Critical Review. Molecular Control of Cell Differentiation and Programmed Cell Death during Digit Development

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1 IUBMB Life, 63(10): , October 2011 Critical Review Molecular Control of Cell Differentiation and Programmed Cell Death during Digit Development Jesús Chimal-Monroy 1, René Fernando Abarca-Buis 1,* Rodrigo Cuervo 1,2, Martha Díaz-Hernández 1, Marcia Bustamante 1, Jesús Alberto Rios-Flores 1, Silvina Romero-Suárez 1 and Alejandro Farrera-Hernández 1 1 Departamento de Medicina Genómica y Toxicología Ambiental, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México Ciudad Universitaria. Apartado Postal México 2 Laboratorio de Biotecnología, Facultad de Ciencias Biológicas y Agropecuarias, Universidad Veracruzana, Carretera Tuxpan-Tampico Km 7.5 Veracruz, México Summary During the hand plate development, the processes of cell differentiation and control of cell death are relevant to ensure a correct shape of the limb. The progenitor cell pool that later will differentiate into cartilage to form the digits arises from undifferentiated mesenchymal cells beneath the apical ectodermal ridge (AER). Once these cells abandon the area of influence of signals from AER and ectoderm, some cells are committed to chondrocyte lineage forming the digital rays. However, if the cells are not committed to chondrocyte lineage, they will form the prospective interdigits that in species with free digits will subsequently die. In this work, we provide the overview of the molecular interactions between different signaling pathways responsible for the formation of digit and interdigit regions. In addition, we briefly describe some experiments concerning the most important signals responsible for promoting cell death. Finally, on the basis that the interdigital tissue has chondrogenic potential, we discuss the hypothesis that apoptotic-promoting signals might also act as antichondrogenic factors and chondrogenic factors might operate as anti-apoptotic factors. Ó 2011 IUBMB IUBMB Life, 63(10): , 2011 Keywords digit development; limb development; programmed cell death; chondrogenesis; Sox9; Wnt; FGF; Bmp; TGFb; Activin. Received 9 May 2011; accepted 18 July 2011 *Present Address: Laboratorio de Tejido Conjuntivo, Centro de Investigación y Atención al Quemado, Instituto Nacional de Rehabilitación. Av. México-Xochimilco 289, México DF, México. Address correspondence to: Dr. Jesús Chimal-Monroy, Departamento de Medicina Genómica y Toxicología Ambiental, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México Ciudad Universitaria, Apartado Postal México DF México. jchimal@unam.mx INTRODUCTION The number of digits varies from five to one in distinct species, and they can be webbed or not. The final shape of the limb is associated with the control of cell fate: to differentiate or to die. During digit formation, the undifferentiated mesenchymal cells underneath the apical ectodermal ridge (AER) receive signals from different signaling pathways that induce the formation of an alternated digit and interdigit pattern in which proliferation, differentiation, or cell death are important processes to determine the final shape of limbs (1). The AER is a specialized region of the ectoderm that rims the distal margin of the limb bud. The undifferentiated cells under the AER receive chondrogenic signals that prefigure digital rays flanked by undifferentiated tissue that will form the prospective interdigits that in species with free digits will subsequently die (2). MAINTENANCE OF UNDIFFERENTIATED STATE During the hand plate development of the vertebrate limb, the maintenance of undifferentiated state of mesenchymal cells underneath the AER is significant to maintain the progenitor cell pool that later will differentiate into cartilage to form the digits. Undifferentiated mesenchymal cells receive signals from ectoderm and AER. The AER releases proteins belonging to the family of fibroblast growth factors (FGFs) and (derived from a combination of wingless and Int1 genes) (WNT) that regulate the size of this cell population, from which the skeletal elements will originate, controlling the maintenance of undifferentiated state of mesenchymal cells, and inhibiting cell differentiation and cell death (3 5). Meanwhile, limb ectoderm releases WNT proteins that induce cell proliferation, mediated by N-Myc, and inhibit cartilage differentiation by stabilizing the activity of bcatenin, which in turn inhibits Sox9 expression (3). Interestingly, the synergistic ISSN print/issn online DOI: /iub.563

2 MOLECULAR INTERACTIONS DURING DIGIT DEVELOPMENT 923 Figure 1. Schematic representation of different signaling pathways during digit development. For more details, see the text. activity of WNT and FGF signaling promotes proliferation of undifferentiated mesenchymal cells, while maintaining their undifferentiated state. WNT signaling induces the expression of its repressor, Axin2, while FGF signaling inhibits its expression, leading to the stimulation of the WNT activity (see Fig. 1). Both signals synergistically induce other genes such as Nmyc and Syndecan, while Sox9 is inhibited (3). When the cells abandon the area of influence of WNT and FGF signaling pathways, two events occur: an arrest of the cell cycle and the onset of differentiation of chondrocyte lineage forming the digital rays (3 5). If the cells that do not enter the chondrogenesis program remain exposed to ectodermal WNT, they undergo a respecification toward connective tissue lineages (3). Therefore, it is important to preserve a balance between the maintenance of an undifferentiated state of mesenchymal cells and the commitment to cell differentiation to ensure a correct shape of the limb. DIGIT DEVELOPMENT As the limb grows, the formation of skeletal elements arises from the progenitor cell pool that is recruited to chondrocyte lineage. Simultaneously, the other mesenchymal cells, which are not recruited to cartilage lineage, remain as undifferentiated cells forming the interdigital membrane. Cartilage differentiation or chondrogenesis initiates when the progenitor cell pool expresses Sox9, promoting the formation of cellular aggregates that prefigure skeletal elements. In addition to Sox9 expression, chondrocyte differentiation is characterized by the expression of Sox5 and Sox6 and consequently the expression of type II collagen, aggrecan, and sulfated proteoglycans (1). After the progenitor cell pool abandons the zone of undifferentiated mesenchymal cells, become responsive to different factors that are able to induce commitment to chondrocyte lineage. Members of the transforming growth factor b (TGFb) superfamily such as Activins A and B and TGFb2 are expressed in the development of digit primordia and act as key signals during the beginning of chondrogenesis (6, 7). The chondrogenic potential of Activin and TGFb has been experimentally evaluated, resulting in the activation of a molecular cascade that culminates with ectopic digit formation in the interdigital membrane (8). Under these conditions, TGFb induces expression of Sox9 after 30 min of treatment and of the bone morphogenetic protein receptor 1b (Bmpr1b) after 6 h. Bone morphogenetic protein (BMP) signaling is mediated by BMPR1A and BMPR1B receptors; when these receptors are inactivated or BMP antagonists are implanted in prechondrogenic mesenchyme of embryonic chick limb, cartilage differentiation is inhibited (9 17). Furthermore, the mutant mice for BMPs have cartilage differentiation deficiencies. In contrast, when BMP signaling is activated in the prechondrogenic mesoderm, cartilage differentiation is observed (6, 12, 15). The border of the growing digital region was called phalanxforming region (PFR) by John Fallon and coworkers (18) and digit crescent (DC) by Juan Hurlé and colleagues (19). DC/PFR presents high levels of signaling activity of BMP/SMAD1/5/8 and TGFb/Activin/SMAD2 (Smad genes are homologues to the sma and mad genes involved in TGFb signaling cascade) pathways. However, the domains of activity of BMP/SMAD1/5/8 are not restricted to DC/PFR, but are also present in the area surrounding undifferentiated mesenchymal cells underneath the AER. In DC/PFR, the activity of BMP/SMAD1/5/8 coincides with Sox9 expression (see Fig. 1). It is remarkable that after an experimental stimulus of BMP at the tip of the developing digit, the activity of BMP/SMAD1/5/8 signaling pathway was apparent in cells underneath the AER and close to the digit. However, expression of Sox9 occurred only in cells that are located in the proximo-lateral region in DC/PFR, far from AER (see Fig. 1). Meanwhile, the cells underneath AER, which did not express Sox9, carried out a process of massive cell death; this point will be discussed below. On the other hand, Juan Hurlé and colleagues (9), to demonstrate the participation of TGFb/Activin signaling in the formation of DC/PFR, induced the formation of an ectopic digit in the interdigital tissue. They found that SMAD2 was activated 30 min after the experimental stimulus by TGFb/Activin, simultaneously with the induction of Sox9 expression. The presence of SMAD2 activated was similar to that found at the tip of developing digits. Furthermore, the expression of Tgfb2 and Activinba in the developing digit coincides with DC/PFR, suggesting a synergistic role for both growth factors. The blockade of the Activin function with Follistatin at the tip of the developing digit inhibited the activity of BMP/SMAD1/5/8 signaling, preceded by the inhibition of SMAD2 activity, and resulting in digit truncation. In addition, TGFb/Activin signaling inhibits the expression of BMP antagonists, Smad6 and Bambi, suggesting that TGFb/Activin/SMAD2 and BMP/SMAD1/5/8 signaling might cooperate to induce the formation of cartilage during digit development. In addition, blocking BMP signaling at the

3 924 CHIMAL-MONROY ET AL. tip of developing digits leads to digit truncation as a consequence of inhibiting Sox9 expression. In fact, blocking BMP signaling coupled with TGFb or Activin treatment in the interdigital tissue resulted in the inhibition of Sox9 expression and, consequently, in delaying ectopic chondrogenesis, and a significant diminishing of the cartilage (19). Altogether, these results suggest that TGFb/Activin signaling induces the expression of Sox9 and later Bmpr1b, making prechondrogenic cells competent to BMP/SMAD1/5/8 signaling that induces Sox9. In contrast, in undifferentiated mesenchymal cells, BMP/SMAD1/ 5/8 signaling is able to induce cell death instead of inducing Sox9 expression (see Fig. 1). Furthermore, BMP treatment in the interdigital tissue induces massive cell death; however, if this treatment is done 3 h after treatment of TGFb, cell death is greatly reduced (8). It was mentioned above that undifferentiated mesenchymal cells under the AER are not committed to chondrocyte lineage because WNT/bcatenin signaling from ectoderm and AER inhibits expression of Sox9 and as a consequence chondrocyte commitment (3 5, 20). During limb outgrowth, once the undifferentiated mesenchymal cells leave the influence of WNT/ bcatenin signaling, Sox9 promotes bcatenin degradation in the chondrogenic-committed cells, resulting in inhibition of this signaling (20) (see Fig. 1). Therefore, it is possible to speculate that a gradient of WNT/bcatenin signaling is present in the region of undifferentiated mesenchymal cells. When the levels of WNT/bcatenin signaling are higher than TGFb/Activin/ SMAD2 signaling, bcatenin inhibits expression of Sox9 or can form a complex with it that causes its degradation, maintaining the undifferentiated state of mesenchymal cells. In contrast, when the levels of TGFb/Activin/SMAD2 signaling are higher than levels of WNT/bcatenin, the chondrogenic-committed cells express Sox9, which in turn promotes bcatenin degradation leading to the formation of DC/PFR. Another mechanism that inhibits WNT/bcatenin signaling occurs by the binding of WNT5A to the receptor tyrosine kinase Ror2 (21); this activation promotes the ubiquitination of bcatenin by Siah2 protein through noncanonical signaling in the limb (22). In addition to Ror2 signaling, Indian hedgehog signaling participates in BMP/ SMAD1/5/8 signaling allowing the elongation of digits (21). It is remarkable that in the absence of Sox9, mesenchymal cells remain in an undifferentiated state. Under these conditions, because there is not a chondrogenic commitment, instead of forming skeletal elements, massive cell death occurs (23). An interpretation of these observations is that in the absence of chondrogenic factors, mesenchymal cells are responsive to cell death signals (24). Furthermore, misexpression of Sox9 in wildtype animals induces extra-formation in developing limb buds and is able to rescue the normal phenotype of animals with hypodactyl (25). However, although we know many details about the digit outgrowth, less is known about how digits regulate the lateral growth toward the interdigits when the interdigit is competent to chondrogenic signals and can give rise to extra-digits. It is Figure 2. Gene Expression Pattern of Sox9 and Raldh2 at early stages of chick digit development. Whole mount in situ hybridization of Sox9 at stages 24HH (A, C), 26HH (B, D). Sox9 expression prefigures the chondrogenic condensations, at stage 24HH (A) the expression is localized in the posterior region of the limb, while at stage 26HH (B) its expression is extended to the anterior region as a continuous that includes the presumptive digits and interdigits. Raldh2 begins its expression in the prospective interdigital tissue at stage 24HH (C) and is more evident at stage 26HH (D). Analysis of Sox9 expression by PCR indicates that the transcripts are present in the interdigital tissue at different developing stages (E). clear that another fate of undifferentiated mesenchymal cells underneath AER that do not develop into cartilage lineage is to become interdigital membrane (1). Nevertheless, during embryonic chick limb development, the expression of Sox9 is observed as a continuous domain that includes the different digital primordia and prospective interdigits. In fact, Sox9 is present in low levels in the interdigital membrane of older stages but before the beginning of cell death (Fig. 2). Therefore, it is possible to speculate that there are factors that inhibit chon-

4 MOLECULAR INTERACTIONS DURING DIGIT DEVELOPMENT 925 drocyte differentiation in the interdigital membrane, and thus they become responsive to cell death signals (23). In addition, chondrogenic factors, such as Activin/TGFb signaling are able to inhibit cell death in embryonic chick limb before the formation of an ectopic digit (6) giving rise to the possibility that mesenchymal cells already committed to chondrocytic lineage are not responsive to cell death signals. Thus, it is possible that apoptotic factors are also antichondrogenic factors, and chondrogenic factors can be antiapoptotic factors. In the following section, we will describe some mechanisms that control programmed cell death (PCD) in the interdigital tissue. PROGRAMMED CELL DEATH IN THE LIMB MESODERM Pentadactyly is the ancestral digit formula in amniotes; however, during evolution many species have had a reduction in digit number that, in developmental studies, has been associated with the spatiotemporal control of PCD of mesenchymal cells (26, 27). In the developing chicken embryo, there are three regions of PCD that are associated with digit morphogenesis and digit number, in the anterior and posterior margins of the limbs, and in the interdigital tissue, called anterior necrotic zone, posterior necrotic zone, and interdigital necrotic zone (ANZ, PNZ, and INZ, respectively) (28, 29). The normal pattern of digit number in avian embryos is three digits for the wing and four for the leg, the absence of ANZ and PNZ in avian mutants, results in a polydactylous pattern (30 32). Formerly, the term necrotic, coined before the discovery of apoptosis, was widely used in the specialized literature; however, on the basis that current literature suggests that the most abundant type of cell death that takes place in these regions is by apoptosis, we suggest that these terms be changed to apoptotic. Therefore in this study, we use the terms anterior apoptotic zone, posterior apoptotic zone, and interdigital apoptotic zone (AAZ, PAZ, and IAZ) instead of ANZ, PNZ, and INZ, respectively. Apoptosis Apoptosis acts by two main pathways. In the first one, the intrinsic pathway, death signals act directly or indirectly on the mitochondria to cause the release of proapoptotic proteins from their intermembrane space (reviewed in 33). Inhibitors of apoptosis belong to the BCL2 family proteins; in survival conditions, these proteins bind to proapoptotic proteins BAX or BAK to avoid their oligomerization (33). Once apoptotic stimulus is triggered, proapoptotic BH3-only proteins BID, BIM, and PUMA activate homo-oligomerization of proapoptotic proteins BAX and BAK that promote the release of cytochrome c from mitochondria to the cytosol (34). Then cytochrome c interacts with datp, APAF-1 and procaspase 9 leading to apoptosome formation and the subsequent activation of Caspase 9, which in turn activates executioner caspases such as Caspase-3 (33). Meanwhile, the protein SMAC/DIABLO released from the mitochondria inhibits inhibitor of apoptosis protein (IAP) allowing the activation of caspases 9 and 3. IAP inhibits the activation of caspase 9 and caspase 3 (33). The intrinsic pathway is also able to act via a caspase-independent machinery, here occurs the release of two mitochondrial proteins, apoptosis inducing factor (AIF) and the endonuclease G (endog) that are translocated to the nucleus to promote chromatin condensation and formation of high-molecular DNA fragments (33). In the absence of the genes that encode BID, BIM, and PUMA, the activation of caspases mediated by cytochrome c does not occur, although diverse apoptotic stimuli were administered and even in the presence of other BH3-only molecules (34). The second pathway of apoptosis is the extrinsic, and requires the activation of death receptors, located on the cell surface, which leads to Caspase-8 activation, then caspase 7 and finally caspase 3, coinciding at this point with the intrinsic pathway (33). However, Caspase 8 activates BID that together with BIM, and PUMA evokes release of cytochrome c from mitochondria (34). Limbs of mice deficient in Bax and Bak or deficient in Bim, Bid, and Puma, lack of interdigital cell death, which leads to softtissue syndactyly (35, 36). On the contrary, mutant mice for any of the caspases that are active in the interdigital membrane (Caspase-2, Caspase-3, Caspase-6, Caspase-7, Caspase-8, or Caspase-9) or when they are suppressed by broad-spectrum caspase inhibitors, such as Z-VAD-FMK in chick limbs, or Apaf-1 2/2 single mutant mice do not exhibit inhibition of interdigital cell death (37 42); this suggests a mechanism of caspase-independent apoptosis. Although the expression of lysosomal cathepsins in mesenchymal cells committed to die has been demonstrated, the loss of the cathepsin function does not inhibit PCD in interdigital membranes (43, 44). Even so, if simultaneous experimental inhibition of both cathepsins and caspases is achieved, inhibition of interdigital cell death occurs, suggesting cooperation between extrinsic and intrinsic pathways mediating interdigital cell death (43). Indeed, overexpression of cathepsin D induces nuclear translocation of AIF in interdigital cell death (43), presumably by promoting permeabilization of the mitochondrial membrane mediated by the activation of BAX (45). Interestingly, in a recent work, Montero et al. (46) establish a model in which the interdigital cell death is initiated by the canonical caspase pathway leading to an initial activation of neutral endonucleases. Then in the second step, the expression of acidic nucleases are up regulated by action of cleavage of Serpin B1, whose gene is expressed at lower levels earlier, and at the beginning of, interdigital cell death, and later the Serpin B1 gene is upregulated, together with the progression of interdigital cell death. Remarkably, these molecular changes correlate with the interdigital ph acidification during tissue regression. In conclusion, interdigital cell death occurs by the coordinated and sequential activation of the caspase and lysosomal degenerative molecular cascades. Molecular cascade that leads to cell death To study the molecular mechanisms of cell death during limb development, it is important to distinguish between direct inducers of apoptosis in the interdigital tissue or other regions

5 926 CHIMAL-MONROY ET AL. of the limb, and inducers of a molecular cascade that ends in apoptosis. BMP signaling is an important regulator of cell death during limb development, as Bmp gene expression overlaps with AAZ, PAZ, and IAZ or precedes apoptosis (10 15). Functional studies show that activation of BMP signaling in the anterior, posterior, and interdigital mesodermal regions of the developing chick limb, promotes the onset and increase of cell death in these regions (6, 12, 15). In contrast, the blockade of BMP signaling results in syndactyly (16, 17, 47). Studies in the mouse conditional mutant BmpRIa 2/2 in the AER and interdigital mesoderm indicate that BMPRIA mediates PCD by negatively regulating Fgf8 in the AER, and/or directly mediates cell death in the interdigital mesoderm (11, 47 49). Besides, at the digit tip, BMP stimulates Sox9 expression in cells that are committed to chondrocyte lineage, but if they do not express Sox9, they carry out a process of massive cell death (23). Under these conditions, Smad8 expression and activation of SMAD1/5/8 signaling occurs in a similar way as it does in mesodermal cells during interdigital regression and in anterior mesoderm after BMP treatment (15, 19, 50). In addition, it was demonstrated that BMP induces activation of caspase 3 and SMAD1/5/8 signaling 10 h after treatment. Remarkably, a 4-h pulse of BMP7 is sufficient to activate caspase 3 in the AAZ with no evident activation of SMAD 1/5/8, which suggests that BMP/SMAD1/5/ 8 signaling activates a molecular cascade that culminates in PCD (50). On the other hand, BMP regulates the expression of Msx2, Bambi, Dkk, Snail, and Fgfr3, in the interdigital tissue or anterior regions of the chick limb, before apoptosis becoming evident (15, 51). Besides BMP signaling, retinoic acid (RA) also plays a pivotal function in the induction of cell death in the interdigital tissue. Two types of nuclear receptors, RAR and RXR (52, 53), mediate RA signaling. Compound mutant mice for these nuclear receptors display inhibition of cell death in IAZ and, in consequence, syndactyly (54, 55). The Hammertoe mutant mouse presents inhibition of cell death and syndactyly in all four limbs, whose normal phenotype that can be rescued by RA treatment of pregnant Hammertoe females (56). The interdigital cell death induced by RA is mediated by BMPs, since simultaneous treatment with RA and the BMP antagonist, Noggin, is unable to induce cell death (57). On the other hand, FGF signaling is a survival factor that protects mesenchyme from cell death (4, 58). Activation of BMP signaling leads to premature cell death, and downregulation of Fgf8 expression in the AER (59). In contrast, inactivation of BMP signaling produces upregulation of Fgf8 in the AER (see Fig. 1). However, BMPs are unable to promote cell death in the interdigital membrane if FGF signaling pathway is inhibited, suggesting that is necessary a cooperative function between BMP and FGF signaling pathways to control interdigital cell death (46). It might be that the presence of FGF signaling maintains mesenchymal cells in an undifferentiated state, where the role of BMP signaling would be to promote cell death. Whereas in the blocking of FGF signaling, the undifferentiated state of mesenchymal cells may be lost, in which case BMP signaling would not be able to induce cell death (46). The study by Hernandez-Martinez et al. (49) suggests that RA and FGF signaling have antagonistic interactions during the process of cell death in the interdigital tissue (see Fig. 1). They suggest that downregulation of Fgf8 in the AER is the first event that triggers cell death. FGF8 signaling antagonizes the RA signaling by inhibiting the expression of retinaldehyde dehydrogenase 2 (Raldh2), and which inhibits the biosynthesis of RA because RALDH2 metabolizes the conversion of retinal to RA. In addition, FGF8 induces the expression of Cy26b1, a member of the cytochrome P450 family, which catalyzes RA oxidation into a wide variety of inactive metabolites (see Fig. 1). In contrast, RA inhibits the expression of Fgfr1 antagonizing FGF signaling evidenced by a reduction of levels of phosphorylated Erk1/2. In addition, RA induces the expression of Bax that encodes the proapoptotic protein that participates in the release of cytochrome c from the mitochondria to cytosol. For a comprehensive review of interdigital cell death, see the works of Montero and Hurle (26) and Hernández-Martínez and Covarrubias (27). The presence of webbed digits observed in species such as the duck or bat is the consequence of cell death inhibition in the interdigital tissue by different mechanisms. In the hindlimb of duck embryos, the BMP signaling is inhibited by the action of Gremlin, a BMP antagonist that is expressed in interdigital mesenchyme (9). However, bat embryos, in addition to Gremlin, use a second mechanism to inhibit cell death in the interdigital membrane in which FGF and SHH signaling have been involved. Fgf8, Shh, and its target Patched are expressed in forelimb interdigital mesenchyme (60). Altogether, this suggests that high levels of FGF signaling and the inhibition of BMP signaling by Gremlin are responsible of the inhibition of cell death in the interdigital membrane. In addition, it has been hypothesized that FGF8 together with SHH contribute to the survival of interdigital tissue (60). CELL DIFFERENTIATION VERSUS INTERDIGITAL CELL DEATH A paradigm in digit development is to know the molecular mechanisms that lead to the establishment of digital and interdigital regions. It was discussed above that at early stages of chick limb development; Sox9 is expressed as a continuous domain that includes the digital primordia and prospective interdigits, while in later stages it gets restricted to the digital primordia, remaining at low levels in the interdigital tissue (Fig. 2). A possible interpretation for these observations is that Sox9-positive cells might be segregated from the non-sox9-positive cells (24). However, the observation that a TGFb stimulus in the interdigital tissue induces the expression of Sox9 30 min after treatment (8) leads to consider alternative interpretations. For example, it is possible to speculate that there are factors that inhibit chondrocyte differentiation in the interdigital

6 MOLECULAR INTERACTIONS DURING DIGIT DEVELOPMENT 927 Figure 3. TGFb inhibits interdigital cell death. The control limb shows cell death (A). Implantation of TGFb soaked beads at concentrations 10ng/ll (B), 25ng/ll (C) or 50ng/ll (D) on the interdigital tissue. Notice that TGFb inhibits cell death in a dose-dependent manner. membrane so the mesenchymal cells can be responsive to cell death signals. In addition, the stimulus of Activin/TGFb in the interdigit inhibits cell death before inducing the formation of an ectopic digit (6, 8). Taking into account this result, it can be proposed that once the mesenchymal cells are induced to chondrocyte lineage, they lose the ability to respond to death signals. Thus, we propose the hypothesis that apoptotic-promoting signals might also act as antichondrogenic factors and chondrogenic factors might operate as antiapoptotic factors. Regarding this idea, the inhibition of RA signaling in the interdigital tissue leads to the inhibition of interdigital cell death and to the induction of an ectopic digit, as it happens with the activation of Activin/TGFb signaling (6, 57). These data suggest that in physiological conditions, RA inhibits the interdigit chondrogenic potential. The mutant mouse model for synpolydactyly supports this idea; this animal model is characterized by the duplication of one or more digits and the fusion of two or more digits (61). This is a consequence of a mutation in the homeotic gene HoxD13 that is expressed during autopod development and that regulates the expression of many genes, included the Raldh2 that is expressed in the interdigital tissue. Consequently, the levels of this enzyme and RA production are low, as well as its downstream targets, such as RARb, dhand, Meis2, and Tbx5. A molecular analysis demonstrates that ectopic expression of Sox9 observed in interdigits was in some cases independent of mesenchymal condensation of digits, while in others the ectopic expression of Sox9 overlaps with the expression of Sox9 of digital primordia. The authors conclude that cartilage formation observed in the mutants does not represent true ectopic digits but rather represents uncontrolled process of chondrocyte differentiation. Significantly, the intrauterine RA treatment of animals with synpolydactyly rescues the normal phenotype (61). The suggestion that the chondrogenic factors might operate as antiapoptotic factors is supported by the observation that when Sox9 is conditionally deleted from undifferentiated mesenchymal cells of limb buds, the result is a loss of digit formation due to the absence of cartilage differentiation. The loss of the skeletal elements is associated with the presence of massive cell death in the region where the digital primordia would have been formed (23), suggesting that apoptotic signals act on cells that never were committed to chondrogenic lineage. Accordingly, in a normal development, if chondrocyte differentiation begins, committed cells turn unresponsive to apoptotic-promoting signals, simply because they have begun the chondrocyte differentiation program. BMP are proteins that induce a molecular cascade that leads either to cell death or to chondrocyte differentiation, inducing Sox9 expression. The dual role of BMP may depend on the differentiation state of the mesenchymal cells and on the type of receptor expressed (see Fig. 1). In the cells that will develop as chondrocytes, TGFb/Activin signaling induces the expression of Sox9 and Bmpr1b, this allows the signaling of BMP/SMAD/1/5/ 8 through this receptor inducing and maintaining the expression of Sox9. In contrast, the mesenchymal cells of the interdigital tissue that will not develop as chondrocytes express the receptor Bmpr1a by which BMP/SMAD1/5/8 promote cell death (see Fig. 1). However, this mechanism does not explain why interdigital tissue is maintained in an undifferentiated stage, assuming that it is beyond the influence of FGF and WNT signaling from AER. Therefore, it is possible that WNT/bcatenin signaling from the dorsal ectoderm in the interdigit region of chick limbs inhibits chondrocyte commitment (3), as demonstrated by its experimental removal that gives rise to ectopic digit formation (62). Another possibility involves a role for RA signaling. It has been reported that RA is able to inhibit Sox9 expression by repressing its transcriptional activity or because it induces the expression of negative regulators of Sox9 expression (63, 64). On the other hand, Zhao et al. (65) demonstrated that Cy26b1 is expressed in developing digits, and suggests that the presence of this enzyme might limit RA action to the interdigital region. Interestingly, the inhibition of CYP26 enzymes in mouse or chick autopods results in massive interdigital cell death (Rios-Flores and Chimal-Monroy manuscript in preparation). However, the question here is what factors induce the

7 928 CHIMAL-MONROY ET AL. expression of Cyp26b1 in developing digits, since it is unlikely that FGF8 from the AER has a role in regulating the induction of this gene. Interestingly, TGFb treatment in interdigital membrane induces Cyp26 and inhibits Raldh2 expression (Rios-Flores and Chimal-Monroy manuscript in preparation), suggesting a protective role of CYP26 in the digit regions against chondrogenesis inhibition by RA (see Fig. 1). Finally, since TGFb inhibits the expression of Raldh2 and induces the expression of Cyp26b1, the presence of high levels of TGFb in the interdigital tissue inhibits cell death, whereas low concentrations are unable to inhibit cell death (Fig. 1). In conclusion, our interpretation of all these results suggests that the development of digits depends of the coordinated interactions between the different signaling pathways that have their role on undifferentiated mesenchymal cells beneath AER to control the final fate of the lineages (Fig. 1). In addition, on the basis that the interdigital tissue has chondrogenic potential, it will be necessary to consider the hypothesis that apoptotic-promoting signals might also act as antichondrogenic factors and chondrogenic factors might operate as anti-apoptotic factors. ACKNOWLEDGEMENTS The authors thank Karen Camargo Sosa for the experiments of the Fig. 2, Donovan Correa Gallegos for his help with the preparation of Fig. 3, Lucía Brito for bibliographical assistance and to all laboratory members for their critical review of the manuscript. This study was partially supported by CONACYT grants 53484, Q, and N, DGAPA-UNAM grants IN220808, IN AND IX to J. C-M. and PAEP-UNAM grant R. F. A-B., M.E. D-H., and A.F-H. were recipients of a scholarship from CONACYT. M.E. D-H. was recipient of a scholarship from RED FARMED. S.R-S. was recipient of a scholarship from the IXTLI Observatorio-UNAM. R.C was recipient of a postdoctoral scholarship from project CONACyT. 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