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.

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.

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.

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.

Dual origin of the floor plate in the avian embryo.

Molecular analysis carried out on quail-chick chimeras, in which quail Hensen's node was substituted for its chick counterpart at the five- to six-somite stage (ss), showed that the floor plate of the avian neural tube is composed of distinct areas: (1) a median one (medial floor plate or MFP) derived from Hensen's node and characterised by the same gene expression pattern as the node cells (i.e. expression of HNF3beta and Shh to the exclusion of genes early expressed in the neural ectoderm such as CSox1); and (2) lateral regions that are differentiated from the neuralised ectoderm (CSox1 positive) and form the lateral floor plate (LFP). LFP cells are induced by the MFP to express HNF3beta transiently, Shh continuously and other floor-plate characteristic genes such as NETRIN: In contrast to MFP cells, LFP cells also express neural markers such as Nkx2.2 and Sim1. This pattern of avian floor-plate development presents some similarities to floor-plate formation in zebrafish embryos. We also demonstrate that, although MFP and LFP have different embryonic origins in normal development, one can experimentally obtain a complete floor plate in the neural epithelium by the inductive action of either a notochord or a MFP. The competence of the neuroepithelium to respond to notochord or MFP signals is restricted to a short time window, as only the posterior-most region of the neural plate of embryos younger than 15 ss is able to differentiate a complete floor plate comprising MFP and LFP. Moreover, MFP differentiation requires between 4 and 5 days of exposure to the inducing tissues. Under the same conditions LFP and SHH-producing cells only induce LFP-type cells. These results show that the capacity to induce a complete floor plate is restricted to node-derived tissues and probably involves a still unknown factor that is not SHH, the latter being able to induce only LFP characteristics in neuralised epithelium.

Axial progenitors with extensive potency are localised to the mouse chordoneural hinge.

Elongation of the mouse anteroposterior axis depends on a small population of progenitors initially located in the primitive streak and later in the tail bud. Gene expression and lineage tracing have shown that there are many features common to these progenitor tissues throughout axial elongation. However, the identity and location of the progenitors is unclear. We show by lineage tracing that the descendants of 8.5 d.p.c. node and anterior primitive streak which remain in the tail bud are located in distinct territories: (1) ventral node descendants are located in the widened posterior end of the notochord; and (2) descendants of anterior streak are located in both the tail bud mesoderm, and in the posterior end of the neurectoderm. We show that cells from the posterior neurectoderm are fated to give rise to mesoderm even after posterior neuropore closure. The posterior end of the notochord, together with the ventral neurectoderm above it, is thus topologically equivalent to the chordoneural hinge region defined in Xenopus and chick. A stem cell model has been proposed for progenitors of two of the axial tissues, the myotome and spinal cord. Because it was possible that labelled cells in the tail bud represented stem cells, tail bud mesoderm and chordoneural hinge were grafted to 8.5 d.p.c. primitive streak to compare their developmental potency. This revealed that cells from the bulk of the tail bud mesoderm are disadvantaged in such heterochronic grafts from incorporating into the axis and even when they do so, they tend to contribute to short stretches of somites suggesting that tail bud mesoderm is restricted in potency. By contrast, cells from the chordoneural hinge of up to 12.5 d.p.c. embryos contribute efficiently to regions of the axis formed after grafting to 8.5 d.p.c. embryos, and also repopulate the tail bud. These cells were additionally capable of serial passage through three successive generations of embryos in culture without apparent loss of potency. This potential for self-renewal in chordoneural hinge cells strongly suggests that stem cells are located in this region.

Cell interactions underlying notochord induction and formation in the chick embryo.

The development of the notochord in the chick is traditionally associated with Hensen's node (the avian equivalent of the organizer). However, recent evidence has shown that two areas outside the node (called the inducer and responder) are capable of interacting after ablation of Hensen's node to form a notochord. It was not clear from these studies what effect (if any) signals from these areas had on normal notochord formation. A third area, the postnodal region, may also contribute to notochord formation, although this has also been questioned. Using transection and grafting experiments, we have evaluated the timing and cellular interactions involved in notochord induction and formation in the chick embryo. Our results indicate that the rostral primitive streak, including the node, is not required for formation of the notochord in rostral blastoderm isolates transected at stages 3a/b. In addition, neither the postnodal region nor the inducer is required for the induction and formation of the most rostral notochordal cells. However, inclusion of the inducer results in considerable elongation of the notochord in this experimental paradigm. Our results also demonstrate that the responder per se is not required for notochord formation, provided that at least the inducer and postnodal region are present, although in the absence of the responder, formation of the notochord occurs far less frequently. We also show that the node is not specified to form notochord until stage 4 and concomitant with this, the inducer loses its ability to induce notochord from the responder. The coincident timing of these changes in the node and inducer suggests that notochord specification and the activity of the inducer are regulated through a negative feedback loop. We propose a model relating our results to the induction of head and trunk organizer activity in the node.

Anti-apoptotic role of Sonic hedgehog protein at the early stages of nervous system organogenesis.

In vertebrates the neural tube, like most of the embryonic organs, shows discreet areas of programmed cell death at several stages during development. In the chick embryo, cell death is dramatically increased in the developing nervous system and other tissues when the midline cells, notochord and floor plate, are prevented from forming by excision of the axial-paraxial hinge (APH), i.e. caudal Hensen's node and rostral primitive streak, at the 6-somite stage ( Charrier, J. B., Teillet, M.-A., Lapointe, F. and Le Douarin, N. M. (1999). Development 126, 4771-4783). In this paper we demonstrate that one day after APH excision, when dramatic apoptosis is already present in the neural tube, the latter can be rescued from death by grafting a notochord or a floor plate fragment in its vicinity. The neural tube can also be recovered by transplanting it into a stage-matched chick embryo having one of these structures. In addition, cells engineered to produce Sonic hedgehog protein (SHH) can mimic the effect of the notochord and floor plate cells in in situ grafts and transplantation experiments. SHH can thus counteract a built-in cell death program and thereby contribute to organ morphogenesis, in particular in the central nervous system.

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.

Specification of the hypaxial musculature.

During development of the amniote embryo, the dorsolateral territory of the somite is destined to give rise to the hypaxial skeletal musculature. To study the mechanisms that lead to the formation of this musculature, we cloned the chick Lbx1 gene that is specific to prospective hypaxial myoblasts at occipital, cervical and limb levels. Using this gene as a marker, we characterised the anatomical structures that produce the signals necessary for the specification of the hypaxial musculature by ablating them or transplanting them to ectopic locations in the chick embryo. In addition, we inserted BMP4 soaked beads medial to the somite. Our data suggest that lateralising signals from intermediate and lateral mesoderm have to synergise with dorsalising signals from the surface ectoderm to induce the formation of the hypaxial musculature. However, the lateralising function of the lateral mesoderm can only in part be mimicked by BMP4.

A role for the roof plate and its resident TGFbeta-related proteins in neuronal patterning in the dorsal spinal cord.

Distinct neuronal cell types are generated at characteristic times and positions in the dorsal horn of the spinal cord. We provide evidence that the identity and pattern of generation of dorsal neurons depend initially on BMP-mediated signals that derive from the epidermal ectoderm and induce dorsal midline cells of the roof plate. Roof plate cells provide a secondary source of TGFbeta-related signals that are required for the generation of distinct classes of dorsal interneurons. These inductive interactions involve both qualitative and quantitative differences in signaling by TGFbeta-related factors and temporal changes in the response of neural progenitor cells.

Inhibitory effects of ventral signals on the development of Brn-3.0-expressing neurons in the dorsal spinal cord.

Brn-3.0, a POU-domain transcription factor, is expressed in specific postmitotic neurons in the dorsal part of the neural tube which are among the first spinal cord neurons to appear in development. In the mature spinal cord, the Brn-3.0 cells form a numerous population of scattered neurons in the intermediate spinal gray. Ablation of the notochord in chick embryos extends the domain of Brn-3.0 expression into the ventral neural tube, while ectopic grafts of notochord tissue suppress Brn-3.0 expression. The notochord effects on Brn-3.0 expression are reproduced in vivo by the implantation of a local source of recombinant Shh protein. The down-regulation of Brn-3.0 expression in the dorsal spinal cord by the notochord and Shh contrasts with the known inductive effects of these ventral signals on the approximately simultaneous development of the spinal motor neurons. In cultured explants of neural plate from the region of the presumptive spinal cord, Brn-3.0 neurons develop in the absence of surface ectoderm and ventral midline tissue, suggesting that the Brn-3.0 phenotype may represent a "default" developmental pathway for early spinal cord neurons. Together these results advance the understanding of the mechanism of the generation of neuronal diversity in the developing vertebrate CNS.

Coordination of early neural tube development by BDNF/trkB.

Neurotrophins signal through members of the trk family of tyrosine kinase receptors and are known to regulate several neuronal properties. Although initially characterized by their ability to prevent naturally occurring cell death of subsets of neurons during development, neurotrophins can also regulate the proliferation and differentiation of precursor cells. Here we report a novel involvement of neurotrophins in early development of the neural tube. We demonstrate that a functional trkB receptor is expressed by motor neuron progenitors in the ventral neural tube and that treatment of ventral neural tube explants with the trkB ligand Brain-Derived Neurotrophic Factor (BDNF) leads to a significant increase in the number of motor neurons. The only BDNF expression detectable at this stage is by a subset of ventrally projecting interneurons in the dorsal neural tube; ablating this region in vivo leads to a reduction of motor neuron numbers. This loss can be prevented by simultaneous treatment with BDNF. We propose that BDNF produced by dorsal interneurons stimulates proliferation and/or differentiation of motor neuron progenitors after anterograde axonal transport and release in proximity to the trkB-expressing motor neuron precursors, thereby coordinating development between dorsal and ventral regions of the neural tube.

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.

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.

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.

Fibroblast growth factor receptor 1 in skeletal and heart muscle cells: expression during early avian development and regulation after notochord transplantation.

Basic fibroblast growth factor (bFGF, FGF-2) mediates several biological functions during embryonic development. With regard to skeletal muscle formation, it has been suggested that FGF-2 is involved in the growth and differentiation of myogenic precursor cells. To identify the FGF-responsive cells we studied the expression of FGF receptor type I (FGFR-1) during early embryonic development of the chick. FGFR-1 immunoreactivity is present at all stages examined (embryonic day [E] 2-E5). Expression of FGFR-1 is found in the somite myotome, limb bud muscle cells, eye and tongue muscle cells, and myocardium. Transplantation of an additional notochord into the paraxial mesoderm, which prevents the formation of a myotome, reveals the absence of FGFR-1 immunoreactivity on the operated side. The distinct expression pattern of FGFR-1 in migrating and differentiating muscle cells indicates that in addition to the stimulation of proliferation of myoblasts, FGF-2 exerts other (nonmitogenic) effects on postmitotic myocytes.

Determination of neuroepithelial cell fate: induction of the oligodendrocyte lineage by ventral midline cells and sonic hedgehog.

Near the floor plate of the embryonic neural tube there is a group of neuroepithelial precursor cells that are specialized for production of the oligodendrocyte lineage. We performed experiments to test whether specification of these neuroepithelial oligodendrocyte precursors, like other ventral neural cell types, depends on signals from the notochord and/or floor plate. We analyzed heterozygous Danforth's short tail (Sd/+) mutant mice, which lack a notochord and floor plate in caudal regions of the neural tube, and found that oligodendrocyte precursors did not appear at the ventricular surface where there was no floor plate. Moreover, oligodendrocytes did not develop in explant cultures of Sd/+ spinal cord in the absence of a floor plate. When a second notochord was grafted into an ectopic position dorsolateral to the endogenous notochord of a chicken embryo, an additional floor plate was induced along with an ectopic focus of oligodendrocyte precursors at the ventricular surface. Oligodendrocytes developed in explants of intermediate neural tube only when they were cocultured with fragments of notochord or in the presence of purified Sonic hedgehog (Shh) protein. Thus, signals from the notochord/floor plate, possibly involving Shh, are necessary and sufficient to induce the development of ventrally derived oligodendroglia. These signals appear to act by specifying the future fate(s) of neuroepithelial cells at the ventricular surface rather than by influencing the proliferation or differentiation of prespecified progenitor cells in the parenchyma of the cord.