Vascularization of the peroneal muscles. Critical evaluation in fibular free flap harvesting.

This anatomical study was carried out in order to discover the etiology of partial necrosis of the peroneus longus and brevis muscles after fibular osteocutaneous flap harvest. The vascular supply to the lateral compartment peroneal muscles was investigated in 10 fresh cadaveric lower limbs. The peroneal muscles are supplied by two principal sources arteries, the anterior tibial artery (ATA) and the peroneal artery (PA). The ATA is the dominant artery and supplies the proximal and middle thirds of these muscles. The PA is considered to be a supplementary vascular source and supplies the distal thirds of these muscles. After harvesting the PA in a fibular flap, regions of the peroneal muscles preoperatively vascularized by its branches become supplied through the anastomotic "choke" vessels between the ATA and the PA. Primary closure of the cutaneous defect increases the possibility of developing a pseudo-compartment syndrome with necrosis of the more precariously vascularized portions of the peroneal muscles. This complication is difficult to diagnosis early and the reason why we advocate that direct skin closure following composite harvest must be avoided. Moreover, conservation of the inferior and superior lateral branches of the ATA is imperative in order to preserve the peroneal muscles vascularization.

Analysis of the molecular cascade responsible for mesodermal limb chondrogenesis: Sox genes and BMP signaling.

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.

Comparative analysis of the expression and regulation of Wnt5a, Fz4, and Frzb1 during digit formation and in micromass cultures.

Previous studies have shown that three members of the Wnt signaling pathway, the ligand WNT5A, the receptor FZ4, and the Wnt antagonist FRZB1, are implicated in the formation and differentiation of the digits. In this study, we have attempted to establish a functional correlation between them by comparing their expression patterns and their regulation by the signals controlling proliferation and differentiation of the limb mesoderm during formation of the avian digits in vivo and in micromass cultures. In vivo Wnt5a and Fz4 are expressed in the undifferentiated mesoderm of the autopod and in the differentiating digit cartilages. In the undifferentiated mesoderm, the expression of both genes is regulated positively by FGFs and negatively by bone morphogenetic proteins (BMPs). As chondrogenic differentiation starts, Fz4 becomes intensely up-regulated in the aggregate and in the developing perichondrium, whereas transcripts of Wnt5a are excluded from the core of the aggregate but maintained in the surrounding mesenchyme and perichondrium. In addition, at this stage, the expression of both genes become positively regulated by BMPs. These changes in expression and regulation are coincident with the induction of Frzb1 in the chondrogenic aggregate, which is expressed under the positive control of BMPs. Our findings fit with a role of Wnt5a/Fz4 negatively regulating in vivo the initiation and progression of cartilage differentiation. In vitro, only Frzb1 is expressed and regulated in a manner resembling that observed in vivo. Wnt5a and Fz4 are both expressed in the differentiating mesenchyme of micromass cultures, but their expression is not significantly regulated by the addition of FGF-2 or BMP-7 to the culture medium.

Role of FGFs in the control of programmed cell death during limb development.

We have investigated the role of FGFs in the control of programmed cell death during limb development by analyzing the effects of increasing and blocking FGF signaling in the avian limb bud. BMPs are currently considered as the signals responsible for cell death. Here we show that FGF signaling is also necessary for apoptosis and that the establishment of the areas of cell death is regulated by the convergence of FGF- and BMP-mediated signaling pathways. As previously demonstrated, cell death is inhibited for short intervals (12 hours) after administration of FGFs. However, this initial inhibition is followed (24 hours) by a dramatic increase in cell death, which can be abolished by treatments with a BMP antagonist (Noggin or Gremlin). Conversely, blockage of FGF signaling by applying a specific FGF-inhibitor (SU5402) into the interdigital regions inhibits both physiological cell death and that mediated by exogenous BMPs. Furthermore, FGF receptors 1, 2 and 3 are expressed in the autopodial mesoderm during the regression of the interdigital tissue, and the expression of FGFR3 in the interdigital regions is regulated by FGFs and BMPs in the same fashion as apopotosis. Together our findings indicate that, in the absence of FGF signaling BMPs are not sufficient to trigger apoptosis in the developing limb. Although we provide evidence for a positive influence of FGFs on BMP gene expression, the physiological implication of FGFs in apoptosis appears to result from their requirement for the expression of genes of the apoptotic cascade. We have identified MSX2 and Snail as candidate genes associated with apoptosis the expression of which requires the combined action of FGFs and BMPs.

The BMP antagonist Gremlin regulates outgrowth, chondrogenesis and programmed cell death in the developing limb.

In this study, we have analyzed the expression and function of Gremlin in the developing avian limb. Gremlin is a member of the DAN family of BMP antagonists highly conserved through evolution able to bind and block BMP2, BMP4 and BMP7. At early stages of development, gremlin is expressed in the dorsal and ventral mesoderm in a pattern complementary to that of bmp2, bmp4 and bmp7. The maintenance of gremlin expression at these stages is under the control of the AER, ZPA, and BMPs. Exogenous administration of recombinant Gremlin indicates that this protein is involved in the control of limb outgrowth. This function appears to be mediated by the neutralization of BMP function to maintain an active AER, to restrict the extension of the areas of programmed cell death and to confine chondrogenesis to the central core mesenchyme of the bud. At the stages of digit formation, gremlin is expressed in the proximal boundary of the interdigital mesoderm of the chick autopod. The anti-apoptotic influence of exogenous Gremlin, which results in the formation of soft tissue syndactyly in the chick, together with the expression of gremlin in the duck interdigital webs, indicates that Gremlin regulates the regression of the interdigital tissue. At later stages of limb development, gremlin is expressed in association with the differentiating skeletal pieces, muscles and the feather buds. The different expression of Gremlin in relation with other BMP antagonists present in the limb bud, such as Noggin, Chordin and Follistatin indicates that the functions of BMPs are regulated specifically by the different BMP antagonists, acting in a complementary fashion rather than being redundant signals.

Regulation by members of the transforming growth factor beta superfamily of the digital and interdigital fates of the autopodial limb mesoderm.

Embryonic limb outgrowth is accomplished by the proliferation of mesodermal cells in the progress zone. In this region, mesodermal cells are maintained in an undifferentiated and proliferating state by the action of the apical ectodermal ridge (AER). Differentiation of these cells into individual skeletal elements occurs when the cells are displaced proximally and leave the influence of the AER as a consequence of the accumulation of cells in that region. Here we review the evidence obtained in the last few years showing that members of the transforming growth factor beta (TGFbeta) subfamily and bone morphogenetic proteins (BMPs) act as proximal signals in the autopod regulating the fate of the progress zone cells towards chondrogenesis or apoptosis. Our findings show that apoptosis is regulated by BMPs while chondrogenesis requires the interaction of TGFbetas and BMPs. Fibroblast growth factors (FGFs) produced by the AER exert an opposite function to both TGFbetas and BMPs, maintaining the progress zone cells in an undifferentiated state.

Control of digit formation by activin signalling.

Major advances in the genetics of vertebrate limb development have been obtained in recent years. However, the nature of the signals which trigger differentiation of the mesoderm to form the limb skeleton remains elusive. Previously, we have obtained evidence for a role of TGFbeta2 in digit formation. Here, we show that activins A and B and/or AB are also signals involved in digit skeletogenesis. activin betaA gene expression correlates with the initiation of digit chondrogenesis while activin betaB is expressed coincidently with the formation of the last phalanx of each digit. Exogenous administration of activins A, B or AB into the interdigital regions induces the formation of extra digits. follistatin, a natural antagonist of activins, is expressed, under the control of activin, peripherally to the digit chondrogenic aggregates marking the prospective tendinous blastemas. Exogenous application of follistatin blocks physiological and activin-induced digit formation. Evidence for a close interaction between activins and other signalling molecules, such as BMPs and FGFs, operating at the distal tip of the limb at these stages is also provided. Chondrogenesis by activins is mediated by BMPs through the regulation of the BMP receptor bmpR-1b and in turn activin expression is upregulated by BMP signalling. In addition, AER hyperactivity secondary to Wnt3A misexpression or local administration of FGFs, inhibits activin expression. In correlation with the restricted expression of activins in the course of digit formation, neither activin nor follistatin treatment affects the development of the skeletal components of the stylopod or zeugopod indicating that the formation of the limb skeleton is regulated by segment-specific chondrogenic signals.

Expression and function of Gdf-5 during digit skeletogenesis in the embryonic chick leg bud.

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.

Bone morphogenetic proteins regulate interdigital cell death in the avian embryo.

The embryonic limb bud provides an excellent model for analyzing the mechanisms that regulate programmed cell death during development. At the time of digit formation in the developing autopod, the undifferentiated distal mesodermal cells may undergo or chondrogenic differentiation or apoptosis depending whether they are incorporated into the future digital rays or into the interdigital spaces. Both chondrogenesis or apoptosis are induced by local BMPS. However, whereas the chondrogenic-promoting activity of BMPs appears to be regulated through the BMPR-1b receptor, the mechanism by which the BMPs execute the death program remains unknown. The BMP proapoptotic activity requires the expression of members of the msx family of closely related homeobox-containing genes and is finally mediated by caspase activation, but the nature of the caspase(s) directly responsible for the cell death is also unknown. Finally, other growth factors present in the developing autopod at the stages of digit formation such as members of the FGF and TGF beta families modulate the ability of BMPs to induce cell death or chondrogenesis.

Morphogenesis of digits in the avian limb is controlled by FGFs, TGFbetas, and noggin through BMP signaling.

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.

Morphological diversity of the avian foot is related with the pattern of msx gene expression in the developing autopod.

The formation of the digits in amniota embryos is accompanied by apoptotic cell death of the interdigital mesoderm triggered through BMP signaling. Differences in the intensity of this apoptotic process account for the establishment of the different morphological types of feet observed in amniota (i.e., free-digits, webbed digits, lobulated digits). The molecular basis accounting for the differential pattern of interdigital cell death remains uncertain since the reduction of cell death in species with webbed digits is not accompanied by a parallel reduction in the pattern of expression of bmp genes in the interdigital regions. In this study we show that the duck interdigital web mesoderm exhibits an attenuated response to both BMP-induced apoptosis and TGFbeta-induced chondrogenesis in comparison with species with free digits. The attenuated response to these signals is accompanied by a reduced pattern of expression of msx-1 and msx-2 genes. Local application of FGF in the duck interdigit expands the domain of msx-2 expression but not the domain of msx-1 expression. This change in the expression of msx-2 is followed by a parallel increase in spontaneous and exogenous BMP-induced interdigital cell death, while the chondrogenic response to TGFbetas is unchanged. The regression of AER, as deduced by the pattern of extinction of fgf-8 expression, takes place in a similar fashion in the chick and duck regardless of the differences in interdigital cell death and msx gene expression. Implantation of BMP-beads in the distal limb mesoderm induces AER regression in both the chick and duck. This finding suggests an additional role for BMPs in the physiological regression of the AER. It is proposed that the formation of webbed vs free-digit feet in amniota results from a premature differentiation of the interdigital mesoderm into connective tissue caused by a reduced expression of msx genes in the developing autopod.

Role of BMP-2 and OP-1 (BMP-7) in programmed cell death and skeletogenesis during chick limb development.

Bone Morphogenetic Protein 2 (BMP-2) and Osteogenic Protein 1 (OP-1, also termed BMP-7) are members of the transforming growth factor beta superfamily. In the present study, we have analyzed the effects of administering them locally at different stages and locations of the chick limb bud using heparin beads as carriers. Our results show that these BMPs are potent apoptotic signals for the undifferentiated limb mesoderm but not for the ectoderm or the differentiating chondrogenic cells. In addition, they promote intense radial growth of the differentiating cartilages and disturb the formation of joints accompanied by alterations in the pattern of Indian hedgehog and ck-erg expression. Interestingly, the effects of these two BMPs on joint formation were found to be different. While the predominant effect of BMP-2 is alteration in joint shape, OP-1 is a potent inhibitory factor for joint formation. In situ hybridizations to check whether this finding was indicative of specific roles for these BMPs in the formation of joints revealed a distinct and complementary pattern of expression of these genes during the formation of the skeleton of the digits. While Op-1 exhibited an intense expression in the perichondrium of the developing cartilages with characteristic interruptions in the zones of joint formation, Bmp-2 expression was a positive marker for the articular interspaces. These data suggest that, in addition to the proposed role for BMP-2 and OP-1 in the establishment of the anteroposterior axis of the limb, they may also play direct roles in limb morphogenesis: (i) in regulating the amount and spatial distribution of the undifferentiated prechondrogenic mesenchyme and (ii) in controlling the location of the joints and the diaphyses of the cartilaginous primordia of the long bones once the chondrogenic aggregates are established.

Role of TGF beta s and BMPs as signals controlling the position of the digits and the areas of interdigital cell death in the developing chick limb autopod.

The establishment of the digital rays and the interdigital spaces in the developing limb autopod is accompanied by the occurrence of corresponding domains of expression of TGF beta s and BMPs. This study analyzes whether these coincident events are functionally correlated. The experiments consisted of local administration of TGF beta-1, TGF beta-2 or BMP-4 by means of heparin or Affi-gel blue beads to the chick limb autopod in the stages preceding the onset of interdigital cell death. When beads bearing either TGF beta-1 or -2 were implanted in the interdigits, the mesodermal cells were diverted from the death program forming ectopic cartilages or extra digits in a dose- and stage-dependent fashion. This change in the interdigital phenotype was preceded by a precocious ectopic expression of ck-erg gene around the bead accompanied by down-regulation of bmp-4, msx-1 and msx-2 gene expression. When BMP-beads were implanted in the interdigital spaces, programmed cell death and the freeing of the digits were both accelerated. Implantation of beads bearing BMP-4 at the tip of the growing digits was followed by digit bifurcation, accompanied by the formation of an ectopic area of cell death resembling an extra interdigit, both morphologically and molecularly. The death-inducing effect of the BMP beads and the chondrogenic-inducing effect of the TGF beta beads were antagonized by the implantation of an additional bead preabsorbed with FGF-2, which constitutes a signal characteristic of the progress zone. It is concluded that the spatial distribution of digital rays and interdigital spaces might be controlled by a patterned distribution of TGF beta s and BMPs in the mesoderm subjacent to the progress zone.

In vivo inhibition of programmed cell death by local administration of FGF-2 and FGF-4 in the interdigital areas of the embryonic chick leg bud.

The formation of the digits in amniote vertebrates is accompanied by a massive degeneration process that accounts for the disappearance of the interdigital mesenchyme. The establishment of these areas of interdigital cell death (INZs) is concomitant with the flattening of the apical ectodermal ridge (AER), but a possible causal relationship between these processes has not been demonstrated. Recent studies have shown that the function of the AER can be substituted for by implantation of beads bearing either FGF-2 or FGF-4 into the apical mesoderm of the early limb bud. According to these observations, if the onset of INZs is triggered by the cessation of the AER function, local administration of FGFs to the interdigital tissue prior to cell death should delay or inhibit interdigit degeneration. In the present study we have confirmed this prediction. Implanting Affi-gel blue or heparin beads pre-absorbed with either FGF-2 or FGF-4 into the interdigital tissue of the chick leg bud in the stages prior to cell death stimulates cell proliferation and causes the formation of webbed digits. Vital staining with neutral red confirmed an intense temporal inhibition of interdigital cell death after FGF treatment. This inhibition of interdigital cell death was not accompanied by modifications in the pattern of expression of Msx-1 or Msx-2 genes, which in normal development display a domain of expression in the interdigital tissue preceding the onset of degeneration.

Mouse embryos lacking RXR alpha are resistant to retinoic-acid-induced limb defects.

Embryonic exposure to the vitamin A metabolite retinoic acid (RA) causes malformations in numerous developing tissues, including the limbs, which serves as a model system of retinoic acid action. RA treatment of wild-type mouse embryos results in digit truncations and long bone reductions. These effects are mediated by products of the retinoic acid and retinoid X receptor genes (RARs and RXRs), members of the nuclear receptor family of ligand-dependent transcription factors. Mouse embryos homozygous for a mutation in the RXR alpha gene appear normal in limb development, although such embryos are phenotypically affected in other tissues. We now describe resistance to limb malformations normally induced by teratogenic RA exposure in the RXR alpha-/- background. RA treatments that cause limb defects in 100% of wild-type embryos fail to elicit malformations in RXR alpha homozygotes, implicating RXR alpha as a component in the teratogenic process in the limbs. Heterozygous embryos are intermediate in sensitivity to RA, suggesting the importance of RXR alpha gene dosage in limb teratogenesis. Expression of the RA-inducible gene RAR beta 2 was equivalent between wild-type and homozygous embryos after RA treatment. RA treatment also did not distinguish between wild-type and RXR alpha -/- embryos in the spatial expression of sonic hedgehog (Shh) and hoxd-12, two other genes implicated in limb development. However, the quantitative level of hoxd-12 expression was elevated in RXR alpha -/- embryos. These observations indicate that transcriptional processes which are inappropriately regulated in the mouse limb by exogenous RA require RXR alpha for their execution, and that specific teratogenic processes, as well as specific normal developmental processes under vitamin A control, occur through individual members of the RXR and RAR families.

Cell death in the embryonic developing limb.

In amniote vertebrates, the development of form and structure of the limb bud is accompanied by precise patterns of massive mesodermal cell death with morphological features of apoptosis. These areas of cell death appear to eliminate undifferentiated cells which are required only for a limited time period of limb development. Predictable skeletal and morphological anomalies of the limb occur when the pattern of cell death is modified in mutant species or under experimental conditions. Most evidence points to the occurrence of local triggering mechanisms to account for the establishment of the areas of cell death and the subsequent activation of cell death genes. Modifications of the extracellular matrix and diminution in the contribution of growth factors by neighbouring tissues appear as the most likely potential candidates for triggering the cell death program. Information on the genetical basis of cell death in the developing limb is very scarce. Among the increasing number of cell death genes identified in other cell death systems, such as p-53 and the ced-3/ICE and ced-9/ bcl-2 gene families, only bcl-2 has been studied in detail during limb development and yet, the information obtained is contradictory. Bcl-2 is not expressed in the areas of cell death of the developing limb, but normal limbs develop in mice with disruption of the bcl-2 gene. Obviously, the clarification of the role of the cell death genes constitute a major task in future studies of cell death in the developing limb.

Pattern regulation in the chick autopodium at advanced stages of embryonic development.

In previous studies we have observed that the interdigital tissue of the chick embryo leg bud during the stages previous to interdigital cell death exhibits a considerable chondrogenic potentiality both in vivo and in vitro. In the present investigation we have carried out a variety of experimental manipulations of the chick leg bud at stage 29 to discover possible mechanisms accounting for interdigital ectopic chondrogenesis and extradigit formation. Our results show that the interdigital tissue is capable of forming an extradigit when temporarily isolated microsurgically and regrafted in its original location and after deletion of one of the adjacent digital primordia, suggesting that developing phalangeal cartilages exercise an inhibitory effect on chondrification in adjoining tissues. Furthermore, and of greater importance, ablation of the primordium of a digit is followed by normal development of the definitive digit if the wound surfaces are suitably apposed. These results reveal a considerable regulatory potential in the autopodium at advanced stages of development.