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). Moreover, in mouse and rat embryos, Shh excised micro-surgically in 5-6 ss chick embryos and replaced by is expressed only in the medial cells (Roelink et al., 1994), their quail counterpart as previously described (Catala et al., 1996). whereas HNF3β transcripts are present in a larger region of the In another series, the presumptive territory of the neural plate, ventral neural tube (Monaghan et al., 1993; Sasaki and Hogan, immediately posterior to the median pit, was replaced by its quail 1993; Ang and Rossant, 1994). counterpart, while Hensen’s node cells remained untouched. These experiments (see Fig. 2E) resulted in the differential quail labelling In the zebrafish, the medial floor plate (MFP) consists of a of the floor plate and of the basal plate neural epithelium, single row of cells flanked on each side by one or two respectively, as previously shown by Catala et al. (Catala et al., additional rows of lateral floor-plate (LFP) cells (Odenthal and 1996). Nusslein-Volhard, 1998; Odenthal et al., 2000). MFP and LFP, although belonging to the same cuboido-epithelial type, differ Graft of quail notochord or floor plate, or of SHH-producing in their gene expression patterns. Cells of the MFP express cells in contact with chick lateral neural tube Netrin1 (Strähle et al., 1997) and members of the Hedgehog Notochords and neural tubes were enzymatically dissociated from E2 (Hh) family: Shh (Krauss et al., 1993) and tiggy-winkle (10-25 ss) quail embryos using pancreatin. Floor-plate fragments were hedgehog (twhh) (Ekker et al., 1995). They also express several dissected from the dissociated neural tubes at the thoracic and cervical forkhead family members: axial and fkd7 (Strähle et al., 1993; levels. Notochord and floor-plate fragments (the latter comprising MFP and LFP components) were grafted between the neural Strähle et al., 1996; Odenthal and Nusslein-Volhard, 1998), epithelium and the segmental plate in 7-25 ss chick embryos, as and the Xenopus Pintallavis homologue fkd4 (Odenthal and described by Pourquié et al. (Pourquié et al., 1993). Clumps of QT6 Nusslein-Volhard, 1998). Netrin1, axial and fkd4 transcripts quail fibroblasts, stably transfected with a construct carrying the chick are in addition present in the LFP (Odenthal et al., 2000). SHH-coding region (Duprez et al., 1998), were implanted into chick Interestingly, in the zebrafish, the Hh paralogues exhibit a hosts in the same situation. Sonic hedgehog induces the lateral floor plate 4787

Transplantation of quail neural tube deprived of node-derived day later, (Fig. 1B-D), caudal neural tubes deprived of node-derived cells into stage-matched chick host cells were isolated by enzymatic dissociation and transplanted into The caudalward movement of Hensen’s node can be prevented by stage-matched chick embryos, after microsurgical excision of a removing its posterior-most region together with the rostral tip of the fragment of their own truncal neural tube and notochord (Charrier et primitive streak forming the axial-paraxial hinge (APH). Under these al., 2001). Quail neural tubes were transplanted either alone or conditions, the posterior neural tube develops without midline cells together with a fragment of E2 chick notochord or floor plate, or with (notochord and floor plate) and abundant apoptosis was observed in clumps of SHH-producing cells (Fig. 1D, parts a-c). the neural epithelium as well as in the paraxial mesoderm of the affected region because of the absence of the sources of SHH, i.e. In situ hybridisation and immunohistochemistry floor plate and notochord (Charrier et al., 1999; Charrier et al., 2001). In situ hybridisation on whole mount or on alternate 7 µm serial APH excisions were performed in 5-6 ss quail embryos (Fig. 1A). One sections was performed as described previously (Charrier et al., 1999) using probes for the following chicken genes: HNF3β (Ruiz i Altaba et al., 1995b), Shh (Riddle et al., 1993), Netrin1 (Kennedy et al., ABExperiment 1 1994), CSox1 (Rex et al., 1997), Pax6 (Goulding et al., 1993), Sim1 (Yamada et al., 1991), Nkx2.2 (Ericson et al., 1997). In each case, adjacent sections were treated overnight with the monoclonal antibody (mAb) QCPN (Developmental Studies Hybridoma Bank), which specifically recognises quail cells. Mounted sections were photographed using Nomarski optics (Leica).

RESULTS

Floor-plate heterogeneity demonstrated by molecular analysis of quail-chick chimeras HN S20 When Hensen’s node is replaced in 5-6 ss chick embryo by its APH C quail counterpart (Fig. 2E, red), floor-plate and notochord cells PS are of quail origin (Fig. 2H,L) from the level of the graft C (thoracic level) down to the posterior end of the spinal cord. V D By contrast, when a region of the prospective neural plate posterior to the node region is replaced by its quail equivalent E 1.5 (Fig. 2E, blue area), cells of the basal plate, but not of the floor plate, carry the quail marker (Fig. 2P). These experiments E 2.5 demonstrated that floor-plate and basal plate cells, in the avian D Experiment 2 neural tube, have distinct embryological origins (Catala et al., 1996). We have now recorded the dynamic expression patterns of several genes expressed in the ventral neural tube in both NT types of quail-chick chimeras. At E3.5, one day after Hensen’s node replacement, Shh and HNF3β were strongly co-expressed both in the node-derived cells and in a fringe of cells deriving from the presumptive neural plate (Fig. 2F-H). From E4 to E5, the HNF3β+ territory Enzyme became progressively restricted to the node-derived cells, whereas Shh gene remained activated both in the node-derived E 2.5 NT graft + NT graft + NT graft + cells, where it was strongly expressed, and in adjacent neural notochord floor plate SHH-producing plate-derived cells, where it was only weakly expressed (not cells shown). This composite character of the floor plate, as defined by Shh expression, was even more obvious at later stages. At E7, the Shh-expressing floor plate was clearly formed by (1) a medial component originating from Hensen’s node that a b c expressed HNF3β and (2) two lateral areas derived from the Fig. 1. (A) Dorsal and lateral views of the sinus rhomboidalis of an original neural plate that are now devoid of HNF3β transcripts E1.5 quail embryo after the excision of the axial-paraxial hinge (Fig. 2J-L). It was noticeable that, in the Hensen’s node- (APH) (V, ventral; D, dorsal). APH is the region encompassing derived part, the floor plate was made up of polarised caudal Hensen’s node (HN) and the rostral primitive streak (PS). cylindrical epithelial cells, whose nuclei were situated in a (B) One day after the operation (at E2.5) the neural tube caudal to basal position (Fig. 2L). This cellular arrangement remains somite 20 (S20) is deprived of midline structures (floor plate and typical of the medial floor plate (MFP) throughout notochord) and is smaller in diameter than normal as seen in cross development. By contrast, the neural plate-derived floor plate section (C). (D) In a second experiment, the quail neural tube (NT) deprived of midline cells is enzymatically isolated and grafted in a was made up of a pseudostratified epithelium like the rest of stage-matched chick embryo in the place of a segment of its own the ventricular epithelium (except for the roof plate). The same neural tube-notochord complex: (a) above a notochord; (b) above a observations were made whether the MFP was derived from floor-plate fragment; and (c) above a layer of SHH-producing cells. the grafted or from the host node (Fig. 2J-L,N-P). 4788 J.-B. Charrier and others

CSox1, the expression of which correlates with the mantle (not shown). Moreover, Netrin1 (Kennedy et al., 1994) formation of the early neural plate (Rex et al., 1997) and transcripts were abundant in the MFP and decreased laterally which has been shown in vitro to be implicated in neural through the LFP areas and beyond in the ventral neural tube determination and differentiation (Pevny et al., 1998), was not (see Fig. 4K, control right side of the neural tube and Fig. 8). detected in the medial node-derived cells, but was found in Thus, between E3.5 and E7, molecular analysis of the Shh+ lateral neural plate-derived cells of the floor plate, as well as territory of the ventral neural tube of quail-chick chimeras in the rest of the ventricular epithelium derived from the neural allows us to distinguish a medial, node-derived region, the plate (Fig. 2I). Interestingly, in situ hybridisation at earlier MFP, which is HNF3β+, Netrin1+, CSox1–, Nkx2.2– and stages showed that CSox1 was never expressed in the node and Sim1– from lateral, neural plate-derived areas, the LFP, which node-derived cells, even at later stages when the node becomes are CSox1+, HNF3β transiently+, Nkx2.2+, Sim1+ and the chordo-neural hinge (CNH) (see Fig. 2B-D). By contrast, Netrin1+. Given this distinction, we examined the molecular CSox1 was highly expressed in the neural epithelium (Fig. 2A- characteristics of supernumerary floor plates induced by grafts C) and to a lesser extent in the neural plate caudal to the node of notochord, floor-plate or SHH-producing cells in the avian (not shown). Moreover, from E5 to E7, transcripts of the embryo in ovo. homeobox transcription factor Nkx2.2 (Ericson et al., 1997) were found in a region corresponding to the Shh+, HNF3β– Induction of an ectopic floor plate by grafting neural epithelium-derived area, that we consider as a lateral notochord, floor-plate or SHH-producing cells lateral floor plate (LFP) (Fig. 2M). At the same time, Sim1 transcripts to the neural tube (Yamada et al., 1991) were confined to two groups of cells Many authors, over the past decade, have shown that a situated in the external area of the Nkx2.2 territory in the fragment of notochord grafted in contact to the neural tube in

Fig. 2. (A) Whole-mount in situ hybridisation of a 15 ss chick embryo with the CSox1 probe. (B,C) Cross-sections of this embryo at the neural tube level (B,C) as indicated in A. CSox1 transcripts are present only in the neuroepithelium and not in the floor plate (FP) or the chordo-neural hinge (CNH), which is present at the posterior neuropore level at this stage. (D) Cross-section of a 15 ss quail-chick chimera (quail Hensen’s node graft as shown in E) at the same level as C, stained with the quail specific mAb QCPN. The CNH is made up of quail cells. (E) Schematic representation of quail-chick grafts of Hensen’s node (in red) and posterior neural plate (in blue) at the 5-6 ss. (F-H) Serial cross-sections of a chimera grafted with a quail Hensen’s node (red in E), 2 days after the operation (E3.5). The expression pattern of HNF3β (F) and Shh (G) genes is wider than the node-derived region revealed by the QCPN mAb (H). (I-M) Serial sections of another Hensen’s node chimera fixed 5.5 days after the graft (E7). CSox1 (I) is not expressed in the quail QCPN+ node-derived region (shown in L), which is where expression of HNF3β is now restricted (J). This region also constitutes the medial floor plate (MFP). Shh (K) is expressed both in the node-derived region and in a neural plate-derived area where Nkx2.2 transcripts (M) are also present. The latter constitutes the lateral floor plate (LFP). (N-P) Serial sections of a quail-chick chimera grafted with a posterior neural plate (blue in E), 5.5 days after the operation (E7). As in Hensen’s node chimeras at the same stage, HNF3β transcripts (N) are localised in the node-derived (host) region as seen in P, while Shh transcripts are distributed over a larger area covering both node-derived (MFP) and QCPN+ neural plate-derived tissues, including the LFP (O). Arrowheads, MFP limits; arrows, LFP lateral limit. Sonic hedgehog induces the lateral floor plate 4789

Donor stage From E5 to E7 (3 to 5 days post operation), MFP and LFP 25 ss characteristics appeared progressively in the induced territory (n=4/7, see Fig. 3) (Fig. 4E-L): HNF3β transcripts were restricted to a medial region (Fig. 4F,J) strongly expressing Shh (Fig. 4E,I); Shh was weakly expressed in a lateral domain 20 where Nkx2.2, Sim1 and CSox1 transcripts were present (Fig. 4G,H,L); CSox1 expression progressively disappeared in the HNF3β+ domain between E6 and E7 (Fig. 4L). Moreover, the chemotropic factor Netrin1, which is known to be expressed 15 in a ventral domain broader than the floor-plate territory No-induced LFP n=3 (Kennedy et al., 1994) was also present in the region of the No-induced MFP n=4 notochord-induced floor plate (Fig. 4K). FP-induced LFP n=8 Thus, a region of the neural epithelium placed in contact 10 FP-induced MFP n=4 with a notochord, can progressively acquire the molecular MFP SHH-induced LFP n=8 characteristics (i.e. be HNF3β +, Shh+ and CSox1–) in addition to the LFP traits (i.e. be Shh+, HNF3β transiently+, CSox1+, SHH producing cells Nkx2.2+ and Sim1+). However, induction of the MFP-like structure was generally observed only over a short length of the grafted notochord. Moreover, floor-plate induction, as already observed by van Straaten et al. (van Straaten et al., 0 1988), occurred only when the graft was performed before 15 5 10 15 20 25 ss ss in the most caudal region of the embryo (see Fig. 3). These Host stage observations suggest that there is a narrow time window when Fig. 3. The distribution of complete floor plate (with medial and this full transformation of a neural epithelium into a MFP can lateral components) (red) or lateral floor-plate cells only (blue) take place. By contrast, induction of LFP characteristics is induced in the lateral neural tube of E5 to E7 chick embryos mapped widespread over the full length of the caudal neural tube as a function of the somitic stage (ss) of the chick hosts (x-axis) and subjected to induction from the grafted notochord, at any stage of the quail donors (y-axis) of inducer tissues [notochord (No; closed of the chick host up to 25 ss. HNF3β and Shh induction by circles) or floor plate (FP; open circles)] at the time of the graft. For SHH-producing cell grafts (squares) only chick host age is exogenous notochord requires at least 2 days of exposure. considered. The number of cases for each experimental series is Moreover, MFP complete differentiation necessitates 4-5 days indicated. It appears that MFP induction can occur only if the chick of exposure and occurs on a shorter length than LFP host is younger than 15 ss when the stage of the quail donor is phenotype. between 10 and 25 ss. Floor-plate grafting Exposure of E2 (9-25 ss) chick posterior neural tube to E2 (12- E2 chick embryos can induce floor-plate-like characteristics in 25 ss) quail floor plates produced various results, generally the neural epithelium (van Straaten et al., 1985; van Straaten depending on the developmental stage of both donor and host et al., 1988; Placzek et al., 1990; Placzek et al., 1991; Placzek tissues (see Fig. 3). For 9-14 ss hosts that received a fragment et al., 1993; Yamada et al., 1991; Pourquié et al., 1993). of floor plate from the cervical or thoracic region of 20-25 ss However, such an induction was not observed in 100% of the donors, a dorsoventral overgrowth of the lateral neural tube cases (our own observation) (van Straaten et al., 1988), and the was seen on the side of the graft one day after the graft (i.e. at variability of the restuls has not been completely explained. We E3; not shown). Two days after the graft (E4), transcripts of decided to explore the response of the neural epithelium to Shh and HNF3β were present in the neural epithelium contact with an inducing tissue in terms of gene activity. immediately opposite to the floor-plate graft (n=3/3) (Fig. 5A,B). At this stage, Shh and HNF3β expression co-existed Notochord grafting with that of CSox1 and Nkx2.2, the latter of which was induced Notochords were dissected enzymatically from 10-21 ss quail by the graft (Fig. 5C,D). Pax6 was not expressed in this region embryos. Notochord fragments two to three somites long were (not shown). Five to 6 days after the operation (E7-8), a typical implanted in 7-23 ss chick embryos (E2), between the neural MFP area (HNF3β +, Shh+, Netrin1+, CSox1–, Nkx2.2–) could epithelium and the segmental plate in the region immediately be observed in four out of 12 cases. The MFP-like structure rostral to the endogenous node. During the first day after the differentiated generally over a short length (about 50 µm) in operation (up to E3), only discreet molecular alterations could embryos operated before 15 ss (see Fig. 3). Only grafts in be detected in the host neural tube but obvious dorsoventral which donor MFP was in close contact to the host neural overgrowth of the tube wall was noticed on the side of the graft epithelium were efficient in MFP induction (see Fig. 5E-H). (not shown). Two days after the graft (E4), the asymmetry of In conclusion, notochord and medial floor plate, both of the neural tube had increased. Moreover, the region of the which are derived from Hensen’s node, are able to induce neural epithelium in contact with the grafted notochord was a supernumerary floor-plate-like structure in the neural thinner than the rest of the neural tube wall (Fig. 4A-D). epithelium, in which the morphological and molecular Transcripts of Pax6, a lateroventral marker of the neural tube characteristics of the natural MFP and LFP can be detected. (Goulding et al., 1993), were not found in this region, while MFP induction can take place only in the posterior-most region Shh and HNF3β began to be expressed (Fig. 4A-C). of the neural plate of embryos younger than 15 ss. 4790 J.-B. Charrier and others

Grafts of SHH-producing cells the expression pattern of several LFP markers including not Grafts of clumps of cells engineered to produce SHH (Duprez only Shh and Netrin1 but also Nkx2.2, Sim1 and the neural et al., 1998) yielded similar results to the grafts of notochord marker CSox. A characteristic MFP devoid of neural markers or floor-plate fragments after the first day of exposure (Fig. 6A- was never observed. C). Later, the grafted cells dispersed and the field resulting from the induction was larger than with notochord and floor- Secondary patterning of neural tube deprived of plate grafts. As a result, after 3 days or more of SHH-exposure node-derived floor-plate cells by caudal Hensen’s (E5-E7), the lateral floor plate of the host embryo was widely node excision enlarged on the side of the graft, as seen in Fig. 6D-K showing The caudalward movement of Hensen’s node was prevented by

Fig. 4. (A-D) Graft of a 9 ss quail notochord (No′) lateral to the caudal neural tube of a 10 ss chick embryo. Serial sections performed 2 days after the graft (E4) show that the neural epithelium has increased in size on the side of the graft when compared with the contralateral side. Shh (A) and HNF3β (B) are expressed ectopically in the region facing the graft. Pax6 (C) is not found in this region. (D) QCPN labelling of the graft. (E-L) Serial sections 4 (E-H) and 5 (I-L) days after the same experiment (E6 and E7) show that the molecular characteristics of a complete ﬂoor plate with its medial and lateral components are progressively acquired in the region close to the graft: wide expression of Shh (E, I) and Netrin1 (K); presence of HNF3β transcripts in a restricted medial region (F,J) where Shh (E,I) and Netrin1 (K) are upregulated and CSox1 is downregulated (L); expression of Nkx2.2 (G) and Sim1 (H) lateral to the HNF3β+ region. Arrowheads, MFP limits; arrows, LFP lateral limit.

Fig. 5. (A-D) Graft of a 15 ss quail floor plate (FP′) lateral to the caudal neural tube of a 14 ss chick embryo. Serial sections collected 2 days after the operation (E4) show that the size of the neural tube has expanded on the side of the graft. At this stage, Shh (A), HNF3β (B) and Nkx2.2 (C) are co- expressed in a region that is still CSox1+ (D), close to the graft. (E-H) Graft of a 12 ss quail FP′ lateral to the caudal neural tube of a 13 ss chick embryo. Serial sections performed 5 days after the operation (E7) hybridised with Shh (E), HNF3β (F), Nkx2.2 (G) and CSox1 (H) probes show that a complete floor plate has differentiated with its medial (Shh+, HNF3β+, Nkx2.2–, CSox1–) and lateral (Shh+, HNF3β–, Nkx2.2+, CSox1+) components. Grafted MFP plus LFP (FP′) generally become circular. A new MFP is induced in the host neural tube close to the MFP part of the graft (HNF3β+). Arrowheads, MFP limits; arrows, LFP lateral limit. Sonic hedgehog induces the lateral floor plate 4791

Fig. 6. (A-K) Graft of SHH-producing cells between the caudal neural tube and the presomitic mesoderm of 10-15 ss chick embryos. (A) Immediately after the graft, clumps of cells are aligned to the left of the enlarged neural tube (between arrowheads). (B) Whole-mount Shh in situ hybridisation 1 day after the graft, at 25 ss (E3). The grafted cells are localised lateral to the neural tube (arrowheads). (C) A cross-section of the whole mount in B shows that the cells are still grouped (arrow) and that the neuroepithelium has expanded dorsoventrally on the side of the graft. (D,E) Serial cross-sections 3 days after the graft (E5) hybridised with Shh (D) and HNF3β (E) probes. SHH- producing cells (arrow) are now dispersed and the Shh+, HNF3β– lateral floor plate has widened. (F-K) Serial cross-sections 5 days after the graft (E7) show that the ventral and lateral neural tube is enlarged and perturbed on the side of the graft. Shh (F), HNF3β (G), Netrin1 (H), Nkx2.2 (I), Sim1 (J) and CSox1 (K) expression patterns in the region facing the SHH-producing cells are characteristic of the lateral floor plate. RP, roof plate. Arrowheads, MFP limits; arrows, LFP lateral limit. excising the APH in 5-6 ss quail embryos (see Fig. 1). One day the notochord, the floor-plate or the SHH-producing cells, after the excision, the midline structures (floor plate and while CSox1 was still uniformly expressed in the transplanted notochord), and consequently the source of SHH in the medial neural tube (not shown). ectodermal and mesodermal layers, were absent. Under these At E7 (5 days after the graft), when the inducer was a circumstances, neither the neural tube nor the paraxial notochord, a complete floor plate with its medial and lateral mesoderm can survive (Charrier et al., 1999). Many of their components (MFP and LFP) could be observed in the region constitutive cells undergo apoptosis within the 24 hours that facing the graft, although over a short length (Fig. 7A-D). The follow the operation. This cell death can, however, be induced MFP exhibited the typical structure of a columnar prevented by the experimental addition of cells producing SHH highly polarised epithelium (Fig. 6A), like a node-derived floor in close proximity to the axial tissues, thus showing that one plate in normal development. It expressed Shh, HNF3β and of the major roles of SHH emanating from the floor plate Netrin1, and failed to exhibit transcripts of the neural marker and/or notochord is to antagonise the built in programmed cell CSox1 (not shown). Moreover, on each side of this MFP-like death of the embryonic tissues that form the neural tube and structure, lateral areas expressing Shh, CSox1, Netrin1 (not the somites (Charrier et al., 2001; Teillet et al., 1998b). shown) and Nkx2.2 (Fig. 7D) were present. Such midline cell-deprived neural tubes taken at E2.5 from When the inducer was either the floor plate (Fig. 7E-H) or operated quail embryos were transplanted into 20-25 ss chick SHH-producing cells (Fig. 7I-L), the ventral neural tube was embryos in a region from which endogenous neural tube and incompletely patterned: CSox1 (not shown) continued to be notochord had been previously removed (Fig. 1D). expressed ventrally in a region where Shh and Nkx2.2 were co- Transplantation was made in three different situations: (1) expressed and where HNF3β became downregulated, thus together with a chick notochord; (2) with a chick floor plate; showing the molecular characteristics of a LFP. (3) with clumps of SHH-producing cells, as already described by Charrier et al. (Charrier et al., 2001). Observation of the transplanted neural tube the first day after the operation showed DISCUSSION a decrease in apoptosis in the three situations when compared with a graft in the absence of these tissues (Charrier et al., The avian floor plate is heterogeneous with medial 2001). No transcripts of the floor-plate markers Shh and and lateral components HNF3β were then observed, although Pax3 and Pax6 We demonstrate in this work that, during normal development transcripts, previously present in the entire neural tube, were of the avian embryo, the floor plate, an epithelial structure downregulated ventrally in contact with the notochord, floor- located ventrally in the neural tube, is heterogeneous and plate or SHH-producing cells [see Fig. 2 by Charrier et al. composed of regions that can be distinguished on the basis of (Charrier et al., 2001)]. However, two days after the graft (E4), their embryological origin and molecular characteristics. The Shh and HNF3β transcripts were present in the region facing medial region of the floor plate (MFP) is formed by cells 4792 J.-B. Charrier and others

Fig. 7. (A-L) Back grafts of quail neural tubes deprived of midline cells by APH excision into chick embryos from which a fragment of their own neural tube and notochord had been previously excised (see Fig. 1). Fixation was 5 days after the operation (E7). (A-D) Grafted over a notochord (No′) the quail neural tube, labelled with the quail specific mAb QCPN (A), develops a typical floor plate with its medial and lateral components distinguished by the characteristic distribution of the Shh (B), HNF3β (C) and (D) NKx2.2 transcripts. (E-H) Grafted over a floor plate (FP′), the QCPN+ quail neural tube (E) weakly expresses Shh (F) and very little HNF3β (G) but strongly expresses Nkx2.2 (H) in the region close to FP′. This pattern recalls that of the lateral floor plate (LFP) as defined in Fig. 2. (I-L) In contact with SHH-producing cells (arrow in J), the quail neural tube deprived of midline cells develops a large LFP-like structure that expresses CSox1 (I), Shh (J), little HNF3β (K) and Nkx2.2 (L). Arrowheads, MFP limits; arrows, LFP lateral limit. originating from the avian organiser, Hensen’s node. During its The genetic profile of medial node-derived cells rostrocaudal movement, which is concomitant with the differs from that of the neuralised ectoderm posterior elongation of the embryo, the node leaves in its wake The mechanisms underlying the neuralisation of the dorsal two closely associated axial structures: the floor plate that ectoderm of the vertebrate embryo and leading to the formation becomes inserted into the ectoderm of the neural plate; and the of the neural plate have been under investigation for almost a notochord that becomes part of the mesoderm (Catala et al., century (see Streit and Stern, 1999; Harland, 2000; Wilson and 1996). These two structures are located midsagittally in the Edlund, 2001). According to the presently accepted view, embryo. Data obtained previously (Charrier et al., 1999; Le neuralisation results from the diffusion by the dorsal organiser Douarin and Halpern, 2001; Le Douarin, 2001) support the (Spemann’s organiser or its equivalent in various vertebrate hypothesis that the notochord and floor plate arise from a species) of dorsalising factors that are able to antagonise population of pluripotent cells present in Hensen’s node, where ventralising influences exerted mainly by members of the they might function as stem cells. At midneurulation (i.e. 5-6 TGFβ and Wnt families of secreted proteins. FGF signalling ss) these cells occupy the posterior end of Hensen’s node (zone could be necessary for neural induction in avian embryo at c) [see Fig. 11 by Charrier et al (Charrier et al., 1999)]. In the earlier stages, before gastrulation (Streit et al., 2000). zebrafish embryo, a common pool of midline precursor cells In the avian embryo, determination of the neural plate has also been hypothesised in the early organiser region occurs very early in development as the expression pattern of designated as the shield (Halpern et al., 1997; Appel et al., a pan-neural marker, CSox1, at the primitive streak stage (Rex 1999). et al., 1997) coincides with the fate map of the neural plate as Lateral to the node-derived MFP, cells of the neural defined by the quail-chick cell marking technique (Garcia- ectoderm acquire molecular and functional characteristics Martinez et al., 1993). The same exact correspondence that allow them to be considered as lateral floor-plate cells between the CSox2 expression domain and the neural tube fate (LFP), analogous to the zebrafish LFP cells (Odenthal et al., map was found at later stages of chick and quail development 2000). [see Fig. 3 by Le Douarin (Le Douarin, 2001)]. Our preceding Sonic hedgehog induces the lateral floor plate 4793

E 2.5 (J.-B. C., F. L., N. M. L. D. and M.-A. T., unpublished) (Duprez et al., 1999). E 1.5 Induction of floor-plate gene activities in the neural ectoderm by the node-derived midline cells The floor plate and the notochord express + Sox1+ Sox1 several genes that were activated in the organiser, such as HNF3β, Shh and Chordin [see Fig. 2 by Charrier et al. (Charrier et al., 1999)]. None of these genes are expressed in Hensen's node HNF3β + Node-derived the neural ectoderm prior to the time floor plate (ND FP) Notochord Shh+ it becomes associated with the node- Chordin+ Dorsal derived midline structures. Moreover, when Noggin+ endoderm insertion of the floor plate is inhibited by Sox1- caudal Hensen’s node excision, the neural tube never expresses Shh or HNF3β E 3.5 E 7 (Charrier et al., 1999). Contact between the node-derived medial cells fated to become the MFP and the adjacent neural ectoderm appears to result in the induction of these genes in cells flanking the MFP that we designate here as lateral floor plate (LFP). Thus, soon after the incorporation of the node-derived cells into the neural ectoderm, HNF3β and Shh transcripts are found laterally in the neuroepithelial LFP. LFP LFP Similarly, Netrin1 transcripts, which encode MFP a chemotropic secreted protein (Serafini et al., 1994) are, like those of Shh, abundant in (ND FP) the MFP and also expressed in the LFP. HNF3β + However, HNF3β, after expanding laterally + Lateral floor plate Shh Lateral floor plate in LFP territories, becomes progressively induced by ND FP Sox1- induced by ND FP restricted to the MFP (see Figs 2, 8). The HNF3β + Netrin1+ HNF3β- presence of CSox1 transcripts distinguishes Shh+ Shh+ the LFP from the MFP which in fact never Sox1+ Sox1+ displays neural traits. Moreover, from E4 Nkx2.2+ onwards, the LFP starts to express Nkx2.2, a Sim1+ transcription factor induced by Shh Netrin1+ and involved in ventral nervous system Fig. 8. Model of avian floor-plate development from E1.5 to E7 at the thoracic level. patterning (Briscoe et al., 1999; Pabst et al., Hensen’s node is schematically represented at 5 ss (E1.5) when it becomes inserted into 2000; Soula et al., 2001). the neural plate which expresses the gene Sox1 (Sox1+). One day later (E2.5) at the same One can therefore distinguish in the level, Hensen’s node has split into floor plate and notochord. The node-derived floor plate avian neural tube a MFP that is CSox1–, (ND FP, in red), like the node itself, expresses HNF3β and Shh. ND FP and perhaps also HNF3β+, Shh+, Netrin1+ and Nkx2.2–, and the notochord, induce adjacent neural plate cells (arrows) to express HNF3β and Shh, a LFP that is CSox1+, HNF3β transiently+, thus forming at E3.5 a lateral floor plate (LFP, in blue), which retains Shh expression Shh+, Netrin1+ and Nkx2.2+. In addition, while loosing HNF3β expression (E7). LFP expresses continuously neural genes (Sox1, LFP cells maintain the pseudostratified Nkx2.2 and Sim1) that are never expressed in the node-derived medial floor plate (MFP). structure of the neuroepithelium and do not Netrin1 is expressed both in MFP and LFP cells. acquire the polarised morphology of the MFP. Genetic analysis has revealed that the results showed that the early neural plate, formed by a planar zebrafish floor plate is also composed of medial and lateral induction in the dorsal ectoderm, lacks a floor-plate territory. components whose patterning is differently regulated. The The latter is intercalated during Hensen’s node regression presence of MFP in sonic-you (syu) mutants showed that the (Catala et al., 1996). Only after the floor plate has been shh zebrafish homologue is not involved in the formation of incorporated into the neural ectoderm is the definitive neural the MFP (Schauerte et al., 1998). By contrast, it is required plate formed. The gene activities of these two domains of the for the induction of LFP cells (Odenthal et al., 2000). definitive neural plate are different: the neural ectoderm Experimental analysis in the zebrafish embryo was pushed one expresses several genes, such as CSox1, CSox2, Msx1, Msx2 step further by the injection of morpholinos directed against and FrzB, that are not expressed in the node derived midline tiggy-winkle hedgehog (twhh), another member of the zebrafish structures (floor plate and notochord) during neurogenesis family of HH-producing genes, into a syu mutant (which is 4794 J.-B. Charrier and others devoid of SHH). In these embryos, the MFP develops normally This work suggests that, in chick, like in zebrafish, SHH is (Etheridge et al., 2001; Lewis and Eisen, 2001), thus showing not involved in specifying the MFP itself but is essential for that its formation does not depend on either SHH or TWHH. inducing the LFP. Experiments using Shh–/– mutant mouse These results are consistent with our observations in the avian embryos (Chiang et al., 1996) and downregulated Shh chick embryo, according to which a floor plate can develop embryos (Ahlgren and Bronner-Fraser, 1999; Britto et al., independently from the notochord (Teillet et al., 1998a). 2002) must be carried out to verify this hypothesis. In conclusion, we have shown that the floor plate, a structure The notochord and the floor plate are able to induce playing an important role in patterning the neural tube of the a MFP in a restricted domain of the E2 normal neural vertebrate embryo, is a composite a structure in an amniote epithelium embryo as it is in lower teleost vertebrates. It is formed by a Induction of a floor-plate-like structure in the lateral neural medial component derived from the organiser, which induces tube of the chick embryo by notochord or floor-plate graft has the adjacent neural ectoderm to develop floor-plate markers been described several times in the past (van Straaten et al., that co-exist with neural epithelial markers (Fig. 8). The SHH 1985; van Straaten et al., 1988; Placzek et al., 1990; Placzek protein plays a key role in inducing the lateral floor plate but et al., 1991; Yamada et al., 1991; Pourquié et al., 1993). In this cannot, by itself, induce the characteristics of a medial floor work we show that fragments of notochord or MFP can induce plate in the neural ectoderm. This can be achieved during a both MFP and LFP if applied to embryos at stages ranging brief window of time by the MFP itself or by the notochord, from 7-15 ss, but not later (see Fig. 3). The full molecular meaning that floor-plate induction in experimental conditions characters of an induced MFP are present only after 5 days of requires factor(s) specific to the organiser or its derivatives. exposure to the notochord or floor-plate graft. Moreover, the MFP is induced only over a short length of the neural tube of We thank P. Brickell, A. Grapin-Botton, P. Gruss, R. Riddle, J. the host located in close vicinity to Hensen’s node at operation Rubenstein, A. Ruiz i Altaba and C. Tabin for the chick nucleic acid time. More rostrally, the graft merely induced LFP- but not probes that they provided. QCPN monoclonal antibody was obtained MFP-type gene activities. from the Developmental Studies Hybridoma Bank. The authors thank Françoise Dieterlen and Heather Etchevers for critical reading of the These results demonstrate that, although the MFP and the manuscript. They are grateful to Bernadette Schuler for technical neural epithelium have different embryological origins in assistance, and to Sophie Gournet, Francis Beaujean and Michel normal development, MFP can be induced in the neural Fromaget for the illustrations. The helpful comments and constructive ectoderm by Hensen’s node-derived tissues. However, the suggestions of the referees are greatly appreciated. This work was neural ectoderm, as it stands in 7-15 ss avian embryos, is able supported by the Centre National de la Recherche Scientifique and the to respond to this induction only over a short period of time Association pour la Recherche contre le Cancer (grant number 5578 and in a region where it is itself in close proximity to the to M.-A. T.). endogenous node of the recipient embryo. Induction of a MFP by an exogenous floor plate can occur only if the donor MFP is in close contact with the neural ectoderm. REFERENCES SHH alone can only induce a LFP but not a MFP in Ahlgren, S. C. and Bronner-Fraser, M. (1999). Inhibition of sonic hedgehog the E2 neural epithelium signaling in vivo results in craniofacial neural crest cell death. Curr. Biol. 9, 1304-1314. In cultures of the 10 ss chick neural plate, SHH has been shown Ang, S. L. and Rossant, J. (1994). 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