Lysosomes, caspase-mediated apoptosis, and cytoplasmic activation of P21, but not cell senescence, participate in a redundant fashion in embryonic morphogenetic cell death.

Micromass cultures of embryonic limb skeletal progenitors replicate the tissue remodelling processes observed during digit morphogenesis. Here, we have employed micromass cultures in an in vitro assay to study the nature of cell degeneration events associated with skeletogenesis. In the assay, "naive" progenitors obtained from the autopod aggregate to form chondrogenic nodules and those occupying the internodular spaces exhibit intense apoptosis and progressive accumulation of larger cells, showing intense SA-ß-Gal histochemical labelling that strictly overlaps with the distribution of neutral red vital staining. qPCR analysis detected intense upregulation of the p21 gene, but P21 immunolabelling showed cytoplasmic rather than the nuclear distribution expected in senescent cells. Semithin sections and transmission electron microscopy confirmed the presence of canonical apoptotic cells, degenerated cell fragments in the process of phagocytic internalization by the neighbouring cells, and large vacuolated cells containing phagosomes. The immunohistochemical distribution of active caspase 3, cathepsin D, and ß-galactosidase together with the reduction in cell death by chemical inhibition of caspases (Q-VAD) and lysosomal cathepsin D (Pepstatin A) supported a redundant implication of both pathways in the dying process. Chemical inhibition of P21 (UC2288) revealed a complementary role of this factor in the dying process. In contrast, treatment with the senolytic drug Navitoclax increased cell death without changing the number of cells positive for SA-ß-Gal. We propose that this model of tissue remodelling involves the cooperative activation of multiple degradation routes and, most importantly, that positivity for SA-ß-Gal reflects the occurrence of phagocytosis, supporting the rejection of cell senescence as a defining component of developmental tissue remodelling.

Regulation of Developmental Cell Death in the Animal Kingdom: A Critical Analysis of Epigenetic versus Genetic Factors.

The present paper proposes a new level of regulation of programmed cell death (PCD) in developing systems based on epigenetics. We argue against the traditional view of PCD as an altruistic "cell suicide" activated by specific gene-encoded signals with the function of favoring the development of their neighboring progenitors to properly form embryonic organs. In contrast, we propose that signals and local tissue interactions responsible for growth and differentiation of the embryonic tissues generate domains where cells retain an epigenetic profile sensitive to DNA damage that results in its subsequent elimination in a fashion reminiscent of what happens with scaffolding at the end of the construction of a building. Canonical death genes, including Bcl-2 family members, caspases, and lysosomal proteases, would reflect the downstream molecular machinery that executes the dying process rather than being master cell death regulatory signals.

Transforming growth factor beta signaling: The master sculptor of fingers.

Transforming growth factor beta (TGFß) constitutes a large and evolutionarily conserved superfamily of secreted factors that play essential roles in embryonic development, cancer, tissue regeneration, and human degenerative pathology. Studies of this signaling cascade in the regulation of cellular and tissue changes in the three-dimensional context of a developing embryo have notably advanced in the understanding of the action mechanism of these growth factors. In this review, we address the role of TGFß signaling in the developing limb, focusing on its essential function in the morphogenesis of the autopod. As we discuss in this work, modern mouse genetic experiments together with more classical embryological approaches in chick embryos, provided very valuable information concerning the role of TGFß and Activin family members in the morphogenesis of the digits of tetrapods, including the formation of phalanxes, digital tendons, and interphalangeal joints. We emphasize the importance of the Activin and TGFß proteins as digit inducing factors and their critical interaction with the BMP signaling to sculpt the hand and foot morphology.

Modeling the Differentiation of Embryonic Limb Chondroprogenitors by Cell Death and Cell Senescence in High Density Micromass Cultures and Their Regulation by FGF Signaling.

Considering the importance of programmed cell death in the formation of the skeleton during embryonic development, the aim of the present study was to analyze whether regulated cell degeneration also accompanies the differentiation of embryonic limb skeletal progenitors in high-density tridimensional cultures (micromass cultures). Our results show that the formation of primary cartilage nodules in the micromass culture assay involves a patterned process of cell death and cell senescence, complementary to the pattern of chondrogenesis. As occurs in vivo, the degenerative events were preceded by DNA damage detectable by γH2AX immunolabeling and proceeded via apoptosis and cell senescence. Combined treatments of the cultures with growth factors active during limb skeletogenesis, including FGF, BMP, and WNT revealed that FGF signaling modulates the response of progenitors to signaling pathways implicated in cell death. Transcriptional changes induced by FGF treatments suggested that this function is mediated by the positive regulation of the genetic machinery responsible for apoptosis and cell senescence together with hypomethylation of the Sox9 gene promoter. We propose that FGF signaling exerts a primordial function in the embryonic limb conferring chondroprogenitors with their biological properties.

Histone Epigenetic Signatures in Embryonic Limb Interdigital Cells Fated to Die.

During limb formation in vertebrates with free digits, the interdigital mesoderm is eliminated by a massive degeneration process that involves apoptosis and cell senescence. The degradation process is preceded by intense DNA damage in zones located close to methylated DNA, accompanied by the activation of the DNA repair response. In this study, we show that trimethylated histone 3 (H3K4me3, H3K9me3, and H3K27me3) overlaps with zones positive for 5mC in the nuclei of interdigital cells. This pattern contrasts with the widespread distribution of acetylated histones (H3K9ac and H4ac) and the histone variant H3.3 throughout the nucleoplasm. Consistent with the intense labeling of acetylated histones, the histone deacetylase genes Hdac1, Hdac2, Hdac3, and Hdac8, and at a more reduced level, Hdac10, are expressed in the interdigits. Furthermore, local treatments with the histone deacetylase inhibitor trichostatin A, which promotes an open chromatin state, induces massive cell death and transcriptional changes reminiscent of, but preceding, the physiological process of interdigit remodeling. Together, these findings suggest that the epigenetic profile of the interdigital mesoderm contributes to the sensitivity to DNA damage that precedes apoptosis during tissue regression.

Cell death in the developing vertebrate limb: A locally regulated mechanism contributing to musculoskeletal tissue morphogenesis and differentiation.

Our aim is to critically review current knowledge of the function and regulation of cell death in the developing limb. We provide a detailed, but short, overview of the areas of cell death observed in the developing limb, establishing their function in morphogenesis and structural development of limb tissues. We will examine the functions of this process in the formation and growth of the limb primordia, formation of cartilaginous skeleton, formation of synovial joints, and establishment of muscle bellies, tendons, and entheses. We will analyze the plasticity of the cell death program by focusing on the developmental potential of progenitors prior to death. Considering the prolonged plasticity of progenitors to escape from the death process, we will discuss a new biological perspective that explains cell death: this process, rather than secondary to a specific genetic program, is a consequence of the tissue building strategy employed by the embryo based on the formation of scaffolds that disintegrate once their associated neighboring structures differentiate.

STAT3 and STAT5B Mutations in T/NK-Cell Chronic Lymphoproliferative Disorders of Large Granular Lymphocytes (LGL): Association with Disease Features.

STAT3 and STAT5B (STAT3/STAT5B) mutations are the most common mutations in T-cell large granular lymphocytic leukemia (T-LGLL) and chronic lymphoproliferative disorders of NK cells (CLPD-NK), but their clinical impact remains unknown. We investigated the frequency and type of STAT3/STAT5B mutations in FACS-sorted populations of expanded T/NK-LGL from 100 (82 clonal; 6 oligoclonal; 12 polyclonal) patients, and its relationship with disease features. Seventeen non-LGL T-CLPD patients and 628 age-matched healthy donors were analyzed as controls. STAT3 (n = 30) and STAT5B (n = 1) mutations were detected in 28/82 clonal T/NK-LGLL patients (34%), while absent (0/18, 0%) among oligoclonal/polyclonal LGL-lymphocytosis. Mutations were found across all diagnostic subgroups: TCD8+-LGLL, 36%; CLPD-NK, 38%; TCD4+-LGLL, 7%; Tαß+DP-LGLL, 100%; Tαß+DN-LGLL, 50%; Tγδ+-LGLL, 44%. STAT3-mutated T-LGLL/CLPD-NK showed overall reduced (p < 0.05) blood counts of most normal leukocyte subsets, with a higher rate (vs. nonmutated LGLL) of neutropenia (p = 0.04), severe neutropenia (p = 0.02), and cases requiring treatment (p = 0.0001), together with a shorter time-to-therapy (p = 0.0001), particularly in non-Y640F STAT3-mutated patients. These findings confirm and extend on previous observations about the high prevalence of STAT3 mutations across different subtypes of LGLL, and its association with a more marked decrease of all major blood-cell subsets and a shortened time-to-therapy.

Confluence of Cellular Degradation Pathways During Interdigital Tissue Remodeling in Embryonic Tetrapods.

Digits develop in the distal part of the embryonic limb primordium as radial prechondrogenic condensations separated by undifferentiated mesoderm. In a short time interval the interdigital mesoderm undergoes massive degeneration to determine the formation of free digits. This fascinating process has often been considered as an altruistic cell suicide that is evolutionarily-regulated in species with different degrees of digit webbing. Initial descriptions of interdigit remodeling considered lysosomes as the primary cause of the degenerative process. However, the functional significance of lysosomes lost interest among researcher and was displaced to a secondary role because the introduction of the term apoptosis. Accumulating evidence in recent decades has revealed that, far from being a unique method of embryonic cell death, apoptosis is only one among several redundant dying mechanisms accounting for the elimination of tissues during embryonic development. Developmental cell senescence has emerged in the last decade as a primary factor implicated in interdigit remodeling. Our review proposes that cell senescence is the biological process identified by vital staining in embryonic models and implicates lysosomes in programmed cell death. We review major structural changes associated with interdigit remodeling that may be driven by cell senescence. Furthermore, the identification of cell senescence lacking tissue degeneration, associated with the maturation of the digit tendons at the same stages of interdigital remodeling, allowed us to distinguish between two functionally distinct types of embryonic cell senescence, "constructive" and "destructive."

UHRF genes regulate programmed interdigital tissue regression and chondrogenesis in the embryonic limb.

The primordium of the limb contains a number of progenitors far superior to those necessary to form the skeletal components of this appendage. During the course of development, precursors that do not follow the skeletogenic program are removed by cell senescence and apoptosis. The formation of the digits provides the most representative example of embryonic remodeling via cell degeneration. In the hand/foot regions of the embryonic vertebrate limb (autopod), the interdigital tissue and the zones of interphalangeal joint formation undergo massive degeneration that accounts for jointed and free digit morphology. Developmental senescence and caspase-dependent apoptosis are considered responsible for these remodeling processes. Our study uncovers a new upstream level of regulation of remodeling by the epigenetic regulators Uhrf1 and Uhrf2 genes. These genes are spatially and temporally expressed in the pre-apoptotic regions. UHRF1 and UHRF2 showed a nuclear localization associated with foci of methylated cytosine. Interestingly, nuclear labeling increased in cells progressing through the stages of degeneration prior to TUNEL positivity. Functional analysis in cultured limb skeletal progenitors via the overexpression of either UHRF1 or UHRF2 inhibited chondrogenesis and induced cell senescence and apoptosis accompanied with changes in global and regional DNA methylation. Uhrfs modulated canonical cell differentiation factors, such as Sox9 and Scleraxis, promoted apoptosis via up-regulation of Bak1, and induced cell senescence, by arresting progenitors at the S phase and upregulating the expression of p21. Expression of Uhrf genes in vivo was positively modulated by FGF signaling. In the micromass culture assay Uhrf1 was down-regulated as the progenitors lost stemness and differentiated into cartilage. Together, our findings emphasize the importance of tuning the balance between cell differentiation and cell stemness as a central step in the initiation of the so-called "embryonic programmed cell death" and suggest that the structural organization of the chromatin, via epigenetic modifications, may be a precocious and critical factor in these regulatory events.

Cell senescence, apoptosis and DNA damage cooperate in the remodeling processes accounting for heart morphogenesis.

During embryonic development, organ morphogenesis requires major tissue rearrangements that are tightly regulated at the genetic level. A large number of studies performed in recent decades assigned a central role to programmed cell death for such morphogenetic tissue rearrangements that often sculpt the shape of embryonic organs. However, accumulating evidence indicates that far from being the only factor responsible for sculpting organ morphology, programmed cell death is accompanied by other tissue remodeling events that ensure the outcome of morphogenesis. In this regard, cell senescence has been recently associated with morphogenetic degenerative embryonic processes as an early tissue remodeling event in development of the limbs, kidney and inner ear. Here, we have explored cell senescence by monitoring ß-galactosidase activity during embryonic heart development where programmed cell death is believed to exert an important morphogenetic function. We report the occurrence of extensive cell senescence foci during heart morphogenesis. These foci overlap spatially and temporally with the areas of programmed cell death that are associated with remodeling of the outflow tract to build the roots of the great arteries and with the septation of cardiac cavities. qPCR analysis allowed us to identify a gene expression profile characteristic of the so-called senescence secretory associated phenotype in the remodeling outflow tract of the embryonic heart. In addition, we confirmed local upregulation of numerous tumor suppressor genes including p21, p53, p63, p73 and Btg2. Interestingly, the areas of cell senescence were also accompanied by intense lysosomal activation and non-apoptotic DNA damage revealed by γH2AX immunolabeling. Considering the importance of sustained DNA damage as a triggering factor for cell senescence and apoptosis, we propose the coordinated contribution of DNA damage, senescence and apoptotic cell death to assure tissue remodeling in the developing vertebrate heart.

Interdigital tissue remodelling in the embryonic limb involves dynamic regulation of the miRNA profiles.

Next-generation sequencing in combination with quantitative polymerase chain reaction analysis revealed a dynamic miRNA signature in the interdigital mesoderm of the chick embryonic hinlimb in the course of interdigit remodelling. During this period, 612 previously known chicken miRNAs (gga-miRNAs) and 401 non-identified sequences were expressed in the interdigital mesoderm. Thirty-six microRNAs, represented by more than 750 reads per million, displayed differential expression between stages HH29 (6 id) and HH32 (7.5 id), which correspond to the onset and the peak of interdigital cell death. Twenty miRNAs were upregulated by at least 1.5-fold, and sixteen were downregulated by at least 0.5-fold. Upregulated miRNAs included miRNAs with recognized proapoptotic functions in other systems (miR-181 family, miR-451 and miR-148a), miRNAs associated with inflammation and cell senescence (miR-21 and miR-146) and miRNAs able to induce changes in the extracellular matrix (miR-30c). In contrast, miRNAs with known antiapoptotic effects in other systems, such as miR-222 and miR-205, became downregulated. In addition, miR-92, an important positive regulator of cell proliferation, was also downregulated. Together, these findings indicate a role for miRNAs in the control of tissue regression and cell death in a characteristic morphogenetic embryonic process based on massive apoptosis.

Sox9 Expression in Amniotes: Species-Specific Differences in the Formation of Digits.

In tetrapods the digit pattern has evolved to adapt to distinct locomotive strategies. The number of digits varies between species or even between hindlimb and forelimb within the same species. These facts illustrate the plasticity of embryonic limb autopods. Sox9 is a precocious marker of skeletal differentiation of limb mesenchymal cells. Its pattern of expression in the developing limb has been widely studied and reflects the activity of signaling cascades responsible for skeletogenesis. In this assay we stress previously overlooked differences in the pattern of expression of Sox9 in limbs of avian, mouse and turtle embryos which may reflect signaling differences associated with distinct limb skeletal morphologies observed in these species. Furthermore, we show that Sox9 gene expression is higher and maintained in the interdigital region in species with webbed digits in comparison with free digit animals.

MRD detection in multiple myeloma: comparison between MSKCC 10-color single-tube and EuroFlow 8-color 2-tube methods.

In patients with multiple myeloma, obtaining posttreatment minimal residual disease (MRD) negativity is associated with longer progression-free survival and overall survival. Here, we compared the diagnostic performance of a single 10-color tube with that of a EuroFlow 8-color 2-tube panel for MRD testing. Bone marrow samples from 41 multiple myeloma patients were tested in parallel using the 2 approaches. Compared with the sum of the cells from the EuroFlow two 8-color tubes, the Memorial Sloan Kettering Cancer Center (MSKCC) single 10-color tube had a slight reduction in total cell number with a mean ratio of 0.85 (range, 0.57-1.46; P < .05), likely attributable to permeabilization of the cells. Percent of plasma cells showed a high degree of concordance (r2 = 0.97) as did normal plasma cells (r2 = 0.96), consistent with no selective plasma cell loss. Importantly, concordant measurement of residual disease burden was seen with abnormal plasma cells (r2 = 0.97). The overall concordance between the 2 tests was 98%. In 1 case, there was a discrepancy near the limit of detection of both tests in favor of the slightly greater theoretical sensitivity of the EuroFlow 8-color 2-tube panel (analytical sensitivity limit of MSKCC single 10-color tube: 6 cells in 1 million with at least 3 million cell acquisitions; EuroFlow 8-color 2-tube panel: 2 cells in 1 million with the recommended 10 million cell acquisitions).

DNA damage precedes apoptosis during the regression of the interdigital tissue in vertebrate embryos.

DNA damage independent of caspase activation accompanies programmed cell death in different vertebrate embryonic organs. We analyzed the significance of DNA damage during the regression of the interdigital tissue, which sculpts the digits in the embryonic limb. Interdigit remodeling involves oxidative stress, massive apoptosis and cell senescence. Phosphorylation of H2AX mediated by ATM precedes caspase dependent apoptosis and cell senescence during interdigit regression. The association of γH2AX with other downstream DNA repair factors, including MDC1, Rad50 and 53BP1 suggests a defensive response of cells against DNA damage. The relative distribution of cells γH2AX-only positive, TUNEL-only positive, and cells double positive for both markers is consistent with a sequence of degenerative events starting by damage of the DNA. In support of this interpretation, the relative number of γH2AX-only cells increases after caspase inhibition while the relative number of TUNEL-only cells increases after inhibition of ATM. Furthermore, cultured interdigits survived and maintained intense chondrogenic potential, even at advanced stages of degeneration, discarding a previous commitment to die. Our findings support a new biological paradigm considering embryonic cell death secondary to genotoxic stimuli, challenging the idea that considers physiological cell death a cell suicide regulated by an internal death clock that pre-programmes degeneration.

The tumor suppressor BTG1 is expressed in the developing digits and regulates skeletogenic differentiation of limb mesodermal progenitors in high density cultures.

In the developing limb, differentiation of skeletal progenitors towards distinct connective tissues of the digits is correlated with the establishment of well-defined domains of Btg1 gene expression. Zones of high expression of Btg1 include the earliest digit blastemas, the condensing mesoderm at the tip of the growing digits, the peritendinous mesenchyme, and the chondrocytes around the developing interphalangeal joints. Gain- and loss-of function experiments in micromass cultures of skeletal progenitors reveal a negative influence of Btg1 in cartilage differentiation accompanied by up-regulation of Ccn1, Scleraxis and PTHrP. Previous studies have assigned a role to these factors in the aggregation of progenitors in the digit tips (Ccn1), in the differentiation of tendon blastemas (Scleraxis) and repressing hypertrophic cartilage differentiation (PTHrP). Overexpression of Btg1 up-regulates the expression of retinoic acid and thyroid hormone receptors, but, different from other systems, the influence of BTG1 in connective tissue differentiation appears to be independent of retinoic acid and thyroid hormone signaling.

Apoptosis during embryonic tissue remodeling is accompanied by cell senescence.

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 ß-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.

Interdigital tissue regression in the developing limb of vertebrates.

Here we have chosen the regression of the interdigital tissue which sculpts the digits from the hand/foot plate in tetrapod embryos to review the most relevant aspects concerning the regulation and biological significance of programmed cell death. We gather abundant information showing that the initiation of the degenerative process is the result of a complex interplay between the different signaling pathways which are also responsible for limb outgrowth and skeletal tissue differentiation, rather than being regulated by a specific signaling pathway. The model further shows that once the death response is triggered, several different routes of cell disruption, including caspase-dependent apoptosis, lysosomal-mediated cell death, and even a cell senescence process, are activated in the interdigits to ensure their elimination. Transcriptional and structural changes accompanying the degenerative process, and their posible contribution to the control of the death process, are also revised in detail. Finally we survey a number of issues still awaiting clarification, such as the functional implication of interdigital cell death as a source of signals acting on the surrounding tissues, as occurs in the so called "regenerative cell death".

Decorin gene expression in the differentiation of the skeletal connective tissues of the developing limb.

Tendons and cartilages are connective tissues of essential importance in the musculoskeletal system. During digit development, cartilage and tendon primordia develop from the undifferentiated mesenchymal cells originated in the lateral plate mesoderm. The specification of these tissues begins with the establishment of cellular aggregates, which prefigure the tendons and phalanges. Transforming growth factor beta proteins (TGFßs) are the inductive signals responsible not only for the initiation of chondrogenesis and tenogenesis during digit formation, but also for joint specification. An early role of this family of secreted proteins during these processes is to promote mesenchymal cell precursors condensation. Here we show that Decorin presents an overlapping pattern of expression with TGFß2 in joint and tendon blastemas of the embryonic digits. Furthermore, Decorin expression is induced by TGFß signaling, and DECORIN promotes aggregation of digit mesenchymal cell precursors. In addition, we provide gene expression studies suggesting that Cadherin-11 may function as an effector of Decorin in this experimental model.

Reelin/DAB-1 signaling in the embryonic limb regulates the chondrogenic differentiation of digit mesodermal progenitors.

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.

Divergent differentiation of skeletal progenitors into cartilage and tendon: lessons from the embryonic limb.

Repairing damaged cartilage and tendons is a major challenge of regenerative medicine. There has been great progress in the past decade toward obtaining stem cells for regenerative purposes from a variety of sources. However, the development of procedures to direct and maintain the differentiation of progenitors into cartilage or tendon is still a hurdle to overcome in regenerative medicine of the musculoskeletal system. This is because connective tissues often lack stable phenotypes and retain plasticity to return to the initial stages of differentiation or to transdifferentiate into another connective tissue cell lineage. This makes it necessary to unravel the molecular basis that is responsible for the differentiation of connective tissue cell lineages. In this review, we summarize the investigations performed in the past two decades to unravel the signals that regulate the differentiation of skeletal cell progenitors into cartilage and tendons during embryonic limb development. The data obtained in those studies demonstrate that Tgfß, BMP, FGF, and Wnt establish a complex signaling network that directs the differentiation of skeletal cell progenitors. Remarkably, in the embryonic digit model, the divergent differentiation of progenitors depends on the temporal coordination of those signals, rather than being specified by an individual signaling pathway. Due to its potential medical relevance, we highlight the importance of the coordinate influence of the Tgfß and BMP pathways in the differentiation of cell progenitors into tendon or cartilage.