The characterization of genes involved in the formation of cartilage is of key importance to improve cell-based cartilage regenerative therapies. Here, we have developed a suitable experimental model to identify precocious chondrogenic events in vivo by inducing an ectopic digit in the developing embryo. In this model, only 12 hr after the implantation of a Tgfβ bead, in the absence of increased cell proliferation, cartilage forms in undifferentiated interdigital mesoderm and in the course of development, becomes a structurally and morphologically normal digit. Systematic quantitative PCR expression analysis, together with other experimental approaches allowed us to establish 3 successive periods preceding the formation of cartilage. The “pre-condensation stage”, occurring within the first 3 hr of treatment, is characterized by the activation of connective tissue identity transcriptional factors (such as Sox9 and Scleraxis) and secreted factors (such as Activin A and the matricellular proteins CCN-1 and CCN-2) and the downregulation of the galectin CG-8. Next, the “condensation stage” is characterized by intense activation of Smad 1/5/8 BMP-signaling and increased expression of extracellular matrix components. During this period, the CCN matricellular proteins promote the expression of extracellular matrix and cell adhesion components. The third period, designated the “pre-cartilage period”, precedes the formation of molecularly identifiable cartilage by 2–3 hr and is characterized by the intensification of Sox 9 gene expression, along with the stimulation of other pro-chondrogenic transcription factors, such as HifIa. In summary, this work establishes a temporal hierarchy in the regulation of pro-chondrogenic genes preceding cartilage differentiation and provides new insights into the relative roles of secreted factors and cytoskeletal regulators that direct the first steps of this process in vivo.

Tendons and cartilage are specialized forms of connective tissues originated from common progenitor cells. Initial stages of differentiation of these tissues are characterized by the formation of cell aggregates, which share many molecular markers. Once differentiated, these cells retain considerable plasticity, and chondral metaplasia of tendon and fibrous connective tissues and eventual ossification often accompany degenerative diseases in the adult musculoskeletal system. While this fact is of great relevance for regenerative medicine and aging biology, its molecular basis remains to be elucidated. Gene expression analysis in several physiological and experimental paradigms suggests that differentiation of tendon and cartilage is regulated by a balance in the expression of chondrogenic versus tenogenic genes in the connective tissue cell precursors. Transforming growth factor b (TGFb) may function both as a profibrogenic or as a prochondrogenic factor for embryonic limb mesoderm and mesenchymal stem cell cultures, but mice that are null for TGFb 2 and 3 lack tendons. Here, we identify big-h3 as a factor downstream TGFb signaling regulated by Smad 2 and 3, which is highly expressed in the differentiating tendons and joint capsules. Furthermore, gain-and loss-of-function experiments using limb mesoderm micromass cultures show that big-h3 downregulates the expression of cartilage master genes, including Sox9, type II collagen, and Hif-1a. Positive regulation of Sox9 and type II Collagen observed in micromass cultures grown under hypoxic conditions is prevented by exogenous administration of bIG-H3, and the antichondrogenic influence of bIG-H3 is lost after Hif-1a silencing with shRNA. Collectively, our findings indicate that big-h3 promotes the fibrogenic influence of TGFb signaling, neutralizing the prochondrogenic influence of the hypoxic-inducible factor 1 activated by the hypoxic microenvironment characteristic of limb mesenchymal aggregates.

Reelin is a bioactive component of some extracellular matrices. Most studies on this signaling glycoprotein have been performed in the developing nervous system, where Reelin binds to the very-low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) of target cells. This induces phosphorylation of the intracellular adaptor protein Disabled-1 (Dab-1), which subsequently activates downstream effectors to regulate important aspects of neuroblast biology. Here, we show that the components of the Reelin signaling pathway exhibit a dynamic expression pattern during the development of the digits in chick and mouse embryonic limbs. Reelin and Dab-1 are highly expressed in the differentiating digit cartilages and tendinous blastemas. Immunolabeling of phospho-Dab-1 indicates that the pattern of gene expression correlates with zones of active signaling. Intense signaling is also present in the early stages of cartilage differentiation in micromass cultures of digit mesodermal progenitors. In this in vitro assay, disruption of the Reelin signaling pathway by gene silencing causes cystoskeletal and cell shape modifications accompanied by reduced chondrogenesis and down-regulation of specific cartilage molecular markers. Of note, Scleraxis and Six2, which are master genes of tendinous blastemas, become up-regulated in these experiments. We further show that the receptors ApoER2 and VLDLR are differentially expressed in cartilage and tendons and that these receptors show temporal expression differences in the micromass cultures. Sox9 and other chondrogenic markers were downregulated in micromass cultures after ApoER2 gene silencing, while gene silencing of VLDLR up-regulates Scleraxis. In summary, our findings provide evidence of a role for Reelin signaling in skeletogenesis that promotes chondrogenesis through ApoER2 and inhibits tenogenic differentiation through VLDLR.

Interdigital cell death is a physiological regression process responsible for sculpturing the digits in the embryonic vertebrate limb. Changes in the intensity of this degenerative process account for the different patterns of interdigital webbing among vertebrate species. Here, we show that Reelin is present in the extracellular matrix of the interdigital mesoderm of chick and mouse embryos during the developmental stages of digit formation. Reelin is a large extracellular glycoprotein which has important functions in the developing nervous system, including neuronal survival; however, the significance of Reelin in other systems has received very little attention. We show that reelin expression becomes intensely downregulated in both the chick and mouse interdigits preceding the establishment of the areas of interdigital cell death. Furthermore, fibroblast growth factors, which are cell survival signals for the interdigital mesoderm, intensely upregulated reelin expression, while BMPs, which are proapototic signals, downregulate its expression in the interdigit. Gene silencing experiments of reelin gene or its intracellular effector Dab-1 confirmed the implication of Reelin signaling as a survival factor for the limb undifferentiated mesoderm. We found that Reelin activates canonical survival pathways in the limb mesoderm involving protein kinase B and focal adhesion kinase. Our findings support that Reelin plays a role in interdigital cell death, and suggests that anoikis (apoptosis secondary to loss of cell adhesion) may be involved in this process.