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First published online August 18, 2003
doi: 10.1242/10.1242/dev.00610

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Secondary chondrocyte-derived Ihh stimulates proliferation of periosteal cells during chick development

¹ Department Craniofacial Development, Guy's, King's and St Thomas' School of Dentistry, Guy's Hospital, London Bridge, London SE1 9RT, UK
² Department of Biology, Dalhousie University, Halifax, NS B3H 4J1, Canada
³ Connective Tissue Biology Laboratories, School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3US, UK

View larger version (107K): [in a new window]   Fig. 1. Initiation of secondary chondrocyte formation. (A-M) quadratojugal/quadrate joints at stage: e10 (A-G), e11 (H-J) and e13 (K-M). (A,D,H,K) Alcian Blue/Chlorantine Fast Red stained controls. (B) Cbfa1 expression is found only in the membranous periosteum of the quadratojugal (QJ). (C) Sox9 expression is only found in the cartilaginous quadrate (Q). (D-G) Comparison of Cbfa1, Gdf5 and Col2 expression. Arrowheads (D-J) indicate future sites of secondary chondrocytes (SCs). (E) Cbfa1 expression is maintained in the periosteum prior to SC formation. Gdf5 (F) is expressed around the quadrate and Col2 (G) is expressed within the quadrate, but neither is expressed within the quadratojugal. (H,I,J) Cbfa1 and Sox9 expression on adjacent sections after formation of the first SCs, adjacent to the germinal region (GR). (I) Cbfa1 is expressed by the periosteum, and by the nascent SCs (arrowed and circled). (J) Sox9 is expressed only by the SCs of the QJ (arrowhead). (K,L,M) Cbfa1 and Sox9 expression at e13. (L) Cbfa1 continues to be expressed in the periosteum and is also expressed by SCs (asterisk). (M) Sox9 is expressed only by SCs (arrowed), not in the periosteum. GR, germinal region; PO, periosteum; Q, quadrate; QJ, quadratojugal; SC, secondary chondrocytes. Scale bars: 100 µm.

View larger version (122K): [in a new window]   Fig. 2. Differentiation of secondary chondrocytes. (A-L) e11, (M-O) e13, (P-R) e14. (A,D,G,J,M,P) Alcian Blue/Chlorantine Fast Red stained controls. (A) SCs forming on the anterior and posterior (arrowhead) aspect of the QJ. (B,C) Adjacent sections to A, showing that Col2 (B) is upregulated as SCs form, and that Col10 (C) upregulation is delayed. (E,F) Adjacent sections to D, showing Ihh expression (E) in the same domain as Col10 (F). (H,I) Adjacent sections to G. (H) Bmp7 expression is observed throughout the periosteum, and (I) Gdf5 expression is restricted to the perichondrium of the Q; neither reveal a change in the germinal region. (K,L) Adjacent sections to J, which show that Sox9 (K) expression is confined to the SCs of the QJ, and that Frzb (L) expression is upregulated by SCs. (N,O) Adjacent sections to M, showing opn expression (N) in osteocytes; ocn expression (O) in osteoblasts adjacent to the PO precedes opn expression. (P,Q,R) At e14, chondroclast activity is evident (asterisk in P), and opn expression (Q) is additionally upregulated by late SCs (yellow arrowhead); ocn expression (R) remains specific for osteoblasts. oC, osteocytes; oB, osteoblasts. Scale bars: 100 µm.

View larger version (26K): [in a new window]   Fig. 3. Proliferation in the QJ at e14, as shown by the percentage of S-phase cells after BrdU labelling for 90 minutes at 0 hours. Figure (inset) indicates regions of the QJ counted for each value. Values shown are mean±s.d. from three QJs, a total of 6 sections. Asterisk indicates a significant difference between the periosteum and the germinal region (P<0.001, Student's t-test).

View larger version (102K): [in a new window]   Fig. 4. BrdU labelling and the origin of secondary chondrocytes at e14. Counterstains: Toluidine Blue (A); (B-G,I) Alcian Blue/Chlorantine Fast Red. BrdU labelled cells are black. (A) Pulse and immediate fix shows cycling cells in the germinal region, and a relative absence of BrdU label within the SCs. (B) Contralateral side to A, with a 24-hour chase following pulse. Doublets found within the SC domain are highlighted with red circles. (B') Higher magnification of image in B. (C) BrdU labelled cells at the boundary between the germinal region and the SCs. (D) Contralateral side to C shows labelled doublets after a 24-hour chase (circled). (D') Higher magnification of image in D. (E) BrdU labelling at 16 hours after explant and immediate fixation labels cells in the germinal region. (F) Contralateral side to E after a 24-hour chase shows no labelled chondrocytes in the SC domain. (G) BrdU label at 0 hour. (H) Adjacent section to G, which shows Col10 expression (yellow) superimposed on, and contained within, Col2 expression (red). Bracket indicates delay in Col10 upregulation. (I) 24-hour chase on contralateral side to G,H results in labelled doublets. (J) Adjacent section to I, showing that the Col10 domain has expanded to coincide with Col2 domain, black arrowhead. Scale bars: 100 µm.

View larger version (114K): [in a new window]   Fig. 8. Mechanical stimulation in secondary chondrogenesis. (A-F) e14, (G-H) e11. (A) BrdU labelled at 0 hours, with a 24-hour chase with mechanical stimulation; labelled doublets are observed. (B) osteocalcin (ocn) expression in an adjacent section: ocn is not seen around the SCs (black arrow). (C) BrdU labelled at 0 hours, with a 24-hour chase without mechanical stimulation; again labelled doublets are found. (D) Upregulation of ocn expression around the SCs on an adjacent section (black arrow). (E) BrdU labelled at 16 hours followed by 24-hour chase without mechanical stimulation; labelled cells are found in the germinal region. (F) As E, but with mechanical stimulation; BrdU labelled cells are found in the chondrocyte zone (circled). (G) e11 BrdU pulse at 0 hours followed by a chase for 48 hours without mechanical stimulation, both chondrocytes (blue circle) and osteoblasts (red circle) are labelled. (H) Adjacent section to G, showing ocn expression in BrdU-labelled cells. Scale bars: E, 100 µm; F, 50 µm.

View larger version (127K): [in a new window]   Fig. 5. Ihh signalling from secondary chondrocytes. (A,D,G,) Alcian Blue/Chlorantine Fast Red stained controls. (A-C) At e11, Ihh is expressed by SCs (B), but, as shown on an adjacent section (C), ptc2 expression is at the limit of detection in the germinal region (white arrowhead); however, it is expressed in the feather buds (FB; inset in C). (D-F) At e12, Ihh is expressed by SCs (E), and ptc2 (F) is expressed in the germinal region (arrowhead). (G-I) At e14, the relationship between Ihh (H) and ptc2 (I) expression is maintained, brackets indicate extent of the germinal region. Scale bars: 100 µm.

View larger version (89K): [in a new window]   Fig. 6. Inhibition of Ihh signalling reduces proliferation in the germinal region at e14. (A) control Q/QJ cultures cultured for 16 hours and labelled with BrdU. Bracket demarcates the germinal region, black arrowhead indicates proliferating cells. (B) Ihh expression in an adjacent section. (C) ptc2 is expressed in the germinal region (white arrowhead). (D-F) Hh blocking-antibody treated contralateral side. (D) The reduction in number of BrdU labelled cells is indicated by the black arrowhead. (E) An adjacent section showing unaffected Ihh expression. (F) An adjacent section showing downregulation of ptc2 expression in the germinal region (arrowhead). Scale bars: 100 µm.

View larger version (98K): [in a new window]   Fig. 7. The response of periosteal cells to exogenous Ihh. (A) Comparison of proliferation rates in control (RCAS-GFP infected) and experimental (RCAS-Ihh infected) micromass cultures after 4 days. Values shown are means±s.d. of BrdU labelled cells per unit area after a 30-minute pulse. (B) Phase contrast image of control micromass after 11 days in culture, showing polygonal osteoblast-like cells, but no nodules. (C) Ihh-infected micromass from the same series as B, again showing polygonal cells at day 11. (D) Same micromass as in C, shown as an example of the precocious bone nodules found in Ihh infected micromasses. (E) Day 11 control micromass displaying alkaline phosphatase (AP) activity (red). (F) Ihh-infected micromass additionally demonstrates mineralised bone matrix (black stain). (G,H,I,J) Cultures at day 14. (G) AP activity is evident at the periphery of the control micormass. (H) AP activity is increased in Ihh-infected cultures. (I,J) Sections stained additionally for von Kossa, demonstrating mineralised bone matrix at the centre of both micromasses; the matrix is increased in Ihh-treated cultures (J). Scale bars: B-D, 50 µm; E-J, 1 mm.

View larger version (24K): [in a new window]   Fig. 9. Two routes to chondrocyte hypertrophy. A common skeletogenic precursor gives rise to both chondrocytes and osteoblasts, a division arguably dictated by Sox9 and Cbfa1/Runx2. Chondrocytes are self-renewing, but differentiate and exit the cell cycle to form pre-hypertrophic chondrocytes, an event that can be mediated by the upregulation of Cbfa1/Runx2. Pre-osteoblasts are also self-renewing, and exit the cell cycle to form osteoblasts; however, when subjected to compression, they undergo chondrogenesis correlated with the upregulation of Sox9, which results in symmetric division followed by exit from the cell cycle and the acquisition of a pre-hypertrophic phenotype. Ihh from both sources of pre-hypertrophic chondrocytes signals to the periosteum: in long bones to induce osteogenesis and in SCs to stimulate proliferation and, in vitro, to promote osteogenesis.

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