[Molecular aspects of vertebral chondrogenesis]. / Aspects moléculaires de la chondrogenèse vertébrale.

In this article, we review the data showing that dorso-ventral polarization of the vertebral cartilage involves two distinct molecular pathways, acting sequentially during development. The first one is mediated by the notochord and the ventral part of the neural tube, by the action of the Sonic Hedghehog protein (SHH): it directs the formation of the cartilaginous pieces located in a deep position within the embryo. The second one is responsible for the differentiation of the dorsal parts of the vertebra, differentiating under the ectoderm; it is initiated by the roof plate of the neural tube, by the action of the secreted protein BMP4. Those two pathways cannot be exchanged, rather they are antagonists. These data allow us to draw a new model for vertebral chondrogenesis.

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.

Two domains in vertebral development: antagonistic regulation by SHH and BMP4 proteins.

It has previously been shown that the notochord grafted laterally to the neural tube enhances the differentiation of the vertebral cartilage at the expense of the derivatives of the dermomyotome. In contrast, the dorsomedial graft of a notochord inhibits cartilage differentiation of the dorsal part of the vertebra carrying the spinous process. Cartilage differentiation is preceded by the expression of transcription factors of the Pax family (Pax1/Pax9) in the ventrolateral domain and of the Msx family in the dorsal domain. The proliferation and differentiation of Msx-expressing cells in the dorsal precartilaginous domain of the vertebra are stimulated by BMP4, which acts upstream of Msx genes. It has previously been shown that the SHH protein arising from the notochord (and floor plate) is necessary for the survival and further development of Pax1/Pax9-expressing sclerotomal cells. We show here that SHH acts antagonistically to BMP4. SHH-producing cells grafted dorsally to the neural tube at E2 inhibit expression of Bmp4 and Msx genes and also inhibits the differentiation of the spinous process. We present a model that accounts for cartilage differentiation in the vertebra.

The role of bone morphogenetic proteins in vertebral development.

This study first shows a striking parallel between the expression patterns of the Bmp4, Msx1 and Msx2 genes in the lateral ridges of the neural plate before neural tube closure and later on, in the dorsal neural tube and superficial midline ectoderm. We have previously shown that the spinous process of the vertebra is formed from Msx1- and 2-expressing mesenchyme and that the dorsal neural tube can induce the differentiation of subcutaneous cartilage from the somitic mesenchyme. We show here that mouse BMP4- or human BMP2-producing cells grafted dorsally to the neural tube at E2 or E3 increase considerably the amount of Msx-expressing mesenchymal cells which are normally recruited from the somite to form the spinous process of the vertebra. Later on, the dorsal part of the vertebra is enlarged, resulting in vertebral fusion and, in some cases (e.g. grafts made at E3), in the formation of a 'giant' spinous process-like structure dorsally. In strong contrast, BMP-producing cells grafted laterally to the neural tube at E2 exerted a negative effect on the expression of Pax1 and Pax3 genes in the somitic mesenchyme, which then turned on Msx genes. Moreover, sclerotomal cell growth and differentiation into cartilage were then inhibited. Dorsalization of the neural tube, manifested by expression of Msx and Pax3 genes in the basal plate contacting the BMP-producing cells, was also observed. In conclusion, this study demonstrates that differentiation of the ventrolateral and dorsal parts of the vertebral cartilage is controlled by different molecular mechanisms. The former develops under the influence of signals arising from the floor plate-notochord complex. These signals inhibit the development of dorsal subcutaneous cartilage forming the spinous process, which requires the influence of BMP4 to differentiate.

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.

Heterogeneity in the development of the vertebra.

Vertebrae are derived from the sclerotomal moities of the somites. Sclerotomal cells migrate ventrally to surround the notochord, where they form the vertebral body, and dorsolaterally to form the neural arch, which is dorsally closed by the spinous process. Precursor cells of the spinous process as well as superficial ectoderm and roof plate express homeobox genes of the Msh family from embryonic day 2 (E2) to E6. The notochord has been shown to be responsible for the dorsoventral polarization of the somites and for the induction of sclerotomal cells into cartilage. Indeed, supernumerary notochord grafted laterally to the neural tube induces the conversion of the entire somite into cartilage. We report here that a mediodorsal graft of notochord prevents the sclerotomal cells migrating dorsally to the roof plate from differentiating into cartilage. Under these experimental conditions, expression of Msx genes is abolished. We thus demonstrate that cartilaginous, differentiation is differentially controlled in the dorsal part of the vertebra (spinous process) and in the neural arch and vertebral body.

[Quox 8, homeogene implicated in the establishment of dorso-ventral organization in vertebrates]. / Quox 8, un homéogène impliqué dans l'établissement de l'organisation dorso-ventrale des vertébrés.

Quox 8 (= quail Msx 2) is an homeo box-containing gene of the msh family; we have been interested in the possible role of this gene in the patterning of somitic tissues around the neural tube. By the combined use of embryonic manipulations and in situ hybridization, we evidence that in embryonic day 2-6 avian embryos, Quox 8 expression in the dorsal mesenchyme depends (1) on the normal dorso-ventral polarization of the neural tube; (2) on tissue interactions between the dorsal neural tube and the overlying ectoderm. The somitic mesenchyme expressing Quox 8 gives rise to the spinous process of the vertebra. Experimental inhibition of Quox 8 expression results in the absence of the spinous process. Thus we conclude that Quox 8 is a step in the cascade of tissue interactions between the neural tube, the ectoderm and the mesenchyme, resulting in the patterning of the dorsal-most part of the vertebra.

A role for Quox-8 in the establishment of the dorsoventral pattern during vertebrate development.

We have been interested in the possible involvement of the gene Quox-8, a member of the Hox-7/msh family of homeobox-containing genes in the patterning of the neural tube and the somitic-derived mesoderm. We demonstrate that in the embryonic day 2-6 avian embryo, the expression of Quox-8 in the roof plate depends upon (i) the normal dorsoventral orientation of the neural primordium and (ii) interactions between the dorsal neural tube and the superficial ectoderm. We also show that Quox-8 is expressed in the dorsal mesenchyme located between the neural tube and the superficial ectoderm that yields the spinous processes of the vertebrae. Experimental inhibition of Quox-8 expression in these areas of the dorsal mesenchyme results in the failure of its differentiation into vertebral cartilage. We conclude, therefore, that this gene is involved in the patterning of vertebral structures through a cascade of tissue interactions primarily occurring between the dorsal neural tube and the overlying ectoderm.