Failure to Maintain Articular Cartilage in Other Joints
In most joints of Bmpr1a conditional knockout mice, embryonic segmentation of skeletal precursors occurred normally. Although Gdf5-Cre-mediated recombination was seen as early as E13.5 in digit interzone regions (see Figure 2C), no changes in cell death or cell proliferation could be seen in the metacarpal-phalangeal or metatarsal-phalangeal joints at E13.5 or E14.5 (unpublished data). Similarly, although clear LACZ expression was seen by E15.5 in interphalangeal joints and periarticular regions (Figure 4D), no difference in morphology or expression of Col2a1, Gdf5, or Bmpr1b was seen in the articular regions of the phalanges at these stages (unpublished data).
At birth, digit joints were generally indistinguishable from those in control animals; chondrocytes were abundant in articular regions and were surrounded by typical cartilage matrix with normal staining by Safranin O, a histological stain for proteoglycans (Figure 5). At this stage, both wild-type and mutant cells in articular regions also expressed high levels of Col2a1 and Aggrecan (Agg), the genes encoding the major structural proteins of cartilage matrix (Figure 5B and 5G) (unpublished data). No alterations in cellular apoptosis or proliferation were observed (unpublished data).
Figure 5  Bmpr1a Is Required to Maintain Expression of ECM Components in Articular Cartilage
In situ hybridization or LACZ staining on near adjacent sections of metacarpal-phalangeal joints (A–C and F–H) and the tarsal 2-metatarsal II joint (D–E and I–J) of P0 mice. At birth, articular cartilage of controls (A–E) and mutants (F–J) appears similar by Safranin O staining (A and F), and Col2 expression (B, G). Mat4 expression confirms that articular cartilage is initially specified in mutants (D andI, brackets). LACZ expression confirms Cre-mediated recombination has occurred in articular cartilage (C, H, E, and J). (K–T) Near adjacent sections of the metacarpal-phalangeal joints of P14 mice. Two weeks after birth, articular cartilage of controls stains with pericellular Safranin O (orange staining, K), and expresses Col2 (L), Agg (M), and SOX9 (N). In contrast, mutant articular cells are smaller and more densely packed, lack pericellular Safranin O staining (P), have reduced expression of Col2 (Q) and Agg (R), but retain normal levels of SOX9 protein (S, brackets; dashed line marks faint edges of articular surfaces). LACZ expression confirms Cre-mediated recombination has occurred in articular cells (O ansd T, brackets). (A and K) Scale bar = 75 μm. To determine whether articular cells were properly specified in mutants, we also analyzed expression of Matrilin-4 (Mat4), a gene expressed specifically in the periarticular and perichondral regions of developing joints (Klatt et al. 2001). In both control and mutant animals, transcription of Mat4 was clearly detectable in the articular cartilage layers of newborn joints (Figure 5D and 5I). In all experiments, expression of LACZ throughout articular regions indicated that Cre-mediated recombination had occurred throughout the articular regions (Figure 5C, 5H, 5E, and 5J). The normal histological appearance, staining properties, and marker gene expression patterns suggest that Bmpr1a is not required for the initial formation or specification of articular cartilage.
By 1 wk after birth, obvious differences began to be detected in the articular regions of mutant animals. The expression of Col2a1 was reduced throughout the articular surfaces of the carpals, metacarpals, and phalanges of the forefeet (unpublished data). Less severe reductions were also seen in articular cells of tarsals and metatarsals in the hindfeet (unpublished data). By 2 wk of age, Col2a1 expression was reduced in most cells of the articular region (Figure 5L and 5Q), accompanied by markedly reduced Safranin O staining (Figure 5K and 5P), and decreased expression of Agg and two genes normally expressed in more mature articular cartilage cells, Collagen 3 (Col3a1) and Collagen 10 (Col10a1) (Figure 5M and 5R) (unpublished data) (Eyre 2002). Inhibition of BMP signaling in cultured chondrocytes has previously been reported to induce Collagen 1 (Col1a1) expression, increase proliferation, and result in cells with flattened, fibroblast-like morphology (Enomoto-Iwamoto et al. 1998). However, we saw no increase in the expression of Col1a1 in mutant articular cartilage, and no proliferation was detected in articular cells of either mutant or control animals (unpublished data). While recombined LACZ marker expression was detected in most articular cartilage cells, it was also observed in scattered subarticular chondrocytes, growth plate chondrocytes, and osteoblasts (Figure 5O and 5T) (unpublished data). Although this implies that BMP signaling was defective in multiple cell types, the observed defects were confined to the articular cartilage. For example, Osteocalcin and Col1a1 expression appeared normal in osteoblasts (unpublished data). Together, these data suggest that BMPR1A activity is required in postnatal joint articular cartilage to maintain expression of many genes encoding structural components of cartilage matrix.
Previous studies have shown that Sox9 is required for normal cartilage differentiation, for expression of cartilage extracellular matrix (ECM) genes including Agg, and is a direct transcriptional regulator of the key cartilage matrix gene Col2a1 (Bell et al. 1997; Lefebvre et al. 1997; Bi et al. 1999; Sekiya et al. 2000). Notably, despite reduced expression of many cartilage matrix marker genes in Bmpr1a mutant mice, the SOX9 protein was present at normal levels in articular regions at all stages examined, including newborn, 2-wk-old, 7-wk-old, and 9-mo-old mice (Figure 5N and 5S) (unpublished data).