Sonic Hedgehog Induces the Lateral Floor Plate

Development 129, 4785-4796 (2002) 4785 Printed in Great Britain © The Company of Biologists Limited 2002 DEV2912 Dual origin of the floor plate in the avian embryo Jean-Baptiste Charrier, Françoise Lapointe, Nicole M. Le Douarin and Marie-Aimée Teillet* Institut d’Embryologie Cellulaire et Moléculaire, CNRS and Collège de France, UMR 7128, 49bis Avenue de la Belle Gabrielle, 94736 Nogent-sur-Marne Cedex, France *Author for correspondence (e-mail: [email protected]) Accepted 30 July 2002 SUMMARY Molecular analysis carried out on quail-chick chimeras, in development, one can experimentally obtain a complete which quail Hensen’s node was substituted for its chick floor plate in the neural epithelium by the inductive action counterpart at the five- to six-somite stage (ss), showed that of either a notochord or a MFP. The competence of the the floor plate of the avian neural tube is composed of neuroepithelium to respond to notochord or MFP signals is distinct areas: (1) a median one (medial floor plate or MFP) restricted to a short time window, as only the posterior-most derived from Hensen’s node and characterised by the same region of the neural plate of embryos younger than 15 ss is gene expression pattern as the node cells (i.e. expression of able to differentiate a complete floor plate comprising MFP HNF3β and Shh to the exclusion of genes early expressed and LFP. Moreover, MFP differentiation requires between in the neural ectoderm such as CSox1); and (2) lateral 4 and 5 days of exposure to the inducing tissues. Under the regions that are differentiated from the neuralised same conditions LFP and SHH-producing cells only induce ectoderm (CSox1 positive) and form the lateral floor plate LFP-type cells. These results show that the capacity to (LFP). LFP cells are induced by the MFP to express HNF3β induce a complete floor plate is restricted to node-derived transiently, Shh continuously and other floor-plate tissues and probably involves a still unknown factor that is characteristic genes such as Netrin. In contrast to MFP not SHH, the latter being able to induce only LFP cells, LFP cells also express neural markers such as Nkx2.2 characteristics in neuralised epithelium. and Sim1. This pattern of avian floor-plate development presents some similarities to floor-plate formation in Key words: Hensen’s node, Lateral floor plate, Medial floor plate, zebrafish embryos. We also demonstrate that, although Notochord, Organiser, Quail/chick chimera, CSox1, HNF3β, MFP and LFP have different embryonic origins in normal Nkx2.2, Shh INTRODUCTION part of the neurectoderm as the floor plate; and a ventral one, which generates the notochord and becomes part of the The early neural tube of the vertebrate embryo is formed of mesodermal germ layer. Underneath, firmly adhering to the two lateral regions in which neurogenesis proceeds, which are notochord during early neurulation, lies a strand of separated by medial structures located in the midsagittal endodermal cells that accompanies Hensen’s node in its plane of the body, the dorsal roof plate and the ventral floor rostrocaudal movement. plate. In the avian embryo, where the morphogenetic In quail-chick chimeras in which chick Hensen’s node has movements through which neurulation takes place have been been replaced by its quail counterpart at the five- to six-somite analysed using cell marking techniques, it is established that stage (5-6 ss), the quail node-derived floor plate exhibits the lateral regions of the neural tube (i.e. the alar plates a characteristic polarised epithelial organisation with basal dorsally and the basal plates ventrally), together with the nuclei. This structure is distinct from the pseudo-columnar roof plate and the neural crest, have an embryological origin neuroepithelium derived from the neural plate itself (Catala et that is different from that of the floor plate (Catala et al., al., 1996) (see Fig. 2). 1995; Catala et al., 1996; Teillet et al., 1998a; Le Douarin, The floor plate of the vertebrate neural tube plays a major 2001). Selective cell labelling by the quail-chick chimera role during neurogenesis as it is a source of the morphogen system revealed that the ventral midline cells of the neural Sonic hedgehog (SHH), a secreted glycoprotein essential for tube, and the notochord, are derived from Hensen’s node: the motoneurone and interneurone specification (Ericson et al., avian organiser. During the posterior elongation of the 1996; Briscoe and Ericson, 1999; Briscoe and Ericson, 2001; embryo, Hensen’s node moves caudally and the active Lewis and Eisen, 2001) and for oligodendrocyte differentiation proliferation of its constitutive cells generates a medial cord (Orentas and Miller, 1996; Poncet et al., 1996; Pringle et al., of cells that are left in its wake. This cell cord promptly splits 1996). In addition, the floor plate provides guidance cues into two components: a dorsal one, which is progressively for outgrowing axons of many neurones (for reviews, inserted within the prospective neural plate and thus becomes see Colamarino and Tessier-Lavigne, 1995; Stoeckli and 4786 J.-B. Charrier and others Landmesser, 1998) (Matise et al., 1999) and participates in dynamic expression in the developing midline. In the early directing neurone migration (de Diego et al., 2002). gastrula, both Shh and twhh are expressed in the organiser In the avian embryo, the floor plate and notochord cells, as region (Ekker et al., 1995), but later on, deep midline cells of well as Hensen’s node (from which they are derived), express the embryonic axis fated to become notochord express only a gene of the Forkhead family of transcription factors, HNF3β, Shh, whereas the overlying cells (i.e. the future floor plate) together with the gene encoding SHH (Charrier et al., 1999). retain only twhh expression (Etheridge et al., 2001). Although Although Shh is transiently downregulated in the floor-plate Shh is re-expressed later in the zebrafish floor plate, as it is in cells as they become segregated from the notochord [over the other vertebrates, the phenotypic analysis of mutations of length of a few prospective somites immediately rostral to this gene (Schauerte et al., 1998; Odenthal et al., 2000) or Hensen’s node at the trunk level (see Marti et al., 1995b)], it downregulation experiments (Etheridge et al., 2001) indicate is considered that these two gene activities characterise the that Shh is not required for development of the MFP. ventral midline cells of the developing embryo (i.e. the floor Furthermore, twhh is not required for floor-plate development plate, the notochord and the cells that constitute Hensen’s either (Etheridge et al., 2001). However, the Hh pathway seems node) (Echelard et al., 1993; Ruiz i Altaba et al., 1993). to be necessary for the formation of the LFP. Thus, in sonic- Grafting experiments carried out in the chick embryo have you (syu) zebrafish mutants lacking the shh gene, LFP cells indicated that floor-plate characteristics can be induced in the are absent (Schauerte et al., 1998). In addition, the gene lateral neural tube by signals arising from the notochord or smoothened, part of the Hh receptor machinery, is required for the floor plate itself (van Straaten et al., 1985; van Straaten induction of LFP (Varga et al., 2001). et al., 1988; Placzek et al., 1990; Placzek et al., 1991; Yamada We aimed to explore the possible existence of a lateral et al., 1991; Pourquié et al., 1993). In vitro cultures have expansion of the floor plate in the neural epithelium of the indicated that this process, as well as the development of avian embryo. We show that the medial floor plate, as defined motoneurones, is mediated by SHH, which is produced by its origin from Hensen’s node, induces a lateral floor plate during neurulation by both these structures. Indeed, HNF3β in which not only floor-plate markers, such as HNF3β and Shh and a motoneurone-specific marker Islet1 are expressed in genes, but also genes that are normally activated in the neural explants of the 10 ss chick posterior neural plate subjected to ectoderm, such as Sox1, are expressed. We also observed that, a culture medium containing the SHH protein (Marti et al., during a short time window, the newly induced neural 1995a; Roelink et al., 1995; Ericson et al., 1996). Moreover, ectoderm can acquire MFP characteristics under the influence in vivo ectopic Shh expression can induce the expression of of an exogenous notochord or MFP. By contrast, SHH alone various floor-plate markers in zebrafish (Krauss et al., 1993), can only induce a LFP, not a MFP. mouse (Echelard et al., 1993) and Xenopus embryos (Ruiz i Altaba et al., 1995a). The description of spatially restricted expression of various MATERIALS AND METHODS immunocytochemical and molecular markers in the ventral neural tube of several vertebrate species has led to the Chick and quail embryos were used as hosts or donors in various in ovo grafting experiments. They were staged according to the number distinction of two cell populations in the floor plate. Thus, in of somites (ss) or embryonic day (E). Operated embryos were the chick ventral neural tube at incubation day 3 (E3), a medial sacrificed between E2.5 and E8. region where the cells express both SC1 and FP1 antigens can be distinguished from lateral areas where the cells express FP1 Microsurgery experiments but not SC1 (Placzek et al., 1991; Yamada et al., 1991). In rat Quail-chick cell labelling of the floor plate embryos, the antigen FP3 is expressed in all floor-plate cells, In a first series of experiments, Hensen’s node cells located at the while FP4 is restricted to medial cells (Placzek et al., 1993; level of the median pit [zone b (see Charrier et al., 1999)] were Roelink et al., 1994).

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