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First published online 24 July 2008
doi: 10.1242/dev.023788

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Embryonic origin and Hox status determine progenitor cell fate during adult bone regeneration

Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, Stanford, CA 94305, USA.

View larger version (123K): [in this window] [in a new window]   Fig. 1. Cranial and appendicular skeletons are derived from a different embryonic origin. (A,B) In an adult Wnt1Cre::Z/EG mouse, mandibular osteocytes and periosteal cells are GFP-positive (neural crest-derived) (A), whereas the adjacent muscle and endothelium (arrowhead) is β-galactosidase-positive (mesoderm-derived) (B). (C,D) Secondary cartilage underlying the skull bone is GFP-positive (C), whereas the hematopoietic system residing in the bone marrow cavity is β-galactosidase-positive (D). (E) The Schwann cells surrounding a peripheral nerve in the hind limb are GFP-positive (arrowheads). (F) Osteocytes and periosteal cells in the tibia stain positive for β-galactosidase. bm, bone marrow; ca, cartilage; cb, cortical bone; mn, mandible; mu, muscle; po, periosteum; tib, tibia. Scale bar: 200 µm in A-C,E; 100 µm in D,F.

View larger version (85K): [in this window] [in a new window]   Fig. 2. Skeletal defects heal through recruitment of progenitor cells of their own origin. (A) Pentachrome staining of the mandibular injury site shows bone matrix deposition (blue-green) at post-surgical day 7. (B) GFP-staining reveals that the majority of the cells in the injury site are immunopositive, indicating their neural crest origin. (C) X-Gal staining (a label for mesoderm) is detectable in the surrounding muscle, but is absent in the defect. (D) After 7 days, the tibial defect exhibits a similar amount of osteoid as the mandible. (E) The bony defect is void of GFP-positive neural crest-derived cells. (F) Instead, the defect is occupied by β-galactosidase-positive cells. cb, cortical bone (outlined); is, injury site. Scale bar: 200 µm.

View larger version (79K): [in this window] [in a new window]   Fig. 3. Bioluminescence imaging demonstrates that cell grafts survive, proliferate and differentiate in vivo. (A) IVIS image of control injury showing no bioluminescence after luciferin injection. (B-E) Time course after L2G85 cell transplantation shows an initial increase of luciferase expression in the injury site, which slowly decreases over time. (F) GFP antibody staining of the injury site at post-surgical day 7 reveals immunopositive osteoblasts and osteocytes. Scale bar: 200 µm in F.

View larger version (113K): [in this window] [in a new window]   Fig. 4. Embryonic origin of the graft influences cell fate decision in a transplantation assay. (A) Pentachrome staining shows that the homotopic transplantation of tibial periosteum into a tibial defect results in robust bone formation through intramembranous ossification at post-surgical day 10. (B) GFP antibody staining reveals that the majority of the regenerate is derived from the grafted periosteum. (C,D) Similarly, the homotopic graft of mandibular periosteum into a mandibular injury induces direct differentiation into bone (C) and, again, the majority of the regenerate is derived from the GFP-positive graft (D). (E) Placement of neural crest-derived periosteum into a mesoderm-derived injury site results in intramembranous bone formation. (F) GFP immunohistochemistry confirmed that the grafted cells are actively committed to the healing response. (G) However, when tibial periosteum is transplanted into a mandible, the cells undergo endochondral ossification. (H) High magnification of the cartilage condensation reveals that the cells are undergoing hypertrophy. (I) Histomorphometry of the four grafting scenarios; see Materials and methods for details. *, # and + indicate significant differences; P0.01. (J,K) Safranin O/Fast Green staining and GFP immunohistochemistry show the spatial correlation of chondrogenesis and the graft. ca, cartilage; is, injury site; mn, mandible; tib, tibia. Scale bar: 200 µm in A,C,E; 100 µm in B,D,F,J,K; 400 µm in G; 50 µm in H.

View larger version (82K): [in this window] [in a new window]   Fig. 5. Neural crest-derived and mesoderm-derived periosteal cells have distinct proliferation and osteogenic differentiation potentials. (A) Skeletal progenitor cells from the mandibular periosteum and GFP-positive tibial periosteum were co-cultured for 0, 3, 5 and 7 days; cell nuclei were labeled with Hoechst and viewed using fluorescent imaging. (B) Quantification of the co-culture showed a linear increase in the number of GFP-positive mesoderm-derived cells and only a minor increase in the number of neural crest-derived cells. (C) BrdU incorporation assay at 3, 5 and 7 days showed higher proliferation rates for mesoderm-derived cells at all time points. (D,E) Alizarin Red staining of mesoderm-derived and neural crest-derived skeletal progenitor cell cultures after 10, 12 and 14 days in osteogenic differentiation media. (F) Quantification of Alizarin Red mineralization showed a statistically significant increase in the amount of mineralized matrix in neural crest-derived samples. (G) qRT-PCR performed on RNA isolated from cell populations after 4 days in vitro. Neural crest-derived cells showed an increase in the expression of Runx2 and Col1a. (H) qRT-PCR performed on RNA isolated from cell populations after 10 days in vitro demonstrated that neural crest-derived cells showed an increase in the expression of all osteogenic markers, including Runx2, osteopontin (op), Col1a and osteocalcin (oc). *P0.01.

View larger version (84K): [in this window] [in a new window]   Fig. 6. Hox-negative neural crest skeletal progenitor cells assume a Hox-positive status when placed into a Hox-positive environment. (A,B) The adult tibial periosteum expresses Hoxa13 and Hoxa11. (C) At post-surgical day 3, the tibial injury site is occupied by Hoxa11-expressing cells. (D,E) By contrast, the mandibular periosteum is devoid of Hoxa11 expression (D), and injury does not induce Hoxa11 expression (E). (F,G) Hox-positive tibial periosteum maintains its expression when transplanted into a Hox-negative mandibular injury site. (H) GFP antibody staining labels Hox-negative mandibular periosteum in a tibial injury site. (I) Adjacent section shows Hoxa11 expression in the transplanted mandibular periosteum (dashed line). (J,K) Skeletal progenitor cells derived from β-actin GFP tibial periosteum express GFP (J) and Hoxa11 (K). (L,M) When these GFP-positive, Hox-positive cells were co-cultured with Hox-negative neural crest-derived cells (L) the formerly Hox-negative mandibular cells begin to express Hoxa11 (M). bm, bone marrow; ca, cartilage; mn, mandible; po, periosteum; tib, tibia. Scale bar: 50 µm in A,B,D,F,G; 100 µm in E; 200 µm in C,H,I.

View larger version (88K): [in this window] [in a new window]   Fig. 7. Scheme of interactions between embryonic origin and Hox status during adult bone regeneration in mouse. (A,B) In a homotopic tibial or mandibular graft, both embryonic origin and Hox code are equal (+), which leads to osteogenic differentiation of osteoprogenitor cells. (C) When mandibular periosteum is transplanted into a tibial injury, the embryonic origin of the graft and recipient are different (-). Hox expression, however, is the same in graft and recipient because the grafted cells, which are initially Hox-negative, turn on Hox expression when placed into the Hox-positive environment. (D) In the mandibular heterotopic graft, the embryonic origin of graft and recipient are different, and the Hox status of the graft and host remain unequal, which results in chondrogenic differentiation of the osteoprogenitor cells. mn, mandible; tib, tibia.

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