluestein, H., 1992b. BEN, a novel surface molecule of the immunoglobin superfamily on avian hemopoietic progenitor cells shared with neural cells. Exp. Cell. Res. 203, 91±99. Corbel, C., PourquieÂ, O., Cormier, F., Vaigot, P., Le Dourin, N.M., 1996. BEN/SC1/DM-GRASP, a homophilic adhesion molecule, required for in vitro myeloid colony formation by avian hemopoietic progenitors. Proc. Natl. Acad. Sci. USA 93, 2844±2849. Ericson, J., Thor, S., Edlund, T., Jessell, T.M., Yamada, T., 1992. Early stages of motoneurons differenciation revealed by expression of homeobox gene Islet-1. Science 256, 1555±1560. Fournier-Thibault, C., PourquieÂ, O., Rouaud, T., Le Douarin, N.M., 1999. BEN/SC1/DM-GRASP expression during neuromuscular development: a cell adhesion molecule regulated by innervation. J. Neurosci. 19, 1382±1392. Hardy, M.H., 1983. Vitamin A and the epithelial-mesenchymal interactions in skin differentiation. In: Sawyer, R.H., Fallon, J.F. (Eds.). EpithelialMesenchymal Interactions in Development, Prayer Press, New York, pp. 163±188. Kanki, J.P., Chang, S., Kuwada, J.Y., 1994. The molecular cloning and characterization of potential chick DM-GRASP homologs in zebra®sh and mouse. J. Neurobiol. 7, 831±845. Karagogeos, D., PourquieÂ, C., Kyriakopoulou, K., Tavian, M., Stallcup, W., PeÂault, B., PourquieÂ, O., 1997. Expression of the cell adhesion proteins BEN/SC1/DM-GRASP and TAG-1 de®nes early steps of axonogenesis in the human spinal cord. J. Comp. Neurol. 379, 415±427. Myat, A., Henrique, D., Ish-Horowicz, D., Lewis, J., 1996. A chick homologue of serrate and its relationship with notch and delta homologues during central neurogenesis. Dev. Biol. 174, 233±247. PourquieÂ, O., Coltey, M., Thomas, J., Le Douarin, N.M., 1990. A widely distributed antigen developmentally regulated in the nervous system. Development 109, 743±752. PourquieÂ, O., Corbel, C., Le Caer, J.P., Rossier, J., Le Douarin, N.M., 1992a. BEN, a surface molecule of the immunoglobin superfamily expressed in a variety of developing systems. Proc. Natl. Acad. Sci. USA 89, 5261±5265. PourquieÂ, O., Hallonet, E.R., Le Douarin, N.M., 1992b. Association of BEN glycoprotein expression with climbing ®ber axogenesis in the avian cerebellum. J. Neurosci. 12, 1548±1557. Prince, J.T., Nishiyama, A., Healy, P.A., Beasley, L., Stallcup, W.B., 1992. Expression of F84.1 glycoprotein in the spinal cord and cranial nerves of the developing rat. Dev. Brain Res. 68, 193±201. Tanaka, H., Obata, K., 1984. Developmental changes in unique cell surface antigens of chick embryo spinal motoneurons and ganglions cells. Dev. Biol. 106, 26±37. Tanaka, H., Matsui, T., Agata, A., Tomura, M., Kubota, J., McFarland, K.C., Kohr, B., Lee, A., Phillips, H.S., Shelton, D.L., 1991. Molecular cloning and expression of a novel adhesion molecule. SC1. Neuron 7, 535±545.
www.elsevier.com/locate/modo
Gene expression pattern
Expression of DM-GRASP/BEN in the developing mouse spinal cord and various epithelia Sandrine Fraboulet a,*, Tessa Schmidt-Petri b, Danielle Dhouailly a, Olivier Pourquie b a
Biologie de la DiffeÂrenciation EpitheÂliale, UMR CNRS 5538 LEDAC, Institut Albert Bonniot, Universite Joseph Fourier, Grenoble, France b Laboratoire de GeÂneÂtique et de Physiologie du DeÂveloppement, CNRS-INSERM-Universite de la MeÂditerraneÂe-AP de Marseille, Developmental Biology Institute of Marseille (IBDM), Marseille, France Received 29 February 2000; received in revised form 27 March 2000; accepted 28 March 2000
Abstract The expression pattern of the immunoglobulin DM-GRASP/BEN gene was studied in the mouse embryo using in situ hybridization. DMGRASP/BEN is expressed in the spinal cord in a subset of motoneurons expressing Islet-1, and non homogeneously in the dorsal root ganglia (DRG). In contrast, it's expression is homogeneous in the vestibulo-cochlear and trigemminal ganglia. DM-GRASP/BEN is also expressed in various epithelia of ectodermal or endodermal origin like the nasal, buccopharyngal and lung epithelia. In upper lip, DM-GRASP/BEN transcripts are present in the epidermal cells of the developing hair vibrissa follicles. First detected in the hair placode, DM-GRASP/BEN expression is localized in the central cells of the epithelial hair peg and then in a thin layer of cells crushed against the outer root sheath by the outgrowth of the hair shaft. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Mouse embryo; BEN/SC1/DM-GRASP; Spinal cord; Motor neuron; Subpopulation; Floor plate; Dorsal root ganglia; Fasciculation; Epithelia; In situ hybridization; Vibrissae; Hair placode; Central cells
BEN/SC1/DM-GRASP is a member of the CAM immunoglobulin (Ig) superfamily and was originally identi®ed by different groups in the chick (SC1: Tanaka and Obata, 1984; Tanaka et al., 1991; BEN: Pourquie et al., 1990, 1992a; DM-GRASP: Burns et al., 1991). DM-GRASP/BEN belongs to a new subfamily of the Ig superfamily which are characterized by an extracellular domain carrying two V-type and three C2-type Ig-like motifs followed by a transmembrane domain and a short cytoplasmic tail. DMGRASP/BEN is transiently expressed during avian embryogenesis by cells of the nervous and hemopoietic systems and by epithelia of various embryonic origins (Pourquie et al., 1990, 1992a; Corbel et al., 1992a,b, 1996). DM-GRASP/ BEN expression in the nervous system suggests that it could play a role in the fasciculation of axons, and in the establishment of synapses between neurons and their targets (Burns et al., 1991; Tanaka et al., 1991; Pourquie et al., 1992b; Fournier-Thibault et al., 1999). Although homologues of this protein have been identi®ed in ®sh, mouse, rat and human, expression of the mouse homologue has been poorly * Corresponding author. BDE, UMR CNRS 5538, Institut Albert Bonniot, 38706 La Tronche Cedex, France. Tel.: 133-4-7654-9573; fax: 133-4-7654-9425. E-mail address: [email protected] (S. Fraboulet).
studied, particularly outside the nervous system. Here we report the expression of the DM-GRASP/BEN mRNA in the developing mouse spinal cord and in a number of non neuronal sites in the embryo. In the mouse embryo, DM-GRASP/BEN mRNA can be detected in the ¯oor plate as early as embyonic day 9.5 (E9.5) and this expression is maintained until E15.5 (Figs. 1 and 2A, and data not shown). As has been reported for the other vertebrate species studied so far, motoneurons are the only mouse neuronal population that express DM-GRASP/ BEN in the spinal cord from E10.5 to E15.5 (Figs. 1B±G and 2A). In contrast to the chick embryo but as documented for rat and human embryos, DM-GRASP/BEN expression occurs too late to play a role in motor and sensory axon pioneering, and is only observed once the peripheral nerves have already fasciculated (Prince et al., 1992; Chedotal et al., 1995; Karagogeos et al., 1997). This suggests that in mammals, DM-GRASP/BEN might play a role in later events such as axon-target recognition and synaptogenesis. Double in situ hybridization using DM-GRASP/BEN and the pan-motoneuronal marker Islet-1 (Ericson et al., 1992) revealed that, at de®ned rostro-caudal positions in the embryo, only subsets of motoneurons express DMGRASP/BEN. At the brachial and lumbar levels (Fig. 1E,F), only a subpopulation of the motoneuronal cell popu-
0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(00)00330-0
222
S. Fraboulet et al. / Mechanisms of Development 95 (2000) 221±224
Fig. 1. BEN expression in mouse spinal cord during development. At E9.5 (A), BEN is expressed in neural crest cells (nc) and can be detected in the ¯oor plate (fp), where it is strongly expressed from day E10.5 (B) to at least day E12.5 (D). At E10.5 (B), BEN expression can be detected in the motoneurons (mn), in which a strong expression is maintained during later development (C±D). At E12.5, Fluorescein-labeled Islet-1 probe in red (empty arrowheads) and digoxigenin-labeled BEN probe in blue (full arrowheads) (E±G), reveal that some motoneuronal subpopulations are devoid of BEN mRNA expression at brachial (Br) (E) and lumbar levels (Lb) (F), whereas both mRNAs are co-expressed in the motoneurons at cervical level (Cerv) (G). Bars: 100 mm.
Fig. 2. BEN expression in different mouse ganglia at E13.5. BEN is expressed in dorsal root ganglia (drg) (A). At higher magni®cation (B) it is clear that this expression involves only a subpopulation of cells (arrowhead), whereas it is homogeneous in trigeminal ganglia (C) as well as in vestibulo-cochlear ganglia (vcg). Note that BEN can also be detected in the periosteum (p) (A) and in the epithelium of the cochlear-duct (cd) (D). Bars: 100 mm (A,C); 50 mm (B,D).
S. Fraboulet et al. / Mechanisms of Development 95 (2000) 221±224
lation which express Islet-1 co-express DM-GRASP/BEN mRNA. At the cervical level, however, all the motoneurons co-express DM-GRASP/BEN and islet-1 mRNA (Fig. 1G). At E9±E10, when neural crest cells located on either side of the neural tube condense to form the DRG, they express DM-GRASP/BEN (Fig. 1A). These cells maintain DMGRASP/BEN mRNA expression from E11.5 to at least E15.5 (Fig. 2A). The `salt and pepper' type of expression pattern observed in these ganglia suggests that not all the cell population expresses DM-GRASP/BEN (Fig. 2B). In contrast, DM-GRASP/BEN appears homogeneously expressed in cranial ganglia, most notably the trigeminal (Fig. 2C) and the vestibulo-cochlear (Fig. 2D). DM-GRASP/BEN was initially isolated from chick bursa of Fabricius epithelium (Pourquie et al., 1990) and its expression pro®le was subsequently described in peritoneal and gut epithelia (Pourquie et al., 1992). Here we show that, in addi-
223
tion, DM-GRASP/BEN is expressed in various epithelia of ectodermal or endodermal origin during mouse development. Expression is detected in the nasal (Fig. 3A), buccopharyngal (Fig. 3B) and lung epithelia (Fig. 3C). When the skin and hair vibrissae follicles form on the mouse upper-lip, from E12.5 to E16.5, DM-GRASP/BEN mRNA is expressed in the epidermal cells of the developing vibrissae. Expression is ®rst homogeneous in the hair placode (stage 1, named from Hardy, 1983) (Fig. 3D). Then, from stage 2±3 (Fig. 3E) to stage 4 (Fig. 3F), DM-GRASP/BEN expression is restricted to the central cells of the epithelial hair peg. At stage 5±6, the outgrowth of the hair shaft and of the inner root sheath push back the central DM-GRASP/BEN expressing cells, which form a plug at the skin surface or are crushed against the outer root sheath (Fig. 3G,H). Here we show that DM-GRASP/BEN is likely to play several roles during mouse development, not only in the
Fig. 3. BEN expression in different epithelia. At E12.5, BEN is speci®cally expressed in the nasal (A), pharynx (B), and lung bronchioles (C) epithelia (e). From E12.5 (D) to E16.5 (H), BEN is expressed in the developing vibrissae, at stage 1, in the hair placodes (pl) (D), and at stages 2±4 in the central cells (cc) of the hair peg (E). At stage 6, a transverse section at two levels (G) and a longitudinal section (H) show that BEN expressing cells are present in the central upper part of the follicle (a) and form a plug (pg) at the skin surface or are crushed against the outer root sheath (ORS). Note that the hair matrix (hm) and some dermal cells (arrowhead), corresponding to the prospective venous cavity, are also stained. d, dermis; e, epidermis; dp, dermal papilla; hs, hair shaft; IRS, inner root sheath. Bars: 100 mm (A±C); 50 mm (D±H).
224
S. Fraboulet et al. / Mechanisms of Development 95 (2000) 221±224
differentiation of motoneuron subpopulations but also during the differentiation of many different epithelial appendages such as the nasal pit, the gut epithelium, the lung bronchioles, and the hair follicles. The obtention of mutant mice (BEN2/2) will allow a better understanding of BEN functions. 1. Experimental procedures OF1 E9.5±E15.5 embryos were ®xed overnight at 48C in 4% paraformaldehyde (PFA), 0.12 M PO4 pH7.4, 0.12 mM CaCl2, 2% sucrose. After washing in 0.12 M PO4, 15% sucrose for at least 24 h at 48C the embryos were embedded in gelatin compound and frozen in nitrogen-chilled isopentane for 1 min at 2708C. Serial sections (20-mm) were stored at 2808C before being used. A mouse BEN cDNA fragment was ampli®ed by RTPCR with oligonucleotides from a previously published sequence (Kanki et al., 1994). cDNA prepared from E12.5 mouse total RNA, was used to amplify a 1 kb cDNA fragment. This fragment was cloned into the pGEMT vector and sequenced. The mouse Islet-1 probe was a gift from Dr C. Henderson. In situ hybridization on cryostat sections was performed as described by Myat et al. (1996). Digoxigenin-labeled BEN probe was detected with NBT/BCIP (Roche diagnostics). For double in situ hybridizations, the ®rst probe labeled with ¯uorescein was revealed with Fast-red tablets (Roche diagnostics), phosphatase reaction was stopped in 100 mM glycine, pH 2.2, 0.1% Tween-20. Slides were ®xed in 4% PFA for 10 min, and washed in PBS before revelation of the second Digoxigenin-labeled probe. Acknowledgements Financial support was provided by the Centre National de la Recherche Scienti®que (CNRS), the Association FrancËaise contre les myopathies (AFM) and a Biotech contract of the European Union attributed to OP. S.F. is a recipient of a fellowship from the Association FrancËaise contre les Myopathies. References Burns, F.R., Von Kannen, S., Guy, L., Raper, J.A., Kaholz, J., Chang, S.,
1991. DM-GRASP, a novel immunoglobulin superfamily axonal surface protein that supports neurite extension. Neuron 7, 209±220. Chedotal, A., PourquieÂ, O., Sotelo, C., 1995. Initial tract formation in the brain of the chick embryo: selective expression of the BEN/SC1/DMGRASP cell adhesion molecule. Eur. J. Neurosci. 7, 198±212. Corbel, C., Bluestein, H., PourquieÂ, O., Vaigot, P., Le Dourin, N.M., 1992a. An antigen expressed by avian neuronal cells is also expressed by activated T lymphocytes. Cell. Immunol. 141, 99±110. Corbel, C., Cormier, F., PourquieÂ, O., Bluestein, H., 1992b. BEN, a novel surface molecule of the immunoglobin superfamily on avian hemopoietic progenitor cells shared with neural cells. Exp. Cell. Res. 203, 91±99. Corbel, C., PourquieÂ, O., Cormier, F., Vaigot, P., Le Dourin, N.M., 1996. BEN/SC1/DM-GRASP, a homophilic adhesion molecule, required for in vitro myeloid colony formation by avian hemopoietic progenitors. Proc. Natl. Acad. Sci. USA 93, 2844±2849. Ericson, J., Thor, S., Edlund, T., Jessell, T.M., Yamada, T., 1992. Early stages of motoneurons differenciation revealed by expression of homeobox gene Islet-1. Science 256, 1555±1560. Fournier-Thibault, C., PourquieÂ, O., Rouaud, T., Le Douarin, N.M., 1999. BEN/SC1/DM-GRASP expression during neuromuscular development: a cell adhesion molecule regulated by innervation. J. Neurosci. 19, 1382±1392. Hardy, M.H., 1983. Vitamin A and the epithelial-mesenchymal interactions in skin differentiation. In: Sawyer, R.H., Fallon, J.F. (Eds.). EpithelialMesenchymal Interactions in Development, Prayer Press, New York, pp. 163±188. Kanki, J.P., Chang, S., Kuwada, J.Y., 1994. The molecular cloning and characterization of potential chick DM-GRASP homologs in zebra®sh and mouse. J. Neurobiol. 7, 831±845. Karagogeos, D., PourquieÂ, C., Kyriakopoulou, K., Tavian, M., Stallcup, W., PeÂault, B., PourquieÂ, O., 1997. Expression of the cell adhesion proteins BEN/SC1/DM-GRASP and TAG-1 de®nes early steps of axonogenesis in the human spinal cord. J. Comp. Neurol. 379, 415±427. Myat, A., Henrique, D., Ish-Horowicz, D., Lewis, J., 1996. A chick homologue of serrate and its relationship with notch and delta homologues during central neurogenesis. Dev. Biol. 174, 233±247. PourquieÂ, O., Coltey, M., Thomas, J., Le Douarin, N.M., 1990. A widely distributed antigen developmentally regulated in the nervous system. Development 109, 743±752. PourquieÂ, O., Corbel, C., Le Caer, J.P., Rossier, J., Le Douarin, N.M., 1992a. BEN, a surface molecule of the immunoglobin superfamily expressed in a variety of developing systems. Proc. Natl. Acad. Sci. USA 89, 5261±5265. PourquieÂ, O., Hallonet, E.R., Le Douarin, N.M., 1992b. Association of BEN glycoprotein expression with climbing ®ber axogenesis in the avian cerebellum. J. Neurosci. 12, 1548±1557. Prince, J.T., Nishiyama, A., Healy, P.A., Beasley, L., Stallcup, W.B., 1992. Expression of F84.1 glycoprotein in the spinal cord and cranial nerves of the developing rat. Dev. Brain Res. 68, 193±201. Tanaka, H., Obata, K., 1984. Developmental changes in unique cell surface antigens of chick embryo spinal motoneurons and ganglions cells. Dev. Biol. 106, 26±37. Tanaka, H., Matsui, T., Agata, A., Tomura, M., Kubota, J., McFarland, K.C., Kohr, B., Lee, A., Phillips, H.S., Shelton, D.L., 1991. Molecular cloning and expression of a novel adhesion molecule. SC1. Neuron 7, 535±545.