Fig 1.
Laser capture microdissection of mouse articular and growth plate cartilage.
(A) Haemotoxylin & eosin stained section of formalin fixed, decalcified 1-wk old mouse proximal tibial cartilage showing the location of zones in the cartilage that were targeted by laser capture microdissection (LCM). (B) Haemotoxylin & eosin stained frozen sections of 1-wk old mouse proximal tibial cartilage after LCM showing regions that were excised for RNA isolation. (C) Relative gene expression (mean ± SEM) of zonal markers used for dissection validation. S, superficial zone; M, middle zone; D, deep zone; R, resting zone; P, proliferative zone; H, hypertrophic zone. Scale bar applies to both (A) and (B).
Fig 2.
Expression of genes involved in BMP signaling in growth plate cartilage.
Relative gene expression (mean ± SEM) of a subset of BMP agonists (top row), BMP functional antagonists (second row), BMP receptors (third row) and downstream signaling modulators (bottom row) in resting zone (R), proliferative zone (P) and hypertrophic zone (H) of 1-wk old mouse tibial growth plate, measured by a multiplex solution hybridization assay (nCounter). A full list of BMP-related gene and expression values were shown in S1 Table.
Fig 3.
Expression of genes involved in BMP signaling in articular cartilage.
Relative gene expression (mean ± SEM) of a subset of BMP agonists (top row), BMP functional antagonists (second row), BMP receptors (third row) and downstream signaling modulators (bottom row) in superficial zone (S), middle zone (M) and deep zone (D) of 1-wk old mouse tibial growth plate, measured by a multiplex solution hybridization assay (nCounter). A full list of BMP-related gene and expression values were shown in S1 Table.
Fig 4.
In situ hybridization for BMP-related genes in articular and growth plate cartilage.
Formalin fixed, decalcified sections of 1-wk old mouse tibial cartilage were hybridized to DIG-labeled riboprobes, producing a purple color and were visualized by scanning the slides with a ScanScope CS digital scanner using bright field microscopy. Left panel, Mason-trichrome stained sections; middle panel, in situ hybridization without counterstaining; right panel, higher magnification views taken from within the rectangular area indicated in the corresponding middle panel. SZ, superficial zone; MZ, middle zone; DZ, deep zone; RZ, resting zone; PZ, proliferative zone; HZ, hypertrophic zone.
Fig 5.
Immunohistochemistry for BMP-related proteins in articular and growth plate cartilage.
Formalin fixed, decalcified sections of 1-wk old mouse tibial cartilage were stained with anti-Bmp2, anti-Bmp6, and anti-Grem1 antibody and signals were developed with DAB substrate (brown color). Tissues were counterstained in methyl green and visualized by scanning with a ScanScope CS digital scanner using bright field microscopy. SZ, superficial zone; MZ, middle zone; DZ, deep zone; RZ, resting zone; PZ, proliferative zone; HZ, hypertrophic zone.
Fig 6.
Immunohistochemistry for SMADs in articular and growth plate cartilage.
Formalin fixed, decalcified sections of 1-wk old mouse tibial cartilage were stained with anti-pSmad1/5/8 and anti-Smad7 antibody. Signals were developed with DAB substrate (brown color) with no counterstaining. pSmad1/5/8 represents activation of BMP intracellular signaling, while Smad7 is an I-SMAD that functions as a functional antagonist of BMP signaling. SZ, superficial zone; MZ, middle zone; DZ, deep zone; RZ, resting zone; PZ, proliferative zone; HZ, hypertrophic zone.
Fig 7.
Model of BMP expression gradients in the articular and growth plate cartilage.
Our current findings suggest BMP gradients in the articular and growth plate cartilages, with higher BMP signaling on the articular surface and the proliferative zone, and lower BMP signaling in the deep and resting zone. Interestingly, although BMP ligands Bmp2 and 6 were most highly expressed in the HZ, BMP signaling (based on pSmad1/5/8) was higheer in the PZ, perhaps because of high Smad7 expression in HZ. The gene names were placed to indicate regions of high expression.