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First published online 1 August 2007
doi: 10.1242/dev.002709

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Regulation of skeletogenic differentiation in cranial dermal bone

Arhat Abzhanov^1,*, Stephen J. Rodda², Andrew P. McMahon² and Clifford J. Tabin^1,

¹ Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
² Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA.

View larger version (43K): [in this window] [in a new window]   Fig. 1. Origin and contribution of dermal bone in cranial skeletal development. (A) Contribution of cranial neural crest cells (orange) and mesoderm (grey) to the cartilage (blue) and bone (red) components of the avian skull (see also Noden, 1986; Couly et al., 1993). Notice the heavy contribution of neural crest to the cranial dermal bones. (B) Bone mineralization in E9-E13 chick embryonic heads as revealed with alizarin red stain. dnt, dentary bone; ep, epidermis; fnt, frontal bone; nt, neural tube; pmx, pre-maxillary bone.

View larger version (113K): [in this window] [in a new window]   Fig. 2. Expression domains of skeletogenic markers in the developing dentary bone. (A-J) HH39 chick embryos; (K-O) 16.5 dpc mouse embryos. Expression of Coll II (A) and Coll IX (B) is detected in cells of both Meckel's cartilage and dentary bone. (C) Expression of Sox9 can be seen in cells of Meckel's cartilage but not in the dentary bone. (D) Expression of Runx2 is limited to the cells of the dentary bone. (E) Osteopontin is expressed in cells of dentary bone but is restricted to the more mature cells. (F) Bmp2,(G) Bmp4, (H) Ihh and (I) strong expression of PTHrP-R are detected in the dentary bone. (J) H&E staining of the dentary bone and Meckel's cartilage from the embryo in A-I. In mice, (K) Coll II, (L) Runx2 and (M) Osx are expressed in the peripheral cells, whereas (N) the more mature osteoblast marker Opn is expressed in cells surrounded by mineralized matrix (H&E stain; O). dnt, dentary bone; MC, Meckel's cartilage. Scale bars: 200 µm.

View larger version (82K): [in this window] [in a new window]   Fig. 3. Fluorescent double in situ hybridizations showing expression of Coll IX, Coll II and Opn relative to each other and to the domains of Runx2, PTHrP-R, Bmp4 and Bmpr1b expression. (A-C) Direct comparison of the Opn and Coll IX expression domains. (C) There are cells that express Coll IX only (red; broad arrows), Opn only (green; arrowheads) or that co-express Opn and Coll IX (yellow; narrow arrows). (D-F,J-O) Considerable overlap is detected between Opn and Bmp4 (D-F), between Runx2 and Coll IX (J-L), and between Coll IX and PTHrP-R (M-O). (G-I) Expression domains of Opn (green) and Runx2 (Cbfa1; red) have very little or no overlap. The same is true for the expression domains of Coll II versus BspII (S-U) and Ihh versus Opn (V-X). Some overlap is detected for the expression domains of Bmpr1b versus Ptc1 (P-R). Scale bar: 200 µm.

View larger version (62K): [in this window] [in a new window]   Fig. 4. Analysis of dissociated frontal bone cells of HH39 chick embryos reveals distinct cell populations defined by the expression of skeletogenic markers. (A-D) Four populations of cells were identified: cells expressing only Coll IX (arrowheads in C), only Opn (broad arrows in C), both Coll IX and Opn (narrow arrows in C), and cells expressing neither marker (arrow in D). FISH revealed a similar situation with Opn and Runx2 (E-H), with Runx2 and Coll IX (I-L), and with PTHrP-R and Coll IX (M-P). (M-P) Despite the high degree of co-expression between Coll IX and PTHrP-R that was observed, some slides showed many cells expressing neither marker (arrows, P), suggesting that many of these cells were early osteoblasts not expressing Coll IX. (Q) Analysis of Col2::Cre3xRosa26::lacZ dentary bones with both alizarin red and ß-galactosidase staining revealed a considerable contribution of Coll II-expressing cells to the developing dentary bone. (R-T) Similar analysis of ß-galactosidase and H&E stainings of the developing frontal bone in 17.5 dpc Col2::Cre3xRosa26::lacZ embryos showed many ß-galactosidase-positive cells within this intramembranous bone. Db, dentary bone; FB, frontal bone. Scale bars: 50 µm.

View larger version (90K): [in this window] [in a new window]   Fig. 5. Analysis of chick frontal bone development with the RISAP replication-incompetent virus. (A-F) Viral injections into condensations in areas `I' and `II' (dark red spots) labeled large portions of the developing frontal bone (A,D), with cells contributing to the anterior (B) and posterior (E) halves of the frontal bone, respectively. Expansion of the two condensations results in a relatively sharp boundary between the anterior and posterior parts, but no suture forms (arrowheads; C,F). Notice that cells from the anterior condensation contribute to the more posterior part (C). (G-L) Skeletal phenotypes in frontal bones after condensations were infected with the RCAS-based viral constructs at E6. Bone and cartilage mineralized structures were revealed with alizarin red (bone) and Alcian blue (cartilage) histological stains. (G) Normal ossification pattern of the posterior frontal bone in E15 embryos. The top, side and back of the skull are covered with membranous bone. Infection with RCAS::noggin led to a loss of bone mineralization in the posterior (H) parts of the frontal bone. By contrast, infection of the posterior (I) and anterior (L) frontal bone with RCAS::Bmp4 led to a loss of mineralized bone material and its replacement with cartilage. (J) RCAS::Ihh misexpression resulted in a significant decrease of frontal bone mineralization, a phenotype that was similar to that of RCAS::PTHrP misexpression (K). Scale bars: 0.7 cm in B,E; 2 mm in G.

View larger version (81K): [in this window] [in a new window]   Fig. 6. Molecular and histological analysis of RCAS::Ihh and RCAS::PTHrP-infected frontal bones. (A-P) Analysis of the RCAS::Ihh-infected frontal bones with molecular markers and H&E staining shows that infected bone failed to produce differentiated osteoblasts (I,J) and to mineralize (M-P). Framed areas in M and N are shown amplified in O and P, respectively. (Q-F1) Misexpression of RCAS::PTHrP resulted in a very similar phenotype, with a loss of Opn expression in the infected skeletal element (Y,Z). Framed areas in C1 and D1 are shown amplified in E1 and F1, respectively. Scale bars: 1 mm.

View larger version (152K): [in this window] [in a new window]   Fig. 7. Analysis of 18.5 dpc Ihh-/- mutant mouse embryos. The skull of wild-type (WT) embryos (A-C) is larger than that of mutant siblings (D-F). Notice the presence of the ossified basal cranial bones in the mutant. Arrows indicate the leading edge of the mineralized dermal calvarial bone. Expression of the early osteoblastic markers, such as Runx2 (G) and Osx (H), is severely reduced in the Ihh-/- condition (K and L, respectively). Red brackets indicate area occupied by undifferentiated proliferating osteoblasts. The expression of Opn (I,M), a marker for more mature osteoblasts, is unaltered or slightly expanded in the mutant condition. Green brackets indicate area occupied by differentiated mature osteoblasts. (J,N) H&E stainings of wild-type and mutant dentary bones, respectively. bas, basisphenoid; boc, basioccipital; dnt, dentary; exo, exoccipital; fnt, frontal bone; int, interparietal; max, maxillary; MC, Meckel's cartilage; nas, nasal; pal, palatal; par, parietal; pte, pterygoid; o.t., os tympanicum. Scale bars: 1 mm in A; 200 µm in G.

View larger version (51K): [in this window] [in a new window]   Fig. 8. Regulation of osteoblastic differentiation in cranial dermal bone. (A) Table summarizing the expression pattern data for the developing dentary and frontal bones. Four major cell types could be distinguished based on skeletogenic markers: proliferating cells at the periphery of the osteogenic front, cells expressing only Runx2, preosteoblastic progenitors and surrounding cells expressing Runx2, Coll II, Coll IX, Ptc1, Gli1, PTHrP-R and Bmp4. (1) These cells differentiate into the CLO (chondrocyte-like osteoblast) cells expressing a unique combination of Opn, Coll II and Coll IX (but not Sox9 or aggrecan) as well as Ihh, Ptc1, Gli1, Bmp4 and PTHrP. As these cells develop into mature osteoblasts, they downregulate Coll II and Coll IX expression and, out of the analyzed markers, they express only Opn, BspII and Ihh. (2) Alternatively, some cells could differentiate directly into mature osteoblasts. (B) During craniofacial development, mesencephalic cranial neural crest cells migrate to populate mesenchyme of the future face and skull. Cells of the early cranial skeletogenic condensations depend on BMP2/4/7 activities to form preosteoblastic progenitors, whereas high levels of BMP2 and/or BMP4 alone induced a chondrogenic fate. Differentiation into the chondrocyte-like osteoblasts is regulated by both IHH and PTHrP activities.