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First published online 14 January 2009
doi: 10.1242/dev.029355

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Hand2 controls osteoblast differentiation in the branchial arch by inhibiting DNA binding of Runx2

¹ Department of Molecular Biology, and Faculty of Dentistry, McGill University, Montreal, Quebec H3A 2B2, Canada.
² Department of Anatomy and Cell Biology, and Faculty of Dentistry, McGill University, Montreal, Quebec H3A 2B2, Canada.
³ Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA.
⁴ Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA.
⁵ Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA.

View larger version (54K): [in this window] [in a new window]   Fig. 1. Abnormal intramembranous ossification in Hand2 mutant mice. (A) Bone staining of P1 mandibles from wild-type (a,c,e) and Hand2BA/BA (b,d,f) mice. (a,b) Ventral views of the mandible. The mandible in Hand2BA/BA is smaller and malformed with multiple sites lacking mineralized bone. Note the wider angle of the mutant mandible. (c,d) Lateral views of the mandibular body. Ectopic boney processes (arrows) and mineralized bone voids (arrowheads) are formed in mutants. (e,f) Lateral views of the mandibular processes. Angular process (agp) is hypoplastic in the mutants. i, lower incisor; crp, coronoid process; cdp, condylar process. Beneath is summarized the hemimandible length (mm) from wild-type and Hand2BA/BA mice (n=6 per genotype) at P1. **P<0.001. (B) Skeletal preparations of mandibles at E14.5 (a,b), E15.5 (c,d) and E16.5 (e-h) from wild-type (a,c,e,g) and Hand2BA/BA (b,d,f,h) embryos. Interruption of Meckel's cartilage is evident in Hand2BA/BA at E14.5 (arrows in b). At E15.5 and E16.5 the mutant mandible is smaller and develops multiple mineralization voids (arrowheads). Brackets indicate mandibular length. (g,h) A lingual view of the mandible. mc, Meckel's cartilage. (C) Micro-CT of hemimandible from wild-type and Hand2BA/BA mice at P1. The mineralized mandible in Hand2BA/BA is smaller, deformed, and has a large void of mineralized tissue (arrowhead). (D) Mineralized tissue volume determined by micro-CT of a hemimandible from wild-type (control) and Hand2BA/BA mice (n=4 per genotype) at P1. The mandibles in Hand2BA/BA are less mineralized than in the control.

View larger version (85K): [in this window] [in a new window]   Fig. 2. Accelerated osteoblast differentiation in Hand2BA/BA embryos. (A) Evaluation of osteoblast differentiation in E12 (a,b) and E13.5 (c,d) mandibles from wild-type (a,c) and Hand2BA/BA (b,d) embryos by alkaline phosphatase (ALP) activity. ALP-positive mandibular primordium (mp, purple and arrows) and the mandibular bone (md and arrows) are expanded in Hand2BA/BA as compared with wild-type embryos. t, tongue. (B) qPCR analysis of Alp and Bsp transcripts in mandibles at E12. Differences observed between wild-type and Hand2BA/BA mandibles were not significant. (C) Detection of osteoblast differentiation markers in wild-type (a-c) and Hand2BA/BA (d-f) mandibles at E13.5. ALP activity (a,d), detected by whole-mount ALP staining, is upregulated in the mandible of Hand2BA/BA embryos as compared with the wild type (arrows), although activity in primordium of temporal bone (tb) is unchanged. Expression of Bsp (b,e) and osteocalcin (c,f), detected by whole-mount in situ hybridization, is expanded in Hand2BA/BA. (D,F) Upregulation of Alp, Bsp, osteocalcin and Runx2 expression in mandibles of Hand2BA/BA embryos at E13.5 as determined by qPCR. *P=0.031 for Alp, 0.043 for Bsp, 0.014 for osteocalcin and 0.0065 for Runx2.(E) The Runx2 expression domain is expanded in E13.5 Hand2BA/BA mandible (c) as compared with the wild type (a) by section in situ hybridization. H&E staining of the adjacent sections is shown (b,d). Scale bars: 200 µm.

View larger version (82K): [in this window] [in a new window]   Fig. 3. Hand2 inhibits osteoblast differentiation. (A) Whole-mount in situ hybridization analysis of wild-type mouse embryos for Runx2 (a), Hand1 (b) and Hand2 (c), and β-galactosidase staining of Hand2-lacZ (d) at E12.5. Runx2, Hand1 and Hand2 are colocalized in the mandibular arch. (B) Section in situ hybridization analysis of Runx2 (a,c,e) and Hand2 (b,d,f) in the branchial arch at E11.5 (a-d) and E12.5 (e,f) from wild-type (a,b,e,f) and Hand2BA/BA (c,d) embryos. At E11.5, Runx2 and Hand2 are partially colocalized in the mandibular primordium (mp) along the medial aspect in the wild-type embryo (arrow in a,b), whereas Hand2 is not expressed in Runx2-expressing cells in Hand2BA/BA embryos (c,d). At E12.5, the overlapping expression domain of Runx2 and Hand2 is decreased in this region of the mandible. mc, Meckel's cartilage. Scale bars: 100 µm. (C) ALP activity of ROS17/2.8 cells stably transfected with empty vector or Myc-tagged Hand2 expression vector. Cells were grown to confluence (day 0) and cultured in the indicated medium for 2, 5 or 16 days, then examined for ALP activity. Note the strong inhibition of the initiation of osteoblast differentiation by Hand2. (D) qPCR analysis of ROS17/2.8 cells cultured for 5 days in β-MEM as indicated in C. Expression of Alp, osteocalcin and Runx2 is significantly reduced in Hand2-overexpressing cells. *P=0.0009 for Alp and osteocalcin, 0.013 for Runx2.

View larger version (29K): [in this window] [in a new window]   Fig. 4. Hand proteins inhibit Runx2 transactivation activity. (A) COS cells were transiently cotransfected with p6OSE2-luc reporter and the indicated amounts of expression vectors. Hand2 and Hand1 inhibited Runx2 transactivation in a dose-dependent manner. The data represent the mean ± s.e.m. (B) p6OSE2-luc reporter construct and Runx2 expression vector were transiently transfected alone or together with expression vectors encoding the indicated constructs. Hand2 and Hand1, Hand2 (F119P), or Hand2 (HLH) inhibited Runx2-dependent transactivation. Paraxis and capsulin did not inhibit the Runx2 activity. Twist1 showed mild inhibition of Runx2 activity. Luciferase data are shown as percentage of Runx2 activation (normalized to 1.0). The data represent the mean ± s.e.m. (C) Co-IP assays. COS cells were transiently transfected with the indicated expression vectors. Flag-tagged Hand1 or Hand2 was immunoprecipitated (IP) from cell lysates with a monoclonal anti-Flag antibody, and co-immunoprecipitated Hand2 or Hand2 (F119P) was detected with anti-Myc antibody (top panel). Five percent of input samples were immunoblotted with an anti-Myc (middle panel) or anti-Flag (lower panel) antibody. The asterisk indicates a non-specific band.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Hand proteins interact directly with Runx2. (A) Co-IP assays. Flag-tagged Runx2 and Myc-tagged Hand1 or Hand2 were used as in Fig. 4C. (B) In vitro binding assays. Glutathione beads conjugated with GST-Runx2, or GST alone, were incubated with MBP-Myc-Hand1 or MBP-Myc-Hand2. Eluates from the beads were immunoblotted with anti-Myc antibody. GST-Runx2 and MBP-Hand2 or MBP-Hand1 showed direct interaction. (C) Subcellular localization of Hand proteins and Runx2. Myc-tagged Hand2 or Hand1, and Flag-tagged Runx2 were detected by immunostaining. Runx2, Hand1 and Hand2 colocalize in the nucleus. (D) p6OSE2-luc reporter was transiently transfected along with Runx2, Hand2 and increasing amounts of CBP or Fhl2 expression vector. The data represent the mean ± s.e.m. (E) Relative luciferase activity of Runx2 with Hand1 or Hand2 in the presence of TSA or ethanol (EtOH) control. The data represent the mean ± s.e.m.

View larger version (38K): [in this window] [in a new window]   Fig. 6. Inhibition of Runx2 DNA-binding function by the N-terminal domain of Hand2. (A) Schematic of Runx2 functional domains and deletion mutants. All mutants contain an N-terminal Flag-epitope tag. Runt, Runt DNA-binding domain; QA, glutamine/alanine repeats; PST, proline/serine/threonine-rich region; NLS, nuclear localization signal; N, nucleus. (B) Mapping the Hand2-interaction domain of Runx2 by Co-IP assays. COS cells were transfected with expression plasmids encoding Flag-tagged Runx2 deletion mutants (as shown in A) and Myc-tagged Hand2. Flag-Runx2 was immunoprecipitated with an anti-Flag antibody, followed by western blot analysis to detect Myc-Hand2 binding. (C) Schematic of Hand2 functional domains and deletion mutants. All mutants contained an N-terminal Myc-epitope tag. b, basic DNA-binding domain; HLH, helix-loop-helix; NLS, nuclear localization signal; N, nucleus; C, cytoplasm, N.D., not determined. Asterisks indicate point mutations. (D) Mapping the Runx2-interaction domain of Hand2 by Co-IP assays. COS cells were transfected with expression vectors encoding Myc-tagged Hand2 deletion mutants (as shown in C) and Flag-tagged Runx2. Myc-Hand2 was immunoprecipitated with an anti-Myc antibody, followed by western blot analysis to detect Flag-Runx2 binding. (E) Subcellular localization of wild-type and mutant Hand2 proteins. Hand2 proteins expressed in COS cells were examined by immunocytochemistry. (F) Identification of the Runx2-inhibition domain of Hand2. p6OSE2-luc reporter was transiently transfected along with Runx2 expression vectors and the various Hand2 deletion mutants (as shown in C). Fold activity measured in the absence of Hand2 was normalized to 1.0. The data represent the mean ± s.e.m. (G) ChIP assays were performed using soluble chromatin prepared from ROS17/2.8 cells stably transfected with Hand2, Hand2 (32-59) or Hand2 (1-90) vector, as indicated. Chromatin was immunoprecipitated with non-immune rabbit IgG or Runx2 antibodies as indicated. Precipitated genomic DNA was analyzed by PCR using primers for the osteocalcin promoter, which contains a Runx2-binding OSE2 site. An input control, in which PCR amplification was performed prior to immunoprecipitation, is also shown. Hand2 specifically perturbs the association of Runx2 with its target sequence on the osteocalcin promoter, whereas Hand2 (1-90) (a) or Hand2 (32-59) (b) shows no effect.

View larger version (28K): [in this window] [in a new window]   Fig. 7. Molecular mechanism of Runx2 regulation by Hand proteins. (A) Hand2 directly binds Runx2 and blocks its transcriptional activity. Hand2 binds the Runt DNA-binding domain of Runx2, thereby preventing access to its target sequence in the promoters of bone-specific genes. N, N-terminal; C, C-terminal; HLH, helix-loop-helix; OSE2, Runx2 binding site. (B) A model for regulation of osteoblast differentiation in the branchial arch by Hand2. Hand2 is required in early development of postmigratory neural crest cells and their derivatives, and subsequently during osteogenesis in the first branchial arch. Function of Runx2, which is necessary and sufficient for osteoblast differentiation and which controls the rate of bone formation by osteoblasts, is transiently inhibited by Hand2.

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