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First published online 30 March 2005
doi: 10.1242/dev.01811

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The homeobox transcription factor Barx2 regulates chondrogenesis during limb development

¹ The Neurosciences Institute 10640 John Jay Hopkins Drive, San Diego, CA 92121, USA
² Department of Neurobiology, The Scripps Research Institute 10550 North Torrey Pines Road, La Jolla, CA 92037, USA

View larger version (41K): [in a new window]   Fig. 7. Schematic of the intronic regulatory region of the Col2a1 gene. (A) Two overlapping segments of 468 and 309 bp have previously been shown to drive chondrocyte specific expression in cell cultures and in transgenic mice (Bell et al., 1997; Zhou et al., 1995). These regions both include a 48 bp element containing three HMG-binding sites (HMG1, HMG2 and HMG3). Two additional HMG motifs (HMG4, HMG5) were identified downstream of this element. Two conserved putative homeodomain binding sites (designated HBS1 and HBS2) were identified by comparison of human and mouse genes. Boxes indicate the sequences used as probes in this study. (B) Sequences of wild-type and mutant probes. The core of binding motifs containing the sequence ATTA is indicated in bold. Mutations are indicated in lower case. (C) Western blot analysis shows that Barx2 and Sox9 are expressed in E12.5 limb nuclear extract.

View larger version (91K): [in a new window]   Fig. 1. Expression of Barx2 and GDF5 during mouse embryogenesis and limb development. Barx2 mRNA expression at E11.5 (A) and E13.5 (B). Barx2 (C,E) and GDF5 (D,F) are expressed in a similar pattern in the prechondrogenic condensations and in the digital rays at E11.5 (C,D) and in phalangeal joints at E13.5 (E,F). ce, conjunctival epithelium; sgp, submandibular gland primordium; vib, vibrissa; ext.e, external ear; fl, forelimb; hl, hindlimb.

View larger version (96K): [in a new window]   Fig. 2. Barx2 (red) and collagen II (green) protein expression during limb development. At E11.0, Barx2 and collagen II are co-expressed in mesenchymal condensations (A,D). Barx2 expression precedes collagen II expression in the chondrogenic front (A, arrows). At E12.5, Barx2 and collagen II continue to be expressed along the developing digit. Barx2 expression is upregulated in the cell layer marking the joint interzone (B,E), whereas collagen II expression is downregulated in the developing joint. Barx2 and collagen II are co-expressed in the developing articular cartilage of carpal (C) and phalangeal (F) joints in newborn mice. (D) A higher magnification of A. (E) A higher magnification of B.

View larger version (33K): [in a new window]   Fig. 3. (A) Morpholino Barx2 antisense ODNs dramatically reduces nodule formation compare to untreated cultures or cultures treated with sense or random control ODNs. (B) Quantitation of Alcian Blue staining in micromass cultures shows that Barx2 antisense ODNs result in a 90% reduction of Alcian Blue staining; n, number of experiments.

View larger version (17K): [in a new window]   Fig. 4. Barx2 increases cell aggregation in Ca+2-independent (A) and Ca+2-dependant (B) aggregation assays.

View larger version (41K): [in a new window]   Fig. 5. Retroviral expression of Barx2 and HD-BBR-C proteins in limb bud micromass cultures increases chondrogenic differentiation. (A) Three different Barx2 proteins encoded in retroviral vectors: the full-length Barx2 protein (Barx2) that contains the homeodomain (HD), Barx basic region (BBR) and an acidic activation domain (AD); the HD-BBR protein contains only the DNA-binding region of Barx2; and the HD-BBR-C protein, which contains the DNA-binding region as well as the C-terminal activation domain. (B) Expression of Barx2 or HD-BBR-C proteins, but not HD-BBR protein, induced chondrogenesis in limb bud micromass cultures as indicated by Alcian Blue staining. (C) Quantitative analyses of Alcian Blue incorporation in retroviral infection experiments; n, number of experiments. (D) Micromass cultures prepared from proximal limb bud mesenchyme show no response to overexpression of control, Barx2, HD-BBR-C or HD-BBR proteins, whereas distal limb bud cultures show a fivefold increase in Alcian Blue staining upon infection with the Barx2 retroviral construct; n, number of experiments.

View larger version (32K): [in a new window]   Fig. 6. Retroviral expression of Barx2 in limb bud micromass cultures increases Col2a1 gene expression. (A) Barx2-retrovirus infection induced approximately fivefold increase in Col2a1 mRNA relative to control infections as measured by real time PCR. n, number of experiments. (B) Expression of HD-BBR-C construct increases collagen II protein expression in micromass cultures.

View larger version (69K): [in a new window]   Fig. 8. Barx2 binds to HBS elements within the Col2a intronic enhancer. (A) In vitro translated Barx2 proteins (HD-BBRC) were tested for binding to the B1 and B2 probes in gel mobility-shift experiments. HD-BBRC protein formed complexes with both the B1 and B2 probes that were completely blocked by anti-Barx2 antibody. HD-BBRC did not bind to B1 and B2 probes in which ATTA sequence was mutated (B1 HBS1 mut and B2 HBS2 mut). No complexes were formed with control pcDNA3 extract. HD-BBRC formed complexes of similar intensity with the B1 and B1 HMG5 mut probes. (B) Binding of the B1,B2 and S1 probes to nuclear extracts from E12.5 embryonic limbs. The B1 probe formed stronger complexes than either the B2 probe or the B1 HMG5 mut probe. Addition of Barx2 antibodies reduced binding to both probes, indicating that Barx2 is present in the complex. Addition of Sox9 antibodies reduced binding to the probes that contain HMG motifs (S1 and B1), suggesting that Sox9 is present in these complexes. Sox9 antibody did not affect binding to the B2 probe. (C) Mutation of HBS motifs in the B1 and B2 probes (B1 HBS1 mut and B2 HBS2 mut) abolished their binding to limb nuclear extracts, whereas, mutation of the HMG motif in the B1 probe (B1 HMG5 mut) reduced binding, suggesting that the HBS and HMG sites can interact.

View larger version (63K): [in a new window]   Fig. 9. Barx2 and Sox9 bind to endogenous Col2a1 enhancer during chondrogenic differentiation. (A) Crosslinked chromatin from limb mesenchymal cells and C3H10T1/2 was precipitated with Barx2 or nonspecific antibody and analyzed by PCR using primers spanning the Col2a1 enhancer. Lanes 1-6, amplification of Col2a1 enhancer: 1, no Barx2 antibody added; 2, amplification of Col2a1 enhancer from input DNA; 3, immunoprecipitation with non-specific antibody; 4, immunoprecipitation with Barx2 antibody; 5, immunoprecipitation with normal rabbit IgG; 6, immunoprecipitation with Barx2 antibody. (B) Barx2 has stronger association with Col2a1 enhancer after the BMP treatment. Equivalent amount of crosslinked chromatin from treated with BMP and untreated limb mesenchymal cells were immunoprecipitated with Barx2 antibody and analyzed by PCR using primers spanning Col2a1 regulatory region. Gapdh control confirming that equivalent amounts of chromatin were used in each ChIP assay. (C,D) During chondrogenic differentiation the Col2a1 enhancer is occupied by both Barx2 (C) and Sox9 (D). D1 cells were simultaneously transfected with Myc-tagged Barx2 and Flag-tagged Sox9 expression vectors and cultured under differentiation conditions. Approximately equivalent amounts of crosslinked chromatin were immunoprecipitated in parallel with antibodies specific for Myc, flag, Phospho-Sox9 or normal rabbit IgG, and Col2a1 enhancer region was amplified by PCR. (C) Lane 1, no antibody; 2, input DNA; 3, rabbit IgG; 4, Myc antibody. (D) Lane 1, negative control; 2, immunoprecipitation with Flag antibody; 3, with Phospho-Sox9 antibody; 4, positive control, input DNA. Arrows indicate Col2a1 product.

View larger version (98K): [in a new window]   Fig. 10. Implantation of GDF5 (B,E,H) and BMP4 (C,F,J) beads (*) into the distal regions of E11.0-12.5 limbs results in ectopic expression of Barx2. mRNA and protein expression were visualized using in situ hybridization (A-C,G,J) and immunohistochemistry (D-F,H), respectively. Nuclei were stained with Oli-green. Arrows indicate ectopic Barx2 expression. Black arrows indicate endogenous Barx2 expression; white arrows indicate ectopic Barx2 expression; en, endogenous expression.

View larger version (27K): [in a new window]   Fig. 11. A model of limb bud skeletal development showing molecules that may influence the various stages of cellular morphogenesis and differentiation. We propose that GDF5, Barx2 and Sox9 act at two stages of skeletal development: cellular aggregation and chondrogenic differentiation. By contrast, BMPs are most probably involved only in the second step of skeletal development – chondrogenic differentiation. Barx2 expression in the limb bud mesenchymal cells first promotes cellular aggregation by inducing the expression of certain cell-adhesion molecules. Later, Barx2 promotes chondrogenic differentiation via the regulation of the expression of collagen II and other genes. The nature of the relationship between Barx2 and Sox9 remains to be determined.