Osteo-chondroprogenitor cells are derived from Sox9 expressing precursors

Abstract

The transcription factor Sox9 is expressed in all chondroprogenitors and has an essential role in chondrogenesis. Sox9 is also expressed in other tissues, including central nervous system, neural crest, intestine, pancreas, testis, and endocardial cushions, and plays a crucial role in cell proliferation and differentiation in several of these tissues. To determine the cell fate of Sox9-expressing cells during mouse embryogenesis, we generated mice in which a Cre recombinase gene preceded by an internal ribosome entry site was inserted into the 3' untranslated region of the Sox9 gene (Sox9-Cre knock-in). In the developing skeleton, Sox9 was expressed before Runx2, an early osteoblast marker gene. Cell fate mapping by using Sox9-Cre;ROSA26 reporter (R26R) mice revealed that Sox9-expressing limb bud mesenchymal cells gave rise to both chondrocytes and osteoblasts. Furthermore, a mutant in which the Osterix gene was inactivated in Sox9-expressing cells exhibited a lack of endochondral and intramembranous ossification and a lack of mature osteoblasts comparable with Osterix-null mutants. In addition, Sox9-expressing limb bud mesenchymal cells also contributed to tendon and synovium formation. By using Sox9-Cre;R26R mice, we also were able to systematically follow Sox9-expressing cells from embryonic day 8.0 to 17.0. Our results showed that Sox9-expressing cells contributed to the formation of all cell types of the spinal cord, epithelium of the intestine, pancreas, and mesenchyme of the testis. Thus, our results strongly suggest that all osteo-chondroprogenitor cells, as well as progenitors in a variety of tissues, are derived from Sox9-expressing precursors during mouse embryogenesis.

Fig. 1.

Comparison of Sox9, Runx2, and Osx expression during embryonic development with lacZ expression in Sox9-Cre;R26R compound heterozygous embryos. (A) Expression of Sox9, Runx2, and Osx during limb bud development. β-gal activity in limb buds of Sox9 heterozygous (Sox9^lacZ/wt) (19), Runx2 heterozygous (Runx2^lacZ/wt) (14), and Osx heterozygous (Osx^lacZ/wt) (15) embryos from E9.5 to E16.5. The arrows indicate the expression of Sox9 in E10.0. The arrowheads indicate the expression of Runx2 in E10.5 and E11.5. (B) Sections of limb buds in Sox9-Cre;R26R compound heterozygous embryos from E10.5 to E17.0 stained with whole-mount β-gal staining. c, resting and proliferating chondrocytes; h, hypertrophic chondrocytes; p, periosteum; ob, osteoblasts; S, synovium; T, tendon. (C) Sections of limb buds in Sox9 heterozygous (Sox9^lacZ/wt) embryos in E13.5 stained with whole-mount β-gal staining. (D) Sections of limb buds in Prx1-Cre;R26R compound heterozygous embryos in E16.5 with whole-mount β-gal staining. The abbreviations are the same as in B.

Fig. 2.

Targeting strategy for conditional inactivation of the Osx gene. (A) Structure of the genomic Osx locus, targeting vector, and the homologous recombined allele. Exons are depicted as filled boxes, and intronic sequences are shown as solid lines. The FRT-flanked PGK-neo bpA and the IRES-EGFP-pA cassettes are depicted as open boxes. DNA fragments revealed in Southern analysis are indicated as arrows with the restriction enzymes and the probes. RI, EcoRI; N, NheI; B, BamHI; X, XbaI; RV, EcoRV. (B) Southern blot analysis of genomic DNA. (C–E) Analysis of skeletal phenotypes in Osx ^flox/lacZ;Sox9-Cre knock-in mice. Skeletons in E17.5 embryos stained with alcian blue followed by alizarin red showed no mineralization of bones in Osx-null and the conditional Osx-null mutants (C). Histological analysis of humerus stained by alcian blue, hematoxylin, and Treosin revealed no bone trabeculae or mineralization in Osx-null and the conditional Osx-null mutants in E17.5 (D). Expression of Oc mRNA was not detected in humerus of E17.5 Osx-null and the conditional Osx-null mutants (E).

Fig. 3.

Comparison of the pattern of cells expressing Sox9 in E17.0 intestine, testis, spinal cord, and pancreas with the pattern of cells that are derived from Sox9-expressing cells in these embryos. (A) Contribution of Sox9-expressing cells during mouse embryogenesis. β-gal activity in Sox9-Cre;R26R compound heterozygous embryos in E8.0 and E10.5. (B) Comparison between the expression of Sox9, assessed by immunohistochemistry (intestine and testis) of wild-type embryos or by β-gal staining (spinal cord and pancreas) of Sox9 heterozygous (Sox9^lacZ/wt) embryos, and the distribution of cells derived from Sox9-expressing cells in E17.0 Sox9-Cre;R26R embryos. The arrowhead indicates enteric neurons.

Fig. 4.

Model of establishment of mesenchymal cell lineages during limb bud development. Prx1-expressing undifferentiated limb bud mesenchyme gives rise to Sox9-expressing osteo-chondroprogenitors, fibroblasts, tendon cells, and synovial cells. These Sox9-expressing osteo-chondroprogenitors form mesenchymal condensations, in which Runx2-expressing osteogenic cells are separated from Sox9-expressing chondrocytes.