The transition from differentiation to growth during dermomyotome-derived myogenesis depends on temporally restricted hedgehog signaling.

The development of a functional tissue requires coordination of the amplification of progenitors and their differentiation into specific cell types. The molecular basis for this coordination during myotome ontogeny is not well understood. Dermomytome progenitors that colonize the myotome first acquire myocyte identity and subsequently proliferate as Pax7-expressing progenitors before undergoing terminal differentiation. We show that the dynamics of sonic hedgehog (Shh) signaling is crucial for this transition in both avian and mouse embryos. Initially, Shh ligand emanating from notochord/floor plate reaches the dermomyotome, where it both maintains the proliferation of dermomyotome cells and promotes myogenic differentiation of progenitors that colonized the myotome. Interfering with Shh signaling at this stage produces small myotomes and accumulation of Pax7-expressing progenitors. An in vivo reporter of Shh activity combined with mouse genetics revealed the existence of both activator and repressor Shh activities operating on distinct subsets of cells during the epaxial myotomal maturation. In contrast to observations in mice, in avians Shh promotes the differentiation of both epaxial and hypaxial myotome domains. Subsequently, myogenic progenitors become refractory to Shh; this is likely to occur at the level of, or upstream of, smoothened signaling. The end of responsiveness to Shh coincides with, and is thus likely to enable, the transition into the growth phase of the myotome.

Negative regulation of midline vascular development by the notochord.

The negative regulation of vascular patterning is one of the least understood processes in vascular biology. In amniotes, blood vessels develop throughout the embryonic disc, except for a midline region surrounding the notochord. Here we show that the notochord is the primary signaling center for the inhibition of vessel formation along the embryonic midline. Notochord ablation in quail embryos results in vascular plexus formation at midline. Implantation of the notochord into paraxial and lateral mesoderm inhibits vessel formation locally. The notochord-expressed BMP antagonists Chordin and Noggin inhibit endothelial cell migration in vitro, and their ectopic expression in vivo results in a local disruption of vessel formation. Conversely, BMP-4 activates endothelial cell migration in vitro, and its ectopic expression along the notochord induces vascular plexus formation at midline. These data indicate an inhibitory role of the notochord in defining an avascular zone at the embryonic midline, in part via BMP antagonism.

Somite differentiation. Sonic signals somites.

Sonic hedgehog, a secreted signalling molecule known to play a role in the patterning of the central nervous system and the limb in vertebrates, also controls differentiation of the somites.

Early steps in neural development.

We studied early neurulation events in vitro by transplanting quail Hensen's node, central prenodal regions (before the nodus as such develops), or upper layer parts of it on the not yet definitively committed upper layer of chicken anti-sickle regions (of unincubated blastoderms), eventually associated with central blastoderm fragments. We could demonstrate by this quail-chicken chimera technique that after the appearance of a pronounced thickening of the chicken upper layer by the early inductive effect of neighboring endophyll, a floor plate forms by insertion of Hensen's node-derived quail cells into the median part of the groove. This favors, at an early stage, the floor plate "allocation" model that postulates a common origin for notochord and median floor plate cells from the vertebrate's secondary major organizer (Hensen's node in this case). A comparison is made with results obtained after transplantation of similar Hensen's nodes in isolated chicken endophyll walls or with previously obtained results after the use of the grafting procedure in the endophyll walls of whole chicken blastoderms.

The developmental relationships of the neural tube and the notochord: short and long term effects of the notochord on the dorsal spinal cord.

Patterning of the ventral half of the neural tube results from the inductive influence of the notochord and of the floor plate. We have studied here the effect of an ectopically grafted notochord on the development of the dorsalmost part of the neural tube i.e. roof plate and alar plates. We show that at their early stages, dorsal genes are repressed by the dorsal graft of a notochord, as shown previously in other studies. We found also that when the notochord is implanted in a mediodorsal position on top of the roof plate (and not laterally as previously performed in other studies) the genes specifics of the floor plate are not induced, and motoneurons do not differentiate. The notochord prevents the formation of the medial septum from roof plate cells and induces their active proliferation between E5 and E7. Roof and dorsal alar plates derived cells start to die from E7 onward leaving a dorsally truncated spinal cord. If the notochord is grafted at 20 degrees-30 degrees from the sagittal plane ventral genes and structures are induced and the roof plate differentiates normally. We conclude that roof plate cells exhibit a specific response to notochord signals, the short range effect of which is thus strikingly demonstrated.

Spatial and temporal effects of axial structures on myogenesis of developing somites.

To elucidate the precise roles of axial structures in the myogenic differentiation of the somite, we have examined the effects of the axial organs' precise spatial position during migration and differentiation of somitic cells by using in vivo transplantation of the neural tube and of the notochord directly into the paraxial mesoderm. Differentiation of myotomal cells was identified through the use of Quox 1 antibody which recognizes specifically a quail homeoprotein Quox 1. We have demonstrated that both ectopic neural tube and notochord are able to influence the myogenesis in somites, but that the spatial position of axial organs and the degree of somite maturation at grafting time are decisive. At the level of the somites which were already formed and developmentally advanced (somites III-VI), both neural tube and notochord promote myogenesis, and the promoting effect of notochord is more efficient than that of the neural tube. In the newly formed somites (I-II) and/or the segmental plate mesoderm, the notochord inhibits the myogenesis of somites, whereas the neural tube plays an evident myogenic promoting role. But the myogenic effect of the neural tube depends not only upon the stage of developing somites and presomitic mesoderm, but also on the developmental maturation of the neural tube. We have demonstrated that the myogenic effect of the rostral part of neural tube is stronger than that of its caudal part. This observation suggests that there is a gradient of myogenic effect along the rostrocaudal axis of the neural tube, which depends on the developmental maturation of neural tube, and that the generation of skeletal muscle during somitogenesis may be in relation with the rostrocaudal gradient of the capacity of the neural tube to stimulate myogenesis since somites are also distributed along an anteroposterior axis.

Induction of oligodendrocyte progenitors in the trunk neural tube by ventralizing signals: effects of notochord and floor plate grafts, and of sonic hedgehog.

Recent evidence indicates that oligodendrocytes originate initially from the ventral neural tube. We have documented in chick embryos the effect of early ventralization of the dorsal neural tube on oligodendrocyte differentiation. Notochord or floor plate grafted at stage 10 in dorsal position induced the development of oligodendrocyte precursors in the dorsal spinal cord. In vitro, oligodendrocytes differentiated from medial but not intermediate neural plate explants, suggesting that the ventral restriction of oligodendrogenesis is established early. Furthermore, quail fibroblasts overexpressing the ventralizing signal Sonic Hedgehog induced oligodendrocyte differentiation in both the intermediate neural plate and the E4 dorsal spinal cord. These results strongly suggest that the emergence of the oligodendrocyte lineage is related to the establishment of the dorso-ventral polarity of the neural tube.

Explanted and implanted notochord of amphibian anuran embryos. Histofluorescence study on the ability to synthesize catecholamines.

The notochord of amphibian anuran embryos contains catecholamines during the early developmental stages. In order to determine if these catecholamines are synthesized in situ, the development of their specific histofluorescence was investigated in the notochord alone or the notochord combined with the lateral somitic mesoderm, both explanted at the neurula stage and cultivated in vitro or implanted into the ventral part of early neurulae endoderm. The histofluorescence evolution, on the other hand, was investigated in the notochord alone or combined with myotomes, both explanted after the beginning of catecholamine biosynthesis and cultivated in vitro for one hour, in order to determine the effect of explantation and culture on the accumulation of notochordal catecholamines. The comparative study of catecholamine histofluorescence in these different samples shows that: the notochord is able to perform, on its own, the entire biosynthesis of the catecholamines stored in it during the early developmental stages. The catecholamines generated from isolated notochords tend to diffuse into the culture medium, probably due to a deficiency in the vesicular storage system usually found in the catecholamine-synthesizing cells. This loss of catecholamines in vitro can be obviated by the presence round the notochord of any embryonal tissue (somitic mesoderm, endoderm).

Differentiation of gut endoderm in dependence of the notochord.

Presumptive intraembryonic endoderm, either isolated or together with adhering mesoderm, from 19-h chick embryos, was grafted to the coelom of 50-h host embryos. The viability of such grafts was low and endodermal differentiation was poor. In a second series the endoderm (with or without adhering mesoderm) was combined with a fragment of notochordal tissue from 48-60-h donor embryos. Then the recovery was much higher, notably after longer periods of in vivo culture. After 10 days of cultivation well-developed entero-endocrine (argyrophilic) cells were found among the regular enterocytes in both series.

The ventralizing effect of the notochord on somite differentiation in chick embryos.

The dorso-ventral pattern formation of the somites becomes manifest by the formation of the epithelially organized dorsal dermomyotome and the mesenchymal ventrally situated sclerotome. While the dermomyotome gives rise to dermis and muscle, the sclerotome differentiates into cartilage and bone of the axial skeleton. The onset of muscle differentiation can be visualized by immunohistochemistry for proteins associated with muscle contractility, e.g. desmin. The sclerotome cells and the epithelial ventral half of the somite express Pax-1, a member of a gene family with a sequence similarity to Drosophila paired-box-containing genes. In the present study, changes of Pax-1 expression were studied after grafting an additional notochord into the paraxial mesoderm region. The influence of the notochord and the floor-plate on dermomyotome formation and myotome differentiation has also been investigated. The notochord is found to exert a ventralizing effect on the establishment of the dorso-ventral pattern in the somites. Notochord grafts lead to a suppression of the formation and differentiation of the dorsal somitic derivatives. Simultaneously, a widening of the Pax-1-expressing domain in the sclerotome can be observed. In contrast, grafted roof-plate and aorta do not interfere with dorso-ventral patterning of the somitic derivatives.

Intervertebral disc regeneration or repair with biomaterials and stem cell therapy--feasible or fiction?

The "gold standard" for treatment of intervertebral disc herniations and degenerated discs is still spinal fusion, corresponding to the saying "no disc - no pain". Mechanical prostheses, which are currently implanted, do only have medium outcome success and have relatively high re-operation rates. Here, we discuss some of the biological intervertebral disc replacement approaches, which can be subdivided into at least two classes in accordance to the two different tissue types, the nucleus pulposus (NP) and the annulus fibrosus (AF). On the side of NP replacement hydrogels have been extensively tested in vitro and in vivo. However, these gels are usually a trade-off between cell biocompatibility and load-bearing capacity, hydrogels which fulfill both are still lacking. On the side of AF repair much less is known and the question of the anchoring of implants is still to be addressed. New hope for cell therapy comes from developmental biology investigations on the existence of intervertebral disc progenitor cells, which would be an ideal cell source for cell therapy. Also notochordal cells (remnants of the embryonic notochord) have been recently pushed back into focus since these cells have regenerative potential and can activate disc cells. Growth factor treatment and molecular therapies could be less problematic. The biological solutions for NP and AF replacement are still more fiction than fact. However, tissue engineering just scratched the tip of the iceberg, more satisfying solutions are yet to be added to the biomedical pipeline.

Induction of an additional floor plate in the neural tube.

The role of the notochord in the morphogenesis of the neural tube was investigated by implanting a notochord fragment laterally to the neural wall of a 1.5 day chick embryo. Embryos were sacrificed at 4 days. In the basal part of the neural tube an additional floor plate was induced in the vicinity of the implant. This floor plate was characterized by a low proliferative activity, a thin wall, spindle-like nuclei crowded peripherally and some neuroblast-like cells. It was either blending with the natural floor plate or separated from it, depending on the exact position of the implant. In the latter case neuroblasts were observed in between both floor plates. The additional floor plate was present only when the implanted notochord was less than 25 micron apart from the neural tube; at larger distance an increase of the ventral horn neuroblast area could be seen. It is concluded that the implanted notochord is able to induce a floor plate at 1.5 days of incubation. The specific influence of the notochord on the morphogenesis of the neural tube, its inductive period as well as the presence of the neuroblast-like cells in the additional floor plate are discussed.

Control of cell pattern in the developing nervous system: polarizing activity of the floor plate and notochord.

Individual classes of neural cells differentiate at distinct locations in the developing vertebrate nervous system. We provide evidence that the pattern of cell differentiation along the dorsoventral axis of the chick neural tube is regulated by signals derived from two ventral midline cell groups, the notochord and floor plate. Grafting an additional notochord or floor plate to ectopic positions, or deleting both cell groups, resulted in changes in the fate and position of neural cell types, defined by expression of specific antigens. These results suggest that the differentiation of neural cells is controlled, in part, by their position with respect to the notochord and floor plate.

The spatial and temporal dynamics of Sax1 (CHox3) homeobox gene expression in the chick's spinal cord.

Sax1 (previously CHox3) is a chicken homeobox gene belonging to the same homeobox gene family as the Drosophila NK1 and the honeybee HHO genes. Sax1 transcripts are present from stage 2 H&H until at least 5 days of embryonic development. However, specific localization of Sax1 transcripts could not be detected by in situ hybridization prior to stage 8-, when Sax1 transcripts are specifically localized in the neural plate, posterior to the hindbrain. From stages 8- to 15 H&H, Sax1 continues to be expressed only in the spinal part of the neural plate. The anterior border of Sax1 expression was found to be always in the transverse plane separating the youngest somite from the yet unsegmented mesodermal plate and to regress with similar dynamics to that of the segregation of the somites from the mesodermal plate. The posterior border of Sax1 expression coincides with the posterior end of the neural plate. In order to study a possible regulation of Sax1 expression by its neighboring tissues, several embryonic manipulation experiments were performed. These manipulations included: removal of somites, mesodermal plate or notochord and transplantation of a young ectopic notochord in the vicinity of the neural plate or transplantation of neural plate sections into the extraembryonic area. The results of these experiments revealed that the induction of the neural plate by the mesoderm has already occurred in full primitive streak embryos, after which Sax1 is autonomously regulated within the spinal part of the neural plate.

Somite formation in the early chick embryo following grafts of Hensen's node.

Quail grafts of Hensen's node were examined for their potential to induce somites in chick blastoderms. The origin of the structures induced depended on the distance of the graft from the host's midline. Nodes placed at the margin of the area pellucida resulted in structures differentiated from the cells of the graft, whereas medially the graft organized host cells to form rows of somites. The results are discussed in terms of competence of graft and host mesenchyme and a positional signal from the node.

Environmental factors modulate the size and the secretory activity of the notochord. A study of the Golgi apparatus in avian embryos.

In this study we examined the Golgi apparatus of avian notochord transplants excised from 2-day-old (E2) chick embryos and grafted isochronically into a chick host either in a medial-ventral position, next to the host notochord, or in a superficial position under the ectoderm laterally or dorsally to the neural tube. The operated embryos were examined from E2 to E8. The diameters, the cytoplasmic vacuolization and the immunostained Golgi apparatus were identical between the endogenous and ventrally grafted notochords, as well as between host and superficially transplanted notochords when observed at E2. In contrast, from E4 to E8, the size of the notochords grafted dorsally or laterally to the neural tube significantly smaller than the host, while the cytoplasmic vacuolization and the degree of fragmentation of the Golgi apparatus were significantly less than in the host notochords. These results show that environmental and position-specific factors influence the developmental program and the secretory activity of the notochordal cells.

Control of dorsoventral patterning of somitic derivatives by notochord and floor plate.

We have examined the effect of implantation of a supernumerary notochord or floor plate on dorsoventral somitic organization. We show that notochord and floor plate are able to inhibit the differentiation of the dorsal somitic derivatives--i.e., axial muscles and dermis--thus converting the entire somite into cartilage, which normally arises only from its ventral part. We infer from these results that the dorsoventral patterning of somitic derivatives is controlled by signals provided by ventral axial structures.

A spinal cord fate map in the avian embryo: while regressing, Hensen's node lays down the notochord and floor plate thus joining the spinal cord lateral walls.

The spinal cord of thoracic, lumbar and caudal levels is derived from a region designated as the sinus rhomboidalis in the 6-somite-stage embryo. Using quail/chick grafts performed in ovo, we show the following. (1) The floor plate and notochord derive from a common population of cells, located in Hensen's node, which is equivalent to the chordoneural hinge (CNH) as it was defined at the tail bud stage. (2) The lateral walls and the roof of the neural tube originate caudally and laterally to Hensen's node, during the regression of which the basal plate anlage is bisected by floor plate tissue. (3) Primary and secondary neurulations involve similar morphogenetic movements but, in contrast to primary neurulation, extensive bilateral cell mixing is observed on the dorsal side of the region of secondary neurulation. (4) The posterior midline of the sinus rhomboidalis gives rise to somitic mesoderm and not to spinal cord. Moreover, mesodermal progenitors are spatially arranged along the rest of the primitive streak, more caudal cells giving rise to more lateral embryonic structures. Together with the results reported in our study of tail bud development (Catala, M., Teillet, M.-A. and Le Douarin, N.M. (1995). Mech. Dev. 51, 51-65), these results show that the mechanisms that preside at axial elongation from the 6-somite stage onwards are fundamentally similar during the complete process of neurulation.