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doi: 10.1242/10.1242/dev.00240

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Pax1 and Pax9 activate Bapx1 to induce chondrogenic differentiation in the sclerotome

Isabel Rodrigo1,*, Robert E. Hill2, Rudi Balling3, Andrea Münsterberg4 and Kenji Imai1,{dagger}

1 GSF-National Research Center for Environment and Health, Institute of Developmental Genetics, Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany
2 MRC Human Genetics Unit, Western General Hospital, Crewe Rd, Edinburgh EH4 2XU, UK
3 German Research Centre for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany
4 University of East Anglia, School of Biological Sciences, Norwich NR4 7TJ, UK
* Present address: Instituto Cajal, CSIC, Dr Arce 37, 28002 Madrid, Spain



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  		Fig. 7. Pax1 and Pax9 bind to regulatory sequences of the Bapx1 promoter. (A) PIP analysis of an 8550 bp mouse Bapx1 genome sequence when compared with an 8600 bp human BAPX1 sequence. Exons are outlined in black and coding parts are gray. Numbers at the top indicate nucleotide positions as defined in the text, and they correspond to 5' ends of intervals included in the series of deletion constructs used in the transactivation assay. The interval between -880 and -748 is outlined in green. PIP scores in % are shown on the right, with 50% identity at the bottom level. Note the presence of several CNS segments outside the coding region. Many of them are located in the interval between -880 and +109. (B) Sequence of the interval between -880 and -731. The different oligonucleotides (S1, S2, B4, B5 and S4) employed in EMSA assays are indicated by lines. Sequence in green (-880 to -748) corresponds to the segment included in the plasmid p0.9Bp-luc, but not in the plasmid p0.7Bp-luc. A potential Pax6-binding site predicted by TFSEARCH and MatInspector is in the region of S1 and indicated in red. (C-F) Results of EMSA experiments. The indicated labeled oligonucleotides (Probe, marked with an asterisk) were incubated with the in vitro translated protein (Prot.) Pax1, Pax9 or luciferase as a control (C), separated and visualized as described in the Materials and Methods. The specific Pax1 (black arrows) and Pax9 (gray arrow in F) complexes are indicated. (C) Pax1 binds to oligonucleotide B4. Cold oligonucleotides e5-5 or e5-3 were added at 250-fold (lanes 2, 4) or 500-fold (lanes 3, 5) molar excess, as indicated. e5-5 specifically competes with B4 for Pax1 binding, whereas mutated e5-3 does not. (D) Specific inhibition of Pax1-B4 complex formation by anti-Pax1 antibody. The indicated antibodies were incubated in the binding reactions. {alpha}P1, anti-Pax1 goat antibody; {alpha}C, anti-mouse goat antibody, used as control. The anti-Pax1 antibody abrogates the formation of the larger complex. (E) Competition experiments employing a 250-fold (lanes 2, 4, 6) or 500-fold (lanes 3, 5, 7) molar excess of cold oligonucleotides B4, S1 or S2. Note that oligonucleotides B4 and S1 compete with B4 for Pax1 binding, whereas oligonucleotide S2 does not. (F) Binding of Pax1 and Pax9 to oligonucleotide S1. S1 or S2 labeled oligonucleotides were incubated with Pax1, Pax9 or luciferase control protein as indicated. Both Pax1 and Pax9 interact with S1 oligonucleotide, but not with S2 oligonucleotide.

 


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  		Fig. 1. Expression of sclerotomal genes in Pax1;Pax9 mutant embryos. Whole-mount in situ hybridization for Meox1 (A,B; E10.5), Meox2 (C,D; E10.5), Nkx3-1 (E,F; E9.5) and Bapx1 (G,H; E11.5) on wild type (w.t.; A,C,E,G) or Pax1-/-, Pax9-/- (B,D,F,H) mouse embryos.

 


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  		Fig. 2. Absence of Bapx1 expression in the sclerotome of Pax1;Pax9-deficient embryos. In situ hybridization for Bapx1 on sagittal (A,C) and transverse (B,D) sections of wild type (w.t.; A,B) or Pax1-/-, Pax9-/- (C,D) E11.5 mouse embryos at the lumbar level. The arrows indicate the notochord (n). Note that in the mutant embryo (C,D) loose mesenchyme of sclerotomal cells around the notochord lack Bapx1 expression.

 


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  		Fig. 3. Bapx1 expression level depends on the Pax1/Pax9 gene dosage. Whole-mount in situ hybridization for Bapx1 on E9.5 (A-D) or E10.5 (E-H) mouse embryos. Genotypes are indicated on top of each panel. w.t., wild type. Note the decrease in Bapx1 staining intensity, as fewer functional copies of Pax1 or Pax9 are present.

 


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  		Fig. 4. Pax1 induces Bapx1 expression and chondrogenesis in explants of chick PSM. Explants of PSM isolated from stage HH10 chick embryos were infected with the control RCAS-A-AP retrovirus (AP; A, lanes 1,3; B, lane 1) or with the RCAS-A-Pax1 retrovirus overexpressing mouse Pax1 (A, lanes 2,4; B, lane 2), and cultured in the absence (A, lanes 1,2; B) or the presence (A, lanes 3,4) of 500 ng/ml Shh. After 5 days of culture, explants were harvested and analyzed by RT-PCR. The PCR products for chick Bapx1, aggrecan, Gapdh and mouse Pax1 are shown.

 


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  		Fig. 5. Pax1 and Pax9 can transactivate Bapx1 promoter. (A) Scheme of the Bapx1 genomic structure and of the p5.3Bp-luc reporter plasmid. The boxes indicate exons of Bapx1 (top) or the luciferase (luc) reporter (bottom). The coding regions of Bapx1 are shown by the black boxes, together with the first ATG codon (position +277) and the stop codon (TGA) in exon 2. The p5.3Bp-luc plasmid contains 5.4-kb genomic sequences from the Bapx1 promoter region and a part of 5' UTR (-5285/+109) upstream of the luciferase reporter. (B) Promoter activities of the p5.3Bp-luc construct alone or co-transfected with the indicated amounts of expression plasmids for Pax1 (pPax1) or Pax9 (pPax9), or both. Numbers inside or above the bars indicate time fold induction with respect to the basal p5.3Bp-luc promoter activity. Lines in the bars indicate s.d.

 


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  		Fig. 6. Pax1 and Pax9 transactivation activity on different Bapx1 promoter deletion constructs. (A) Bapx1 promoter deletion plasmids used in transient transfection assays. (B) Luciferase activity driven by the Bapx1 promoter deletion constructs or control vector (pGL3-Basic), alone or co-transfected with the indicated amounts of expression plasmids for Pax1 (pPax1) or Pax9 (pPax9), or both. Numbers inside or above the bars indicate the time fold induction with respect to the basal activity of the same construct in the absence of transfected Pax1 and Pax9. Lines in the bars indicate s.d.

 


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  		Fig. 8. Model for regulatory pathways involved in sclerotome differentiation. Ovals indicate genes with a proposed role in axial skeletal development. See text for details.

 

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