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...

The progress zone (PZ) is a specialized area at the distal margin of the developing limb where mesodermal cells are kept in proliferation and undifferentiated, allowing limb outgrowth. At stages of digit morphogenesis the PZ cells can undergo two possible fates, either aggregate initiating chondrogenic differentiation to configure the digit blastemas, or to die by apoptosis if they are incorporated in the interdigital mesenchyme. While both processes are controlled by bone morphogenetic proteins (BMPs) the molecular basis for such contrasting differential behavior of the autopodial mesoderm remains unknown. Here we show that a well-defined crescent domain of high BMP activity located at the tip of the forming digits, which we termed the digit crescent (DC), directs incorporation and differentiation of the PZ mesenchymal cells into the digit aggregates. The presence of this domain does not correlate with an exclusive expression domain of BMP receptors and its abrogation by surgical approaches or by local application of BMP antagonists is followed by digit truncation and cell death. We further show that establishment of the DC is directed by Activin/TGFbeta signaling, which inhibits Smad 6 and Bambi, two specific BMP antagonists expressed in the interdigits and progress zone mesoderm. The interaction between Activin/TGFbeta and BMP pathways at the level of DC promotes the expression of the chondrogenic factor SOX9 accompanied by a local decrease in cell proliferation. Characteristically, the DC domain is asymmetric, it being extended towards the posterior interdigit. The presence of the DC is transitorily dependent of the adjacent posterior interdigit and its maintenance requires also the integrity of the AER.

This study re-examined the dying process in the interdigital tissue during the formation of free digits in the developing limbs. We demonstrated that the interdigital dying process was associated with cell senescence, as deduced by induction of β-gal activity, mitotic arrest, and transcriptional up-regulation of p21 together with many components of the senescence-associated secretory phenotype. We also found overlapping domains of expression of members of the Btg/Tob gene family of antiproliferative factors in the regressing interdigits. Notably, Btg2 was up-regulated during interdigit remodeling in species with free digits but not in the webbed foot of the duck. We also demonstrate that oxidative stress promoted the expression of Btg2, and that FGF2 and IGF1 which are survival signals for embryonic limb mesenchyme inhibited Btg2 expression. Btg2 overexpression in vivo and in vitro induced all the observed changes during interdigit regression, including oxidative stress, arrest of cell cycle progression, transcriptional regulation of senescence markers, and caspase-mediated apoptosis. Consistent with the central role of p21 on cell senescence, the transcriptional effects induced by overexpression of Btg2 are attenuated by silencing p21. Our findings indicate that cell senescence and apoptosis are complementary processes in the regression of embryonic tissues and share common regulatory signals.

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.