Transforming growth factor ␤ (TGF␤) signaling has an increasing interest in regenerative medicine as a potential tool to repair cartilages, however the chondrogenic effect of this pathway in developing systems is controversial. Here we have analyzed the function of TGF␤ signaling in the differentiation of the developing limb mesoderm in vivo and in high density micromass cultures. In these systems highest signaling activity corresponded with cells at stages preceding overt chondrocyte differentiation. Interestingly treatments with TGF␤s shifted the differentiation outcome of the cultures from chondrogenesis to fibrogenesis. This phenotypic reprogramming involved downregulation of Sox9 and Aggrecan and up-regulation of Scleraxis, and Tenomodulin through the Smad pathway. We further show that TGF␤ signaling up-regulates Sox9 in the in vivo experimental model system in which TGF␤ treatments induce ectopic chondrogenesis. Looking for clues explaining the dual role of TGF␤ signaling, we found that TGF␤s appear to be direct inducers of the chondrogenic gene Sox9, but the existence of transcriptional repressors of TGF␤ signaling modulates this role. We identified TGF-interacting factor Tgif1 and SKI-like oncogene SnoN as potential candidates for this inhibitory function. Tgif1 gene regulation by TGF␤ signaling correlated with the differential chondrogenic and fibrogenic effects of this pathway, and its expression pattern in the limb marks the developing tendons. In functional experiments we found that Tgif1 reproduces the profibrogenic effect of TGF␤ treatments. TGF␤s4 form a small family of multipotent regulatory polypeptides that gives the name to a large cytokine superfamily, which also includes Activins and bone morphogenetic proteins, characterized by structural and signaling similarities. In mammalians and birds the family is composed of three highly homologous isoforms, although the avian homologue of the mammalian TGF␤1 was initially named TGF␤4 (1). Signaling by these factors is mediated by ligand binding with type II and type I receptors to form heteromeric complexes, which phosphorylate Smad2 and -3 proteins. The activated p-Smads associate with Smad4 prior to translocation into the nucleus, where they regulate the expression of a variety of genes (see Ref. 2). TGF␤s may also signal through the mitogen-activated protein kinase (MAPK) transduction pathway (3).TGF␤s are considered major regulators of the differentiation and growth of the skeletal connective tissues, with a promising future in regenerative medicine as tools to modulate the differentiation of stem cells for reconstruction of cartilage, tendon, or bone. In the long appendicular bones TGF␤s are expressed in the chondrocytes of the growth plate and regulate the rate of differentiation of prehypertrophic chondrocytes to hypertrophic chondrocytes (4 -7). In embryonic systems, TGF␤s are expressed in the developing limb forming well defined domains in the digit chondrogenic aggregates, in the developing joints and in the differentiating tendon blastema...

Here, we have studied how Sox genes and BMP signaling are functionally coupled during limb chondrogenesis. Using the experimental model of TGFbeta1-induced interdigital digits, we dissect the sequence of morphological and molecular events during in vivo chondrogenesis. Our results show that Sox8 and Sox9 are the most precocious markers of limb cartilage, and their induction is independent and precedes the activation of BMP signaling. Sox10 appears also to cooperate with Sox9 and Sox8 in the establishment of the digit cartilages. In addition, we show that experimental induction of Sox gene expression in the interdigital mesoderm is accompanied by loss of the apoptotic response to exogenous BMPs. L-Sox5 and Sox6 are respectively induced coincident and after the expression of Bmpr1b in the prechondrogenic aggregate, and their activation correlates with the induction of Type II Collagen and Aggrecan genes in the differentiating cartilages. The expression of Bmpr1b precedes the appearance of morphological changes in the prechondrogenic aggregate and establishes a landmark from which the maintenance of the expression of all Sox genes and the progress of cartilage differentiation becomes dependent on BMPs. Moreover, we show that Ventroptin precedes Noggin in the modulation of BMP activity in the developing cartilages. In summary, our findings suggest that Sox8, Sox9, and Sox10 have a cooperative function conferring chondrogenic competence to limb mesoderm in response to BMP signals. In turn, BMPs in concert with Sox9, Sox6, and L-Sox5 would be responsible for the execution and maintenance of the cartilage differentiation program.

In the final stages of limb morphogenesis, autopodial cells leaving the progress zone differentiate into cartilage or undergo apoptotic cell death, depending on whether they are incorporated into the digital rays or interdigital spaces. Most evidence indicates that these two opposite fates of the autopodial mesoderm are controlled by BMP signaling. However, the molecular basis for these two distinct actions of BMPs, including the receptors involved in the process, is controversial. In this study we have addressed this question by exploring the presence in the developing autopod of diffusible signals able to modulate BMP function and by analyzing the effects of their exogenous administration on the pattern of expression of BMP receptor genes. Our findings show that tgfbeta2 and noggin genes are expressed in the condensing region of the developing digital rays in addition to the well-known distribution in the autopodial tissues of FGFs (apical ectodermal ridge, AER) and BMPs (AER, progress zone mesoderm, and interdigital regions). Exogenous administration of all the factors causes changes in the expression of the bmpR-1b gene which are followed by parallel alterations of the skeletal phenotype: FGFs inhibit the expression of bmpR-1b compatible with their function in the maintenance of the progress zone mesoderm in an undifferentiated state; and TGFbetas induce the expression of bmpR-1b and promote ectopic chondrogenesis, compatible with a function in the establishment of the position of the digital rays. In addition we provide evidence for the occurrence of an interactive loop between BMPs and noggin accounting for the spatial distribution of bmpR-1b which may control the size and shape of the skeletal pieces. In contrast to the bmpR-1b gene, the bmpR-1a gene is expressed at low levels in the autopodial mesoderm and its expression is not modified by any of the tested factors regardless of their effects on chondrogenesis or cell death. Finally, the role of BMPs in programmed cell death is confirmed here by the intense inhibitory effect of noggin on apoptosis, but the lack of correlation between changes in the pattern of cell death induced by treatment with the studied factors and the expression of either bmpR-1a or bmpR-1b genes suggest that a still-unidentified BMP receptor may account for this BMP function.

Bone morphogenetic proteins (BMPs) constitute a large family of secreted signals involved in the formation of the skeleton but the specific function of each member of this family remains elusive. GDF-5 is a member of the BMP family which has been implicated in several skeletogenic events including the induction and growth of the appendicular cartilages, the determination of joint forming regions, and the establishment of tendons. Here, we have studied the function of GDF-5 in digit skeletogenesis by analyzing the effects of its local administration in the developing autopod of embryonic chick and the regulation of its pattern of gene expression by other signals involved in digit morphogenesis. As reported in the mouse, the gdf-5 gene exhibits a precise distribution in the joint-forming regions of the developing chicken digital rays. GDF-5 beads implanted at the tip of the digits promote intense cartilage growth and fail to induce morphological or molecular signs of joint formation. Furthermore, GDF-5 beads implanted in the interdigits inhibit the formation of joints in the adjacent digits. These data suggest that the role of GDF-5 in joint formation is the control of growth and differentiation of the cartilage of the epiphyseal regions of the phalanges rather than accounting for the differentiation of the sinovial joint tissues. The interdigital mesoderm in spite of its potential to form ectopic digits with their tendinous apparatus failed to form either ectopic cartilages or ectopic tendons after the implantation of GDF-5 beads in the stages preceding cell death. At difference with other BMPs, GDF-5 exhibited only a weak cell death promoting effect. The BMP antagonist Noggin binds to GDF-5 and is able to inhibit all the observed effects of this growth factor in vivo. Potential interactions of GDF-5 with other signals involved in digits morphogenesis were also explored. BMP-7 regulates negatively the expression of gdf-5 gene in the joint forming regions and local treatment with Noggin induces the ectopic expression of gdf-5 in the interdigital mesoderm. Retroviral-induced misexpression of Indian or Sonic Hedgehog genes in the developing digits leads to the formation of digits without joints in which gdf-5 expression occurs throughout the entire perichondrial surface. In conclusion, this study indicates that GDF-5 is a signal regulated by other BMPs which controls the growth and differentiation of the epiphyses of the digital cartilages acting in close relationship with Hedgehog signaling.