Mutations in human SOX9 are associated with campomelic dysplasia (CD), characterised by skeletal malformation and XY sex reversal. During chondrogenesis in the mouse, Sox9 is co-expressed with Col2a1, the gene encoding type-II collagen, the major cartilage matrix protein. Col2a1 is therefore a candidate regulatory target of SOX9. Regulatory sequences required for chondrocyte-specific expression of the type-II collagen gene have been localized to conserved sequences in the first intron in rats, mice and humans. We show here that SOX9 protein binds specifically to sequences in the first intron of human COL2A1. Mutation of these sequences abolishes SOX9 binding and chondrocyte-specific expression of a COL2A1-driven reporter gene (COL2A1-lacZ) in transgenic mice. Furthermore, ectopic expression of Sox9 trans-activates both a COL2A1-driven reporter gene and the endogenous Col2a1 gene in transgenic mice. These results demonstrate that COL2A1 expression is directly regulated by SOX9 protein in vivo and implicate abnormal regulation of COL2A1 during, chondrogenesis as a cause of the skeletal abnormalities associated with campomelic dysplasia.

The developmental potency of cells in the proximal and distal regions of the epiblast of pre- and early-primitive-streak-stage mouse embryos was assessed by their differentiation in the host embryo following orthotopic and heterotopic cell transplantation. Normally, cells in the distal epiblast differentiate predominantly into neuroectoderm and surface ectoderm. However, when they were transplanted to proximal regions of the epiblast, distal epiblast cells behaved like proximal epiblast cells: they colonised the extraembryonic mesoderm and other mesodermal tissues in the posterior region of the host embryo. In addition, about 3.7% of the transplanted distal epiblast cells differentiated into primordial germ cells. This proportion is comparable to the 3.9% of orthotopically transplanted proximal epiblast cells that became primordial germ cells. When proximal epiblast cells were transplanted heterotopically to distal sites, their descendants were generally absent from the extraembryonic mesoderm and the germ cell population of the host embryo. Like cells in the distal epiblast, they mostly colonised the neural plate and surface ectoderm. This plasticity of cell fate suggests that the epiblast cells are not irreversibly allocated to any specific lineages, including the germ line. The adoption of developmental fate that is typical of the cell population at the site of transplantation suggests that the specification of cell lineages is subject to certain site-specific influences in the epiblast. Allocation of cells to the ectodermal and germ cell lineages may be subject to local tissue interactions and the restriction of morphogenetic tissue movement of different epiblast cell populations during gastrulation.

There are conflicting views on whether collagen X is a purely structural molecule, or regulates bone mineralization during endochondral ossification. Mutations in the human collagen α1(X) gene (COL10A1) in Schmid metaphyseal chondrodysplasia (SMCD) suggest a supportive role. But mouse collagen α1(X) gene (Col10a1) null mutants were previously reported to show no obvious phenotypic change. We have generated collagen X deficient mice, which shows that deficiency does have phenotypic consequences which partly resemble SMCD, such as abnormal trabecular bone architecture. In particular, the mutant mice develop coxa vara, a phenotypic change common in human SMCD. Other consequences of the mutation are reduction in thickness of growth plate resting zone and articular cartilage, altered bone content, and atypical distribution of matrix components within growth plate cartilage. We propose that collagen X plays a role in the normal distribution of matrix vesicles and proteoglycans within the growth plate matrix. Collagen X deficiency impacts on the supporting properties of the growth plate and the mineralization process, resulting in abnormal trabecular bone. This hypothesis would accommodate the previously conflicting views of the function of collagen X and of the molecular pathogenesis of SMCD.

The developmental potential of parthenogenetic embryonic stem (P-ES) cells was studied in teratomas and mouse chimaeras. Teratomas derived from P-ES cells contained a mixture of tissue types with variable proportions of specific tissues. Three of the eight P-ES cell lines analysed showed high proportions of striated muscle in teratomas, similar to teratomas from normal embryos or ES cell lines derived from fertilised embryos (F-ES cells). Our study also revealed that one P-ES cell line showed little lineage restriction in injection chimaeras. Descendants of the P-ES cells contributed to most tissues of chimaeric fetuses in patterns similar to F-ES cells. Normal colonisation of muscle, liver and pancreas was found in adult chimaeras. P-ES cells also showed similar haematopoietic differentiation and maturation as F-ES cells. However, extensive P-ES cell contribution was associated with a reduction in body size. These findings suggest that, while P-ES cells display more extensive developmental potential than the cells of parthenogenetic embryos from which they were derived, they only retained properties related to the presence of the maternal genome. To elucidate the molecular basis for the lack of lineage restriction during in vivo differentiation, the expression of four imprinted genes, H19, Igf2r, Igf2 and Snrpn was compared among five P-ES and two F-ES cell lines. Expression levels of these genes varied among the different ES cell lines, both in undifferentiated ES cells and in embryoid bodies.