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First published online October 27, 2004
doi: 10.1242/10.1242/dev.01414

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The AP1 transcription factor Fra2 is required for efficient cartilage development

Research Institute of Molecular Pathology (I.M.P.), Dr Bohr-Gasse 7, 1030 Vienna, Austria

View larger version (56K): [in a new window]   Fig. 1. Reduced zones of hypertrophic chondrocytes in Fosl2–/– embryos and newborn mice. (A) Skeletal staining of a Fosl2–/– newborn and wild-type littermate at P0. (B) Length of mineralized regions of knock-out and wild-type littermates at P0; bars represent mean value±s.e.m.; n=4. (C) Genotyping-PCR of DNA from wild-type, heterozygous and Fosl2-null newborn mice. (D) Expression of Fosl2 in primary rib cage chondrocytes and its absence in mutant cells. Primary chondrocytes were cultured for 3 days prior to RNA isolation and RNase protection assay. GAPDH was used as loading control. (E) Real-time PCR of cartilage markers. Relative expression of type X collagen (colX), aggrecan (agg) and Ihh is shown. Expression levels were normalized to tubulin expression. The mean of two independent measurements is shown. (F) In situ hybridization on sections of embryonic and postnatal femoral growth plates of Fosl2–/– mice and littermate controls using a type X collagen antisense probe as a marker for hypertrophic zones. Pictures were taken at 200x (E13.5), 100x (E14.5, E16.5, E18.5) and 50x (P2, P4) magnification.

View larger version (57K): [in a new window]   Fig. 2. Impaired chondrocyte differentiation and matrix deposition in vivo. (A) In vivo BrdU labeling of E19.0 embryos. The labeling index (BrdU positive cells/total cells) in zones of proliferating chondrocytes is shown. Bars represent mean value±s.d.; n=2; *P<0.001. (B) Ki67-positive cells in zones of proliferating chondrocytes at E14.5, E16.5 and E18.5. (C) Von Kossa staining of calcified matrix of E15.5 and E17.5 Fosl2–/– embryos and littermate controls. Arrowheads indicate stained matrix in the zones of hypertrophic chondrocytes. Pictures were taken at 50x magnification.

View larger version (66K): [in a new window]   Fig. 3. Delayed development of vertebral columns in Fosl2–/– embryos. (A) Hematoxylin and Eosin staining, in situ hybridization using type II collagen (col II) and type X collagen (col X) antisense probes, and Ki67 immunohistochemistry on transversally sectioned primordial arches of developing vertebrae at E14.5. Arrowheads indicate Ki67-stained cells. (B) Hematoxylin and Eosin staining, in situ hybridization using osteocalcin (oc, arrowheads) antisense probe, Ki67 immunohistochemistry and von Kossa staining on transversally sectioned primordial arches of developing vertebrae at E16.5. Arrowheads indicate Ki67-stained cells. Pictures were taken at 100x (Haematoxylin and Eosin) and 50x (in situ hybridization, Ki67 immunohistochemistry, von Kossa staining) magnification.

View larger version (35K): [in a new window]   Fig. 4. Impaired differentiation and delayed senescence of primary Fosl2–/– rib cage chondrocytes. (A) Formation of cartilage nodules by primary chondrocytes. Primary chondrocyte cultures stained with Alcian Blue (left panel). Quantification of Alcian Blue bound to sulfated proteoglycans in the chondrocyte ECM (right panel). Bars represent mean value±s.d.; n=4; *P<0.05. (B) Cumulative cell number assay of primary rib cage chondrocytes. Experiments were carried out in triplicate and repeated three times. One representative experiment is shown. (C) BrdU-labeling of proliferating primary chondrocytes after 2 days in culture. (D) TUNEL staining of apoptotic primary chondrocytes after 2 days in culture. (E) Semi-quantitative RT-PCR analysis of cartilage markers and Fosl2 in primary rib cage chondrocytes at day 3, 6 and 9 of differentiation. Tubulin was used as a loading control (n=3).

View larger version (41K): [in a new window]   Fig. 5. Growth retardation in coll2a1-Cre, Fosl2f/f mice. (A) X-ray analysis of a 23-day-old coll2a1-Cre, Fosl2f/f mouse and littermate control (arrowhead indicates kyphosis). (B) Decreased body size and body weight of coll2a1-Cre, Fosl2f/f mice at P10. Bars represent mean value±s.d.; n=5; *P<0.05. (C) PCR for Fosl2 deletion in tissues and organs of a 6-day-old coll2a1-Cre, Fosl2f/f mouse. The same deletion efficiency was observed in older mice. he, heart; li, liver; lu, lung; ki, kidney; sp, spleen; br, brain; th, thymus; ln, lymph node; mu, muscle; sk, skin; st, stomach; in, intestine; bm, bone marrow; sm, sternum; cv, calvariae.

View larger version (68K): [in a new window]   Fig. 6. Impaired hypertrophic differentiation but normal proliferation in coll2a1-Cre, Fosl2f/f mice. (A,B) In situ hybridization of femoral growth plates of P5, P10 and P23 coll2a1-Cre, Fosl2f/f mice and littermate controls using type X collagen (A) and type II collagen (B) antisense probes. Arrowheads indicate size of proliferating zone. (C) Ki67-positive cells in femoral growth plates of Fosl2–/–, coll2a1-Cre, Fosl2f/f and control mice at P2, P5 and P10. Bars represent mean value±s.e.m.; n=3. (D) Immunohistochemical detection of Ki67 in P23 coll2a1-Cre, Fosl2f/f mice and P23 coll2a1-Cre, Fosl2f/+ controls. Pictures were taken at 50x (col X, P10 and P23; col II, P5 and P10) and 100x (col X, P5; col II, P23; Ki67) magnification.

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