Ihha induces hybrid cartilage-bone cells during zebrafish jawbone regeneration

Abstract

The healing of bone often involves a cartilage intermediate, yet how such cartilage is induced and utilized during repair is not fully understood. By studying a model of large-scale bone regeneration in the lower jaw of adult zebrafish, we show that chondrocytes are crucial for generating thick bone during repair. During jawbone regeneration, we find that chondrocytes co-express genes associated with osteoblast differentiation and produce extensive mineralization, which is in marked contrast to the behavior of chondrocytes during facial skeletal development. We also identify the likely source of repair chondrocytes as a population of Runx2(+)/Sp7(-) cells that emanate from the periosteum, a tissue that normally contributes only osteoblasts during homeostasis. Analysis of Indian hedgehog homolog a (ihha) mutants shows that the ability of periosteal cells to generate cartilage in response to injury depends on a repair-specific role of Ihha in the induction as opposed to the proliferation of chondrocytes. The large-scale regeneration of the zebrafish jawbone thus employs a cartilage differentiation program distinct from that seen during development, with the bone-forming potential of repair chondrocytes potentially due to their derivation from osteogenic cells in the periosteum.

Keywords: Bone regeneration; Chondrocyte; Chondroid bone; Ihha; Jaw; Osteoblast; Zebrafish.

Fig. 1.

Regeneration of the lower jawbone in adult zebrafish. (A) Whole-mount views of adult zebrafish heads before and after resection stained with Alizarin Red and Alcian Blue to label bone and cartilage. Arrows show resection sites. (B) Dissected lower jaws show the time course of cartilage and bone formation during regeneration. Arrow denotes the repair cartilage that has contracted somewhat during mounting. Arrowheads indicate regeneration of the anguloarticular prominence. (C) Qualitative assessment of cartilage and bone formation during lower jaw regeneration in individual animals. The y-axis shows the amount of cartilage/bone in the lesion from none (0) to full spanning (5). (D) H&E staining on sections of the lower jawbone before and after resection. An extensive cartilage callus (yellow arrowheads) is seen at 10 dpr, including at the anterior cut site (blue arrow) devoid of Meckel's cartilage (m). Dashed lines show resection sites. (E) Bone µCT images show ventral views of lower jawbones from un-injured (uninj) and regenerated animals. Arrowheads indicate resection sites. See also Movie 1. Scale bars: 1 mm in A,B and 100 μm in D.

Fig. 2.

Co-expression of chondrocyte and osteoblast genes in repair cartilage. (A) Colorimetric in situ hybridization shows gene expression in the cartilage callus anterior to the cut site of the lower jawbone. The chondrocyte markers sox9a and col2a1a are seen in the cartilage callus by 6 dpr, with a peak of expression at 8 dpr. Expression of the hypertrophic chondrocyte marker col10a1 begins at 8 dpr. The osteoblast markers runx2b and col1a1a are expressed in mesenchyme and early cartilage at 6 dpr and beyond. By 10 dpr, expression of the osteoblast marker spp1 is evident in the cartilage callus. (B) Two-color fluorescent in situ hybridization shows co-expression of col2a1a with col1a1a or runx2b at 10 dpr. Dashed lines show resection sites and magnified regions (white boxes) demonstrate that individual cells co-express both markers. Hoechst labels nuclei in blue. (C) Summary of the time course of expression within the cartilage callus. Scale bars: 100 μm.

Fig. 3.

Mineralization and osteoblastic maturation of repair chondrocytes. (A) Trichrome staining at 8 dpr reveals the presence of collagen-rich osteoid-like material surrounding individual chondrocytes of the regenerating jaw. Inset shows a magnified view of several chondrocytes. Note the similar appearance of collagen staining (blue) between the unresected jawbone (arrow) and the mineralizing cartilage. (B) Alizarin Red staining of the regenerating jawbone in live adult zebrafish shows mineralization of col2a1a^BAC:GFP-expressing chondrocytes. (C) Fluorescent in situ hybridization together with anti-GFP immunohistochemistry shows a subset of col2a1a^BAC:GFP⁺ chondrocytes co-expressing the osteoblast marker spp1. (D) Schematic showing that GFP protein perdures in cells long after endogenous col2a1a mRNA ceases to be produced. In the fluorescent in situ images of regenerating jawbone at 30 dpr, cells of the left callus still express col2a1a whereas cells of the right callus have largely ceased col2a1a expression and now express high levels of bglap (osteocalcin). Retention of GFP protein shows that bglap⁺ cells arise from cells that previously expressed col2a1a^BAC:GFP. Areas in the white boxes are magnified and presented as merged and individual channels. Scale bars: 100 μm.

Fig. 4.

Development of growth plates in juvenile zebrafish. (A) Trichrome staining of a coronal section through the juvenile fish jaw. (B) Coronal section of a 28 dpf juvenile jaw shows osteoblast precursors labeled by sp7:GFP, col10a1-expressing hypertrophic chondrocytes and BrdU⁺ proliferating cells. Hoechst labels nuclei in white. The ceratohyal cartilage (yellow box) displays a mammalian-like growth plate architecture. BrdU⁺ and col10a1-expressing zones are also observed in other cartilages, e.g. Meckel's (arrowhead). (C) Double fluorescent in situ hybridization at 14 dpf shows sox9a or col2a1a expression (green) in growth plate chondrocytes at either end of the ceratohyal cartilage and col1a1a expression (red) in the periosteum and a subset of chondrocytes at the hypertrophic borders. spp1 expression (red) is confined to the periosteum. At 21 dpf, col2a1a continues to be expressed in two zones of chondrocytes and col1a1a is now largely confined to the periosteum. At 28 dpf, bglap is expressed exclusively in the periosteum, and col10a1 is expressed in two stripes of hypertrophic chondrocytes surrounding each zone of col2a1a⁺ chondrocytes. A comparison of col2a1a expression with BrdU reactivity shows that col2a1a labels proliferating chondrocytes. (D) At 16 dpr, mineralization labeled by Alizarin Red occurs exclusively within the periosteum. By 49 dpf, two growth zones of col2a1a^BAC:GFP⁺ chondrocytes persist, yet Alizarin Red⁺ mineralization remains confined to a layer covering the ceratohyal cartilage. Scale bars: 100 μm.

Fig. 5.

Mobilization of the periosteum in response to jaw resection. (A,B) H&E staining shows the posterior site of jaw resection before and during the first week of regeneration. In the uninjured animal, the lower jawbone is lined by a thin layer of periosteum (arrowheads, see inset B). Immediately after resection (0 dpr), a small portion of Meckel's cartilage (m) remains and soft tissue collapses into the section. By 2 dpr, the periosteum (arrowheads) thickens and covers the cut surface of the bone (see inset B). The periosteum continues to expand into the resected region by 4 dpr, and 2 days later, mesenchymal cells are seen throughout the resected area and early chondrocytes can be distinguished (yellow arrows). (C) Fluorescent in situ hybridization for col1a1a (green) combined with BrdU staining (red) shows col1a1a⁺ cells lining the uninjured jawbone periosteum (arrows), as well as the skin. At 4 dpr, col1a1a expression increases in the periosteum and many col1a1a⁺ cells are seen in the mesenchyme within the resection zone. While col1a1a⁺ cells within the uninjured periosteum are largely negative for BrdU (in contrast to those within the skin), many BrdU⁺, col1a1a⁺ cells are seen in the mesenchyme near the resection site (see insets for magnified images, merged and single channels). (D) Section of an uninjured adult jaw shows sp7:mCherry (detected by anti-mCherry antibody) in osteoblasts lining bone and RUNX2:GFP (detected by anti-GFP antibody) in sparse patches of periosteum. (E) Magnification of boxed region in D shows RUNX2:GFP expression in periosteal cells underneath sp7:mCherry⁺ osteoblasts. A few cells co-express both transgenes (arrowhead), consistent with early differentiating osteoblasts. (F) After resection, RUNX2:GFP⁺ cells are found in the periosteum (arrow) overlying the jawbone and in expanding mesenchyme at 4 dpr, but not in Meckel's cartilage or its associated periochondrium (arrowheads). By 7 dpr, both mesenchymal and early chondrocytes express RUNX2:GFP. By contrast, sp7:GFP labels the periosteum but not mesenchymal cells or chondrocytes at 4 and 7 dpr. Hoechst labels nuclei in blue. Dashed lines indicate resection sites. Scale bars: 100 μm.

Fig. 6.

Requirement of ihha in the generation of repair cartilage. (A) In situ hybridization of the regenerating jawbone shows mesenchymal expression of ptc2 at 6 dpr, co-expression of sox9a and ptc1 in the cartilage callus at 8 dpr, and ihha and gli1 in the callus at 10 dpr. Dashed lines show resection sites. (B) Compared with size-matched wild-type siblings, ihha^−/− adults show a lack of cartilage at 14 dpr. Whereas the wild type bridges the resection site with thick bone by 28 dpr, ihha^−/− mutants have reduced and hollow bone. (C) Bone µCT shows reduced mineralization within the repair region (arrowheads). Top images are ventral views of the lower jaw and boxes show magnified images below. See also Movies 2 and 3. (D) Quantification of the area of repair bone and cartilage in the wild type and ihha mutants at 28 dpr. A Student's t-test showed statistical differences between groups. Standard errors of the mean are shown. (E) ihha mutants and their wild-type siblings have similar numbers of BrdU⁺ cells in the resected regions (left of the dashed lines). (F) BrdU incorporation (red) and col2a1a^BAC:GFP labeling of chondrocytes (green, detected by anti-GFP antibody) shows reduced cartilage but similar proliferation rates in ihha mutants. (G) Quantification of labeled BrdU⁺ nuclei in wild type and mutants. A Student's t-test showed no statistical differences at either stage. (H) Antibody staining in wild type and ihha mutants carrying the col2a1a^BAC:GFP transgene shows fewer chondrocytes expressing Sox9 (red) and GFP (green) in mutants versus siblings. Insets show that the few cartilage cells that form in mutants express Sox9 protein. Hoechst labels nuclei in blue. Scale bars: 1 mm in B and 100 μm in A,E.

Fig. 7.

Model of jawbone regeneration. (A) During endochondral bone development, chondrocyte (blue) and osteocyte (red) lineages are largely distinct. By contrast, cells co-express chondrocyte and osteoblast gene programs during lower jawbone regeneration in adult zebrafish, with chondrocyte-like cells rapidly mineralizing and maturing into osteoblasts. (B) During bone homeostasis, periosteal cells (gray layer) self-renew and contribute to new osteoblasts (black). In response to jawbone resection, Ihha signaling induces the chondrogenic differentiation of periosteal cells, with these repair chondrocytes (blue) maturing into bone-producing cells.