Heads, Shoulders, Elbows, Knees, and Toes: Modular Gdf5 Enhancers Control Different Joints in the Vertebrate Skeleton

Abstract

Synovial joints are crucial for support and locomotion in vertebrates, and are the frequent site of serious skeletal defects and degenerative diseases in humans. Growth and differentiation factor 5 (Gdf5) is one of the earliest markers of joint formation, is required for normal joint development in both mice and humans, and has been genetically linked to risk of common osteoarthritis in Eurasian populations. Here, we systematically survey the mouse Gdf5 gene for regulatory elements controlling expression in synovial joints. We identify separate regions of the locus that control expression in axial tissues, in proximal versus distal joints in the limbs, and in remarkably specific sub-sets of composite joints like the elbow. Predicted transcription factor binding sites within Gdf5 regulatory enhancers are required for expression in particular joints. The multiple enhancers that control Gdf5 expression in different joints are distributed over a hundred kilobases of DNA, including regions both upstream and downstream of Gdf5 coding exons. Functional rescue tests in mice confirm that the large flanking regions are required to restore normal joint formation and patterning. Orthologs of these enhancers are located throughout the large genomic region previously associated with common osteoarthritis risk in humans. The large array of modular enhancers for Gdf5 provide a new foundation for studying the spatial specificity of joint patterning in vertebrates, as well as new candidates for regulatory regions that may also influence osteoarthritis risk in human populations.

The Role of Bmp2 in the Maturation and Maintenance of the Murine Knee Joint

“…In comparison, Gdf5 was expressed earlier in knee joint morphogenesis at E14 in the interzone in a pattern similar to its previously reported localization (Fig. E ) . At E16, Gdf5 was expressed in a broad domain in the E16 knee, which included the meniscus, articular cartilage, and forming cruciate ligaments (Fig.…”

“…1E). (4,25) At E16, Gdf5 was expressed in a broad domain in the E16 knee, which included the meniscus, articular cartilage, and forming cruciate ligaments ( Fig. 1F-H).…”

Micro-pellet culture reveals that bone marrow mesenchymal stromal cell (BMSC) chondrogenic induction is triggered by a single day of TGF-β1 exposure

“…ACh expressed higher levels of PTHLH, a known modulator of cartilage hypertrophy [25], than BMSC, in the presence of TGF-b1, but not following its withdrawal. Chondrocyte-associated factors from the TGF-b superfamily, GDF5, GDF6 [26] and TGFBR3 [27], were consistently expressed in ACh, but were underexpressed by TGF-b1 exposure in BMSCs. BMP antagonists GREM1 and CHRD were expressed by both BMSCs and ACh prior to induction (Day 0) and in the presence of TGF-b1, with higher relative expression in ACh, and decreased in both cell types following TGF-b1 withdrawal.…”

Expression analysis of limb element markers during mouse embryonic development

“…This difference may be due to strain differences in embryonic development, which can exhibit differences in gestation periods (Degenkolbe et al, 2013; Huang et al, 2016). Gdf5 is expressed across developing joint sites, including interphalangeal joints, from E14.5 (Chen et al, 2016; Houweling et al, 2001; Huang et al, 2016; Sohaskey et al, 2009). Our findings for Scleraxis expression are in agreement with previously published analyses of Scleraxis , indicating expression in the autopod and in developing digit rays and cartilage condensations at E12.5, as well as defined expression in developing tendons at E13.5 (Huang et al, 2015; McGlinn et al, 2005; Schweitzer et al, 2001) and along developing digits, with strongest expression across sites of developing interzones at E14.5 (Murchison et al, 2007; Schweitzer et al, 2001).…”

“…Distinct aspects of Gdf5 expression have been reported in several publications that combined have shown Gdf5 expression between E11.5 and E14.5 in the presumptive sites of joint development and the forming joint interzone (Chen et al, 2016; Degenkolbe et al, 2013; Hellman et al, 2012; Houweling et al, 2001; Huang et al, 2016; Meech et al, 2005; Ota et al, 2007; Perez et al, 2010; Seemann et al, 2005; Sohaskey et al, 2008). Uniquely, we have shown side‐by‐side Gdf5 expression across a range of key stages of development in both the forelimb and hindlimb.…”