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First published online November 10, 2005
doi: 10.1242/10.1242/dev.02097

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Gli3 acts as a repressor downstream of Ihh in regulating two distinct steps of chondrocyte differentiation

Max-Planck-Institute for Molecular Genetics, Berlin, Germany and Department for Developmental Biology, Center for Medical Biotechnology, University Duisburg-Essen, Essen, Germany

View larger version (125K): [in a new window]   Fig. 1. Expression pattern of the Gli family of transcription factors. Sections of E14.5 (A,C,E,G,I) and E16.5 (B,D,F,H,J) wild-type limbs were hybridized with antisense riboprobes as indicated. At E14.5 Gli1 (C), Gli2 (E) and Gli3 (G) are expressed in proliferating chondrocytes distal to the Ihh (A) expression domain. In the perichondrium low levels of Gli2 and Gli3 and strong levels of Gli1 expression can be detected (yellow arrow, C,E,G). In elbow and carpal joints Gli3 is strongly expressed and Gli2 weakly expressed, overlapping with PTHrP (I) expression. At E16.5 Gli1 is uniformly expressed in proliferating chondrocytes (D). By contrast there is a decreasing gradient of Gli2 and Gli3 expression from distal (red arrows F,H,) to central. All three Gli genes are additionally expressed in the chondro-osseus junction (green arrow, D,F,H) and in the perichondrium. Ulna is up and radius is down.

View larger version (128K): [in a new window]   Fig. 2. Loss of Gli3 in Ihh-deficient limbs rescues chondrocyte proliferation and the onset of hypertrophic differentiation, but not bone collar formation. (A-L) Hematoxylin and Eosin staining of E16.5 wild-type (A,D,G,H), Ihh–/– (B,E,I,J) and Ihh–/–;Gli3–/– (C,F,K,L) limb sections. (A-C) Compared with Ihh–/– mutants (B) the size of the skeletal elements is increased and hypertrophic chondrocytes are restricted to the center of the cartilage anlagen in Ihh–/–;Gli3–/– double mutants (C). D-F are higher magnifications of the boxed regions in A-C; G-L show the chondrocyte morphology of distal (G,I,K) and central (H,J,L) regions indicated by the boxes in D-F. In Ihh–/– limbs no columnar chondrocytes can be detected, whereas wild-type and Ihh–/–;Gli3–/– cartilage anlagen have a zone of distal (D,F, red bar) and columnar (D,F, blue bar) chondrocytes. (M-O) The chondrocyte proliferation rate in Ihh–/–;Gli3–/– limbs (O) is similar to wild-type levels (M) and strongly increased compared to Ihh–/– limbs (N). (P-U) Van Kossa staining detects mineralization of chondrocytes of E16.5 wild-type (P), Ihh–/– (Q, red arrow in the humerus) and Ihh–/–;Gli3–/– limbs (R). S-U are higher magnifications of the boxed regions in P-R. Wild-type limbs form a bone collar adjacent to hypertrophic chondrocytes (S, red arrow), which is missing in Ihh–/– (T) and Ihh–/–;Gli3–/– mutants (U, red arrow). In Ihh–/–;Gli3 –/– limbs, bone collar-like structures form in restricted regions (U, black arrowhead).

View larger version (107K): [in a new window]   Fig. 3. Molecular characterization of Ihh–/–;Gli3–/– limbs. Serial sections of E16.5 wild-type (A,D,G,J), Ihh–/– (B,E,H,K) and Ihh–/–;Gli3–/– limbs (C,F,I,L) were hybridized with riboprobes as indicated. (B) In Ihh–/–;Gli3–/–-deficient mice Col10a1 expression is restricted to the center of the skeletal elements, indicating a delayed onset of hypertrophic chondrocyte differentiation compared to Ihh–/– mice. (E,H,K) The Ihh target genes Ptch, Gli1 and PTHrP are not expressed in Ihh–/– mice. (F,I,L) In Ihh–/–;Gli3–/– limbs the expression of Ptch and PTHrP, but not Gli1, is partially rescued. (F) Ptch is weakly expressed in proliferating chondrocytes. (J,L) PTHrP expression can be detected in the perichondrium (red arrow) and in the carpal joints (green arrowhead). No PTHrP expression can be detected in the distal chondrocytes (yellow arrowhead).

View larger version (99K): [in a new window]   Fig. 4. Gli3–/– limbs are delayed in endochondral ossification. Serial sections of E16.5 wild-type (A,C,E,) and Gli3–/– (B,D,F) limbs were hybridized with riboprobes as indicated. (A-D) Expression of Ihh and Col10a1 reveals normal regions of prehypertrophic and hypertrophic chondrocytes. (A,B) The overall size of the radius in wild-type mice (light-green bar) is reduced in Gli3–/– mutants (dark-green bar, P=0.0036), and the ossification is slightly delayed (compare dark- and light-blue double-headed arrows in wild-type and Gli3–/– limbs, respectively). (E,F) The expression of Ptch is unchanged in Gli3–/– limbs.

View larger version (66K): [in a new window]   Fig. 5. Gli3 regulates distal chondrocyte differentiation. Two sets of serial sections, A,B,D,E and G,H,J,K, of E16.5 wild-type (A,D,G,J) and Gli3–/– (B,E,H,K) limbs were hybridized with riboprobes as indicated. Red dashed lines indicate the distal border of the Ihh expression domain. (C,F,I,L) Models of gene expression domains. (A-C) Gli3–/– mice display a reduced zone of proliferating chondrocytes compared to wild-type mice (red arrow). (D-F) PTHrP is expressed at similar levels in Gli3–/– and wild-type limbs, however its domain of expression is shifted towards the distal end of the cartilage anlagen in Gli3–/– limbs. (G-L) In Gli3–/– limbs the Fgfr1 expression domain (G-I) is reduced (green arrow) and the Fgfr3 expression domain (J-L) is shifted towards the end of the skeletal elements (blue arrow), indicating an accelerated differentiation of distal chondrocytes. (M) The zone of proliferating chondrocytes (red arrow in A-C) is shortened in Gli3–/– limbs (***P=0.00003, n=6). The PTHrP-expressing zone of distal chondrocytes (yellow arrow in D-F) is reduced in Gli3–/– limbs (*P=0.00008, n=6). The distance between the Ihh expression domain and the central end of the PTHrP expression domain (gray arrow in D-F) is of similar size in both, Gli3–/– and wild-type limbs (**P=0.304, n=6). (N) The zone of Fgfr1-expressing distal chondrocytes (green arrow in G-I) is reduced in Gli3–/– mice (*P=0.0002, n=5), whereas the zone of Fgfr3 expression (blue arrow in J-L) is of similar size (**P=0.19, n=5).

View larger version (137K): [in a new window]   Fig. 6. (A-D) In Ihh–/– limbs distal chondrocytes differentiate directly into prehypertrophic chondrocytes. Sections of E14.5 wild-type (A,C) and Ihh–/– (B,D) limbs were hybridized with Fgfr1 (A,B) and Pthr1 (C,D) riboprobes. In Ihh–/– mutants Fgfr1 is expressed in a stripe of distal chondrocytes, which is flanked by Pthr1 expressing hypertrophic chondrocytes. Black dashed lines indicate the border between Fgfr1 and Pthr1 expression domains; red dashed lines indicate the end of the cartilage elements. (E-H) Loss of Gli3 restores columnar chondrocyte differentiation in Ihh–/– mice. Sections of E16.5 wild-type (E,G) and Gli3–/–;Ihh–/– (F,H) limbs were hybridized with riboprobes for Fgfr1 (E,F) and Pthr1 (G,H). In Gli3–/–;Ihh–/– mutants a reduced zone of Fgfr1 expressing distal chondrocytes is separated from the Pthr1 expression domain by a population of columnar chondrocytes. B,D and F,H display serial sections.

View larger version (124K): [in a new window]   Fig. 7. Differentiation of distal chondrocytes is accelerated in Col2a1-Ihh mice. Sections of E16.5 wild-type (A,C) and Col2a1-Ihh (B,D) limbs were hybridized with Fgfr1 (A,B) and Fgfr3 (C,D) riboprobes. (A1-D1) are higher magnifications of boxed regions in A-D, the dashed line in B,B1 demarcates the end of the radius, which is partially fused with the carpal navicular lunare in Col2a1-Ihh mice. The domain of Fgfr1 expression is reduced in Col2a1-Ihh mice (A,B, green arrow). The domain of Fgfr3 expression is broadened and shifted towards the end of the skeletal elements in Col2a1-Ihh mice (C,D, blue arrow in C1,D1). (E) The Fgfr1 expression domain (green arrow in A,B) is reduced in Col2a1-Ihh limbs (*P=0.003, n=3) whereas the domain of Fgfr3 expression (blue arrow in C,D) is strongly expanded compared to that of wild-type limbs (**P=0.007, n=3).

View larger version (115K): [in a new window]   Fig. 8. Loss of Gli3 partially rescues the Col2a1-Ihh phenotype. Serial sections of E16.5 Gli3+/– (A,E,I,M,Q; control), Gli3–/– (B,F,J,N,R), Col2a1-Ihh;Gli3+/– (C,G,K,O,S) and Col2a1-Ihh;Gli3–/– (D,H,L,P,T) limbs of the same litter were hybridized with riboprobes as indicated. E-H are higher magnifications of the distal radii (boxed) in A-D. (E-H) The size of the zone of proliferating chondrocytes (red arrow) is reduced in Gli3–/– mice and strongly increased in Col2a1-Ihh;Gli3+/– mice. Compared to Col2a1-Ihh;Gli3+/– mice, the zone of proliferating chondrocytes is decreased in double transgenic Col2a1-Ihh;Gli3–/– mice. (I-L) The size of the domain of distal chondrocytes, demarcated by the Fgfr1 expression domain (green arrow), is slightly reduced in Col2a1-Ihh;Gli3+/– and strongly reduced in Gli3–/– and Col2a1-Ihh;Gli3–/– mice. (M-P) The Fgfr3 expression domain is expanded in Col2a1-Ihh;Gli3+/– limbs, whereas in double transgenic Col2a1-Ihh;Gli3–/– mice the Fgfr3 expression domain is of similar size to that in wild-type and Gli3–/– limbs. (Q-T) The upregulated level of PTHrP expression in Col2a1-Ihh;Gli3+/– mice seems to be downregulated in Col2a1-Ihh;Gli3–/– mice to similar levels as in control or Gli3–/– limbs.

View larger version (34K): [in a new window]   Fig. 9. Gli3 regulates two steps of chondrocyte differentiation: the transition from (1) distal into columnar and from (2) columnar into hypertrophic chondrocytes. Left half: Gli3 regulates the onset of hypertrophic differentiation by regulating PTHrP (yellow) expression. In distal chondrocytes Gli3 acts as a strong repressor of PTHrP expression. Ihh signaling inhibits the repressor function of Gli3 (Gli3-R) and possibly activates PTHrP expression by Gli3-mediated activation (Gli3-A). As columnar chondrocytes loose the competence to express PTHrP its expression domain is restricted to distal chondrocytes. In the most distal cells, which do not receive sufficient Ihh, the Gli3 repressor cannot be inactivated and therefore inhibits the expression of PTHrP, resulting in a stripe of PTHrP expression. Right half: Gli3 negatively regulates the differentiation of Fgfr1 expressing, distal (green) chondrocytes into columnar cells independent of PTHrP. Ihh antagonizes the repressor function of Gli3 and promotes this differentiation process.

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