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Requirement for Pbx1 in skeletal patterning and programming chondrocyte proliferation and differentiation

¹ Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, 650-723-5471, USA
² Nina Ireland Laboratory of Developmental Neurobiology, University of California San Francisco, San Francisco, CA, USA
³ Department of Biochemistry, The University of Hong Kong, Hong Kong, China
⁴ Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA

View larger version (46K): [in a new window]   Fig. 1. Targeted inactivation of Pbx1 and gross morphology of wild-type and mutant embryos. (A) Schematic representation of the mouse Pbx1 cDNA, genomic locus, targeting vector and mutated allele following homologous recombination. In the Pbx1 cDNA, sequences derived from exon 3 and the homeodomain (HD) are shown as black and gray boxes, respectively. Approximately 20 kb of the Pbx1 locus flanking exon 3 (black box) are depicted along with mapped restriction sites. The targeting construct carries a PGK-neo cassette, inserted into the unique NheI site of Pbx1 exon 3, and the HSV-tk gene, shown as solid white boxes. The transcriptional orientations of the Pbx1 arms of homology are opposite to that of the PGK-neo cassette. The 5' and 3' external probes used for Southern blot analyses are shown as solid black boxes below the mutated allele. Restriction enzyme sites: E, EcoRI; N, NheI; S, SspI; X, XhoI. XmaI and NotI sites are in the targeting vector; the SspI site (S) was introduced to facilitate diagnostic analysis. (B,C) Southern blot analysis of Pbx1 alleles. DNA from cell lines (B) or mouse tissues (C) was analyzed with probes and enzymes indicated beneath the panels. Wild-type (16 and 6.5 kb) and mutant (7.2 and 5.5 kb) Pbx1 alleles for XmaI and SspI digests, respectively, are indicated to the right of each panel. TL, ES cell line used for Pbx1 gene targeting; clones 38 and 176, targeted ES cells that passed the Pbx1 mutation through the germ line; MEFs, mouse embryonic fibroblasts. (D) Western blot analysis of Pbx1b expression. Protein extracts of E16 embryos were subjected to western blot analysis using a monoclonal antibody specific for the Pbx1b isoform. Genotypes determined by Southern blotting are listed at the top. Right lane contains in vitro translated Pbx1b. Migrations and sizes (kDa) of molecular mass standards are indicated to the right. (E,F) Gross morphology of wild-type and Pbx1-/- embryos at E13.5 (E) and E16 (F). Mutant embryos display massive subcutaneous edema (yellow arrowhead), pallor, slender thorax and abdomen, hypoplastic pinna (red arrowhead) and atypical hunched posture with abnormal orientation of both hind- and forelimbs (black arrowheads).

View larger version (126K): [in a new window]   Fig. 2. Pbx1 expression and skeletal structures in wild-type and mutant embryos. (A-D) Immunohistochemical analysis of Pbx1b localization visualized with DAB (brown staining) on tissues counter-stained with Hematoxylin. Diffuse nuclear Pbx1b expression is present in the epidermis and mesenchymal tissues of the embryo at E13.5 (A), most intensely in mesenchyme that is condensing to form rib (black arrowheads) and vertebral (red arrowheads) cartilage (B). Abundant Pbx1b expression in proliferating rib chondrocytes at E14.5 (C), which is nuclear (C, inset), is reduced to undetectable levels in hypertrophic chondrocytes at E16 when ossification is imminent (D). BA2, second branchial arch; vert, vertebrae. (E,F) Differential bone and cartilage staining of skeletal elements. E16 embryos were stained with Alcian Blue and Alizarin Red to visualize cartilage and bone, respectively, in the forming skeletal structures. Lateral views demonstrate agenesis of the ventral ribs, hypoplasia of the sternum and clavicles, together with rib malformations and fusions (red arrowhead) in the Pbx1-/- embryo (E, right). There is also thinning, compressions (black arrows) and fusions (red-tipped arrows) of the vertebral bodies and neural arches in the Pbx1-/- embryo (F, bottom). The styloid process and hyoid lesser horns (green arrows) are malformed, as are the structures of the occipital arch. Costal cartilage, Cc; clavicle, cv; exoccipital, EO; otic capsule, OC; ossified rib, orb; supraoccipital arch, SOA; sternum, stn. Asterisks indicate malformed structures.

View larger version (77K): [in a new window]   Fig. 3. Transformation of second branchial arch cartilages into structures resembling mandibular first arch cartilages in Pbx1-/- embryos as determined from anatomic, histological and molecular analyses. (A) Immunohistochemical analysis shows extensive Pbx1b expression in mesenchyme of the second branchial arch and first pharyngeal groove of E11 and E13.5 wild-type embryos (transverse section). Auricular pinna, AP; first branchial arch, BA1; second branchial arch, BA2; Meckel’s cartilage, MC; mandibular arch, MdA; maxillary arch, MxA; optic cup, opc; otic capsule, OC; first pharyngeal groove, PG1; styloid process, SP; spinal cord, spc; tongue, Tg. (B) Lateral views of wild-type (top) and mutant (bottom) E16 head skeleton. The lesser horns of the mutant are greatly elongated, resembling Meckel’s cartilage (or BA2 homologs of non-mammalian vertebrates). They extend from the hyoid body and end adjacent to the styloid process. The stapes is absent, and the styloid process is also dysmorphic, being shorter, thicker and having an ectopic flange (yellow arrowhead). Its shape resembles, in parts, a malleus. Proximocaudal madibular arch structures are slightly dysmorphic, as exemplified by the caudal border of the malleus. There is a small, ectopic cartilaginous spicule near the malleus (green arrowhead). (C) Inferior views of the wild-type (left) and mutant (right) head showing the branchial arch defects, in addition to malformations (red arrowhead) of the cranial base. Green and yellow arrowheads denote structures described in B. (D) Morphologies of dissected hyoid and mandible cartilages. Asterisks indicate malformed structures. Abbreviations: atlas, at; dentary, dnt; exoccipital, eo; greater horn of hyoid, gh; hyoid body, hb; incus, in; lesser horn of hyoid, lh; transformed lesser horn of hyoid bone, lh*; malleus, ma; Meckel’s cartilage, MC; nasal capsule, nc; otic capsule, OC; supra capsular fissura, scf; styloid process, sp. (E) Histology of H and E-stained transverse sections of wild-type and Pbx1-/- embryos at E16. The transformed and elongated lesser horns of the hyoid bone are indicated by an asterisk. greater horn of hyoid, gh; lesser horn of hyoid, lh; transformed lesser horn of hyoid bone, lh*; Meckel’s cartilage, MC; oropharynx, o. (F) Same as E, magnified 100x. The transformed lesser horn of the hyoid consists of tightly packed, proliferating chondrocytes. (G,H) Whole-mount in situ hybridization analysis of Lhx6 and dHAND expression in wild-type and Pbx1-/- embryos at E10.5.

View larger version (34K): [in a new window]   Fig. 4. Malformations of proximal but not distal appendicular skeletal structures in Pbx1-/- embryos. (A) Immunohistochemical analysis using anti-Pbx1b (Pbx1b) or anti-Meis (Meis) antibodies on transverse sections of limb buds from E11.5 wild-type and Pbx1-/- embryos. Diffuse nuclear expression (brown) of Pbx1b and Meis is detected in superimposable domains confined to the proximal limb buds of wild-type embryos. In limb buds of Pbx1-/- embryos, both nuclear and cytoplasmic expression of Pbx1b is absent, while Meis protein expression is unperturbed. (B) Skeletal structures in right forelimb of wild-type (left) and Pbx1-/- (right) E16 embryos. The morphologies of the limbs with associated scapulae and clavicles dissected free from embryos are shown in lateral views. In the Pbx1-/- embryo, there is hypoplasia of the superior scapular border (1), acromial spine and process (2), and coracoid process (3). The clavicle is shortened and attenuated (4). The humerus is shorter, distorted and thinned proximally; its head is fused to the glenoid cavity (5) and coracoid process (3) and continuous with the inferior border of the scapula (7). The cartilaginous core of the deltoid tuberosity is nonconjoined (6). The cartilages of the distal forelimb are spared. acromial process, ap; acromial spine, as; coracoid process, cp; clavicle, cv; deltoid tuberosity, dt; glenoid process, gf; humerus, h; humeral head, hh; manus, ms; radius, r; scapula, scp; ulna, u. (C) Lateral views show the morphology of the pelvic girdle, with malformed and rudimentary ilium (2), ischium (3) and pubis (4), in the Pbx1-/- hindlimb (right). The os coxae-femur articulation (1) is fused. The hindlimb malformation also affects the femur, which is shorter and distorted proximally (5) in the Pbx1-/- embryo, while the cartilaginous structures of the distal hindlimb are spared. acetabular fossa, af; femoral head, fh; femur, f; fibula, fl; greater trocanter, gt; ilium, il; pubis, pb; pes, ps; patella, pt; tibia, t.

View larger version (117K): [in a new window]   Fig. 5. Accelerated chondrocyte maturation in malformed skeletal structures of Pbx1-/- embryos. Histologic analysis shows H and E and von Kossa-stained transverse sections of wild-type and Pbx1-/- embryos. Aggregations of small chondrocytes are present in the ribs of wild-type (A,B) and Pbx1-/- (C,D) embryos at E13.5. In Pbx1-/- embryos, the center of the growing rib cartilage (green arrows) shows subtle morphologic features of more advanced chondrocyte maturation evidenced by the presence of more cells with small nuclei and large lipid droplets (black arrowheads). At E15.5, the Pbx1-/- rib (G,H) is prematurely comprised exclusively of hypertrophic chondrocytes, some clearly undergoing autolysis (red arrowhead, inset in H), and surrounded by extensive perichondral bone formation (blue arrows). In contrast, normal chondrocyte maturation appears to be present in the distal forelimbs of wild-type (I, J) and Pbx1-/- (K, L) embryos. At E 15.5, von Kossa staining shows the presence of extensive calcium deposits in Pbx1-/- rib cartilage (N), that are practically absent from wild-type cartilage (M). Magnifications: A,C,E,G,I,K (20x); B,D,F,H,J,L (40x); M, N and insets in F,H (100x).

View larger version (138K): [in a new window]   Fig. 6. Accelerated differentiation and diminished proliferation of chondrocytes in Pbx1-/- embryos, demonstrated by cell-type specific gene expression and cellular proliferation assays. (A-H) Immunohistochemistry was performed on serial sections of wild-type (left panels) and Pbx1-/- (right panels) cartilage at E15.5 using antibodies specific for markers of chondrocyte differentiation (A-F) and antibodies specific for cycling and dividing cells (G-J). Representative data are shown for embryos that were extensively sectioned and analyzed. (A,B) Collagen type II; (C,D) collagen type X; (E,F) stromolysin-1 (MMP3); (G,H) immunodetection of PCNA shows strong nuclear staining in numerous cells of wild-type but not Pbx1-/- rib cartilage at E15.5; (I,J) BrdU in vivo labelling shows a marked difference in the percentage of BrdU-positive nuclei (dark brown) between wild-type and Pbx1-/- rib cartilage at E13.5; (K) BrdU incorporation calculated as a percentage of BrdU-positive nuclei in rib cartilages at different developmental stages. Black bars, wild type; grey bars, Pbx1-/-. Percentage PCNA staining of wild-type (black) and Pbx1-/- (grey) at E15.5 is also shown. Bars represent means + s.d.

View larger version (98K): [in a new window]   Fig. 7. Appropriate expression of chondrogenic regulators at early days of embryonic skeletogenesis and precocious bone formation in Pbx1-/- embryos. (A-R) In situ hybridizations were performed on sections of wild-type and Pbx1-/- rib cartilage at E13.5 and 15.5. (A-L) Digoxigenin-labeled riboprobes specific for candidate chondrogenic regulators were utilized on frozen sections. (A-D) Ihh; (E-H) Sox9; (I-L) Fgfr3. (D,H,L) Expression of these genes was physiologically lost in Pbx1-/- rib cartilage at E15.5, when many more hypertrophic chondrocytes are present, most of which are undergoing accelerated autolysis and precocious mineralization, compared to wild type (C,G,K). Rib cartilage boundaries in Pbx1-/- embryos at E15.5 are indicated by dotted lines in D,H and L. (M,P) Bright-field photos of serial sections consecutive/adjacent to those where in situ hybridization was performed (N-R). 35S-labeled riboprobes for Col1a1, a marker of bone, and Col10a1, a marker of hypertrophic chondrocytes, were utilized on wax sections. In Pbx1-/- rib the presence of hypertrophic chondrocytes and scarce proliferating chondrocytes (P), is associated with advanced expression of Col1a1 in the perichondrium of the rib (Q), indicative of the forming bone collar (green arrow). Col10a1 expression is diminished in the middle of the mutant rib (R) presaging ossification and bone deposition. By contrast, in wild-type rib, proliferating chondrocytes (black arrow in M) are still present in large numbers and Col1a1 is not expressed (N). However, Col1a1 is expressed in the developing calvarial bones of both the wild-type and Pbx1-/- mutants (M and P: insets). Col10a1 is expressed at high levels in wild-type rib (O). Representative data are shown for embryos that were extensively sectioned and analyzed.

View larger version (44K): [in a new window]   Fig. 8. Functional domains and relationships of the Pbx1 and Hox genes in axial and appendicular skeletal morphogenesis. (A,B) Coloured lines indicate the anatomical regions affected by mutations in the Pbx1 or Hox genes (paralog groups 9-13 in the limb and 2-4 in the ectomesenchymal component of the branchial arches). In the embryonic limb, Meis protein expression is spatially coincident with Pbx1-dependent regions and unperturbed in a Pbx1 mutant background. B is adapted from Couly et al. (Couly et al., 1996). (C) Schemata depicting chondrocranial morphologies in wild-type, Pbx1-/- and Hoxa2-/- embryos at E16. The latter represents authors’ interpretation of Rijli et al. (Rijli et al., 1992). Although not phenocopies, both Pbx1-/- and Hoxa2-/- mutants exhibit mandibular arch-like morphologies in the hyoid arch (BA2). Dark blue indicates BA1 structures, yellow, wild-type BA2 structures, and red, mutant BA2 structures at E16. Lateral views, upper panels; basal views, lower panels. ala temporalis, at; greater horn of hyoid, gh; hyoid body, hb; incus, in; lesser horn of hyoid, lh; malleus, ma; Meckel’s cartilage, MC; styloid process, sp; stapes, st.