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In this commentary we focus on the function of FGFs during limb development and morphogenesis. Our goal is to understand, interpret and, when possible, reconcile the interesting findings and conflicting results that remain unexplained. For example, the cell death pattern observed after surgical removal of the AER versus genetic removal of the AER-Fgfs is strikingly different and the field is at an impasse with regard to an explanation. We also discuss the idea that AER function may involve signaling components in addition to the AER-FGFs and that signaling from the non-AER ectoderm may also have a significant contribution. We hope that a re-evaluation of current studies and a discussion of outstanding questions will motivate new experiments, especially considering the availability of new technologies, that will fuel further progress toward understanding the intricate ectoderm-to-mesoderm crosstalk during limb development. Developmental Dynamics 246:208-216, 2017. © 2016 Wiley Periodicals, Inc.

The limb forms from a bud of mesoderm encased in a hull of ectoderm that grows out from the flank of the embryo. Coordinated signaling between the limb mesoderm and ectoderm is critical for normal limb outgrowth and patterning. The apical ectodermal ridge (AER), found at the distal tip, is a rich source of signaling molecules and has been proposed to specify distal structures and maintain the survival of cells in the underlying distal mesoderm. The dorsal and ventral non-AER ectoderm is also a source of signaling molecules and is important for dorsal-ventral patterning of the limb bud. Here we determine if this ectoderm provides cell survival signals by surgically removing the dorsal or ventral ectoderm during early chicken limb bud development and assaying for programmed cell death. We find that, similar to the AER, removal of the dorsal or ventral non-AER ectoderm results in massive cell death in the underlying mesoderm. In addition, although a re-epithelialization occurs, we find perturbations in the timing of Shh expression and, for the case of the dorsal ectoderm removal, defects in soft tissue and skeletal development along the proximal-distal axis. Furthermore, ectoderm substitution experiments show that the survival signal produced by the dorsal limb ectoderm is specific. Thus, our results argue that the non-AER ectoderm, like the AER, provides a specific survival signal to the underlying mesoderm that is necessary for normal limb development and conclusions drawn from experiments in which the non-AER ectoderm is removed, need to take into consideration this observation. © 2013 Elsevier Inc., This work was supported by grant BFU2011-24972 from the Spanish Ministry of Science (M.R.) and by the University of Southern California (F.M.).

In this commentary we focus on the function of FGFs during limb development and morphogenesis. Our goal is to understand, interpret and, when possible, reconcile the interesting findings and conflicting results that remain unexplained. For example, the cell death pattern observed after surgical removal of the AER versus genetic removal of the AER-Fgfs is strikingly different and the field is at an impasse with regard to an explanation. We also discuss the idea that AER function may involve signaling components in addition to the AER-FGFs and that signaling from the non-AER ectoderm may also have a significant contribution. We hope that a re-evaluation of current studies and a discussion of outstanding questions will motivate new experiments, especially considering the availability of new technologies, that will fuel further progress toward understanding the intricate ectoderm-to-mesoderm crosstalk during limb development. Developmental Dynamics 246:208-216, 2017. © 2016 Wiley Periodicals, Inc.

El pdf del artículo es el manuscrito de autor (PMCID: PMC3258580).-- et al., Two broad classes of models have been proposed to explain the patterning of the proximal-distal axis of the vertebrate limb (from the shoulder to the digit tips). Differentiating between them, we demonstrate that early limb mesenchyme in the chick is initially maintained in a state capable of generating all limb segments through exposure to a combination of proximal and distal signals. As the limb bud grows, the proximal limb is established through continued exposure to flank-derived signal(s), whereas the developmental program determining the medial and distal segments is initiated in domains that grow beyond proximal influence. In addition, the system we have developed, combining in vitro and in vivo culture, opens the door to a new level of analysis of patterning mechanisms in the limb., This work was supported by an NIH grant, R37HD032443, to C.T. and by BFU2008-00397, from the Spanish Ministry of Science and Innovation to M.R.

Cell death and cell proliferation are basic cellular processes that need to be precisely controlled during embryonic development. The developing vertebrate limb illustrates particularly well how correct morphogenesis depends on the appropriate spatial and temporal balance between cell death and cell proliferation. Precise knowledge of the patterns of cell proliferation and cell death during limb development is required to understand how their modifications may contribute to the generation of the great diversity of limb phenotypes that result from spontaneous mutations or induced genetic manipulations. We have performed a comprehensive analysis of the patterns of cell death, assayed by terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL), and cell proliferation, assayed by anti-phosphorylated histone H3 immunohistochemistry, in consecutive sections of forelimbs and hindlimbs covering an extensive period of chick and mouse limb development. Our results confirm and expand previous reports and show common and specific areas of cell death for each species. Mitotic cells were found scattered in a uniform distribution across the early limb bud, with the exception of the areas of cell death in which mitotic cells were scarce. At later stages, mitotic cells were seen more abundantly in the digital tips. The aim of the present study was to satisfy the need for organized data sets describing these processes, which will allow the side-by-side comparison between the two major model organisms of limb development, i.e., the mouse and the chick.

Congenital malformations of the limbs are among the most frequent congenital anomalies found in humans, and they preferentially affect the distal part--the hand or foot. The presence of extra digits, a condition called polydactyly, is the most common limb deformity of the human hand and is the consequence of disturbances in the normal program of limb development. However, despite the extensive use of the developing limb as a classical developmental model, the cellular and genetic mechanisms that control the number and identity of the digits are not completely understood. The aim of this review is to introduce the reader to the current state of knowledge in limb development and to provide the necessary background for an understanding of how deviations from the normal developmental program may lead to polydactyly.

Removal of the posterior wing bud leads to massive apoptosis of the remaining anterior wing bud mesoderm. We show here that this finding correlates with an increase in the level of the repressor form of the Gli3 protein, due to the absence of the Sonic hedgehog (Shh) protein signaling. Therefore, we used the anterior wing bud mesoderm as a model system to analyze the relationship between the repressor form of Gli3 and apoptosis in the developing limb. With increased Gli3R levels, we demonstrate a concomitant increase in Bmp4 expression and signaling in the anterior mesoderm deprived of Shh signaling. Several experimental approaches show that the apoptosis can be prevented by exogenous Noggin, indicating that Bmp signaling mediates it. The analysis of Bmp4 expression in several mouse and chick mutations with defects in either expression or processing of Gli3 indicates a correlation between the level of the repressor form of Gli3 and Bmp4 expression in the distal mesoderm. Our analysis adds new insights into the way Shh differentially controls the processing of Gli3 and how, subsequently, BMP4 expression may mediate cell survival or cell death in the developing limb bud in a position-dependent manner.

The recombinant limb is a model system that has proved fruitful for analyzing epithelial-mesenchymal interactions and understanding the functional properties of the components of the limb bud. Here we present an overview of some of the insights obtained through the use of this technique. Among these are the understanding that fore or hind limb identity is inherent to the limb bud mesoderm, that the apical ectodermal ridge (AER) is a permissive signaling center and that the limb bud ectoderm plays a central role in the control of dorsoventral polarity. Recombinant limb studies have also allowed the identification of the affected tissue component in several limb mutants. More recently this model has been applied to the study of regulation of gene expressions related to patterning. In this report we use recombinant limbs to analyze pattering of the Pax3 expressing limb muscle cell lineage in the early stages of limb development. In recombinant limbs made without the zone of polarizing activity (ZPA), myoblasts appear intermingled with other mesodermal cells at the beginning of the recombinant limb development. Rapidly thereafter, the muscle precursors segregate and organize around the central forming chondrogenic core of the recombinant. Although this segregation is reminiscent of that occurring during normal development, the myoblasts in the recombinant fail to proliferate appropriately and also fail to migrate distally. Consequently, the muscle pattern in the recombinant limb is defective indicating that normal patterning cues are absent. However, recombinant limbs polarized with a ZPA exhibited a larger mass of muscle cells and a more normal morphogenesis, supporting a role for this signaling center in limb muscle development. Finally, we have ruled out host somite contributions to recombinant limbs by grafting chick recombinant limbs to quail hosts. This initial report demonstrates the value of the recombinant limb model system for dissecting the environmental cues required for normal muscle limb patterning.

The Apical Ectodermal Ridge (AER) is one of the main signaling centers during limb development. It controls outgrowth and patterning in the proximo-distal axis. In the last few years a considerable amount of new data regarding the cellular and molecular mechanisms underlying AER function and structure has been obtained. In this review, we describe and discuss current knowledge of the regulatory networks which control the induction, maturation and regression of the AER, as well as the link between dorso-ventral patterning and the formation and position of the AER. Our aim is to integrate both recent and old knowledge to produce a wider picture of the AER which enhances our understanding of this relevant structure. © UBC Press., This work was supported by grant BFU2005-09309-CO2-01 from the Spanish Ministry of Education and Science.

eHAND is a bHLH transcription factor with important functions during embryogenesis. Here, we report that eHAND has a dynamic pattern of expression during limb development. In chick embryos, eHAND expression is first observed in the ventral mesoderm of the emerging limb. Its expression is then restricted to an anteroventral area of mesoderm at mid-level in the proximodistal axis. At later stages, expression is observed in the autopod encompassing the ventral tendons of the digits. In mouse embryos, only the anteroventral domain of expression is conserved, the early ventral expression not being detectable and the late pattern of expression differing clearly from that in the chick. A constant feature of all areas of expression is their ventral and anterior localization. Respecification of the anterior mesoderm as occurs secondarily to Sonic hedgehog (SHH) or retinoic acid application to the anterior border leads to down-regulation of eHAND expression. Accordingly, eHAND expression is not detectable in talpid(2) mutant limbs, which are considered to be posteriorized limbs. However, eHAND expression is little modified in oligozeugodactyly, a chick mutant that lacks Shh signaling in the limb but retains certain anteroposterior polarity. Interestingly, eHAND expression is also linked to the ventral identity of the mesoderm and is repressed by the dorsal ectoderm. It is also positively regulated by bone morphogenetic protein signaling, which is also known to participate in dorsoventral patterning. We suggest that eHAND expression may be related to the anteroventral identity of the mesoderm. However, in overexpression experiments using retroviral vectors, only a low percentage of cases (5%) showed phenotypic alterations, consisting of a duplication of digit 2.

We have analyzed a new limb mutant in the chicken that we nameoligozeugodactyly (ozd). The limbs of this mutant have a longitudinal postaxial defect, lacking the posterior element in the zeugopod(ulna/fibula) and all digits except digit 1 in the leg. Classical recombination experiments show that the limb mesoderm is the defective tissue layer in ozd limb buds. Molecular analysis revealed that theozd limbs develop in the absence of Shh expression, while all other organs express Shh and develop normally. NeitherPtc1 nor Gli1 are detectable in mutant limb buds. However,Bmp2 and dHAND are expressed in the posterior wing and leg bud mesoderm, although at lower levels than in normal embryos. Activation ofHoxd11-13 occurs normally in ozd limbs but progressively declines with time. Phase III of expression is more affected than phase II,and expression is more severely affected in the more 5′ genes. Interestingly, re-expression of Hoxd13 occurs at late stages in the distal mesoderm of ozd leg buds, correlating with formation of digit 1. Fgf8 and Fgf4 expression are initiated normally in the mutant AER but their expression is progressively downregulated in the anterior AER. Recombinant Shh protein or ZPA grafts restore normal pattern toozd limbs; however, retinoic acid fails to induce Shh in ozdlimb mesoderm. We conclude that Shh function is required for limb development distal to the elbow/knee joints, similar to the Shh-/-mouse. Accordingly we classify the limb skeletal elements as Shh dependent or independent, with the ulna/fibula and digits other than digit 1 in the leg being Shh dependent. Finally we propose that the ozd mutation is most likely a defect in a regulatory element that controls limb-specific expression of Shh.

The bHLH transcription factor dHAND is required for establishment of SHH signaling by the limb bud organizer in posterior mesenchyme, a step crucial to development of vertebrate paired appendages. We show that the transcriptional repressor GLI3 restricts dHANDexpression to posterior mesenchyme prior to activation of SHH signaling in mouse limb buds. dHAND, in turn, excludes anterior genes such asGli3 and Alx4 from posterior mesenchyme. Furthermore, genetic interaction of GLI3 and dHAND directs establishment of the SHH/FGF signaling feedback loop by restricting the BMP antagonist GREMLIN posteriorly. These interactions polarize the nascent limb bud mesenchyme prior to SHH signaling.

dHAND is a basic helix-loop-helix (bHLH) transcription factor essential for cardiovascular development. Here we analyze its pattern of expression and functional role during chick limb development. dHAND expression was observed in the lateral plate mesoderm prior to emergence of the limb buds. Coincident with limb initiation, expression of dHAND became restricted to the posterior half of the limb bud. Experimental procedures that caused mirror-image duplications of the limb resulted in mirror-image duplications of the pattern of dHAND expression along the anterior-posterior axis. Retroviral overexpression of dHAND in the limb bud produced preaxial polydactyly, corresponding to mild polarizing activity at the anterior border. At the molecular level, misexpression of dHAND caused ectopic activation of members of the Sonic hedgehog (Shh) pathway, including Gli and Patched, in the anterior limb bud. A subset of infected embryos displayed ectopic anterior activation of Shh. Other factors implicated in anterior-posterior polarization of the bud such as the most 5′ Hoxd genes and Bmp2 were also ectopically activated at the anterior border. Our results indicate a role for dHAND in the establishment of anterior-posterior polarization of the limb bud.

Recombinant limbs were performed by ensembling dissociated-reaggregated wing bud mesoderm inside an ectodermal hull. The zone of polarizing activity was excluded from the mesoderm used to perform the recombinant limbs (non-polarized recombinants), and grafted when desired (polarized recombinants). Reorganization of patterning progressively occurred in the newly formed progress zone under the influence of the apical ectodermal ridge (AER), explaining the proximo-distal gradient of morphogenesis observed in developed recombinant limbs. The AER, without the influence of the polarizing region (ZPA), was sufficient to direct outgrowth and appropriate proximo-distal patterning, as observed in the expression of the Hoxa-11 and Hoxa-13 genes. The development of the recombinant limbs coursed with symmetric AER and downregulation of Bmp expression in the mesoderm supporting a negative effect of Bmp signaling upon the apical ridge. The recombinant ectoderm maintained previously established compartments of gene expressions and organized a correct dorso-ventral patterning in the recombinant progress zone. Finally, the ZPA effect was only detected on Bmp expression and pattern formation along the antero-posterior axis.

During heart development in the chick some of the endocardial cells that cover the cushion areas leave the cushion endocardium, seed the underlying cardiac jelly, and are transformed into mesenchyme. Cushion mesenchymal (CM) cells migrate from the endocardium toward the myocardium using the cardiac jelly as substratum. Developing cushions have been microinjected with fibronectin (FN), antifibronectin antibodies (AbFN), and four synthetic peptide probes. Two of these peptides (P7 and P10) contained the sequence Arg-Gly-Asp-Ser (RGDS), while the other two (P15 and PColl) did not. Cushion area, individual cell area, cell density, cell orientation and a factor of form were evaluated in both experimental and control cushions. CM cell migration was inhibited by FN and AbFN, only partially inhibited by P10 and unaffected by P7. Cushions injected with P15 and PColl were unaffected. These results can be explained by steric modifications of the extracellular matrix, that may render cardiac jelly nonpermissive for CM cell migration, or by interaction of the substances injected at the endocardial cell surface. Migrating CM cells do not present any preferential orientation in any particular direction. CM cell migration seems to depend upon intrinsic migratory behaviour and the presence of FN at the CM cell surface. The enforcement of the direction of CM cell migration does not appear to rely upon matrix signals but be the result of randomly migrating cells becoming distributed more evenly in the matrix.

Normal embryonic development is not a linear process of cell and tissue growth and differentiation (Hurle, 1988). There are many examples in which an early embryonic structure undergoes a reshaping process involving a substantial phenomenon of cell death. In some cases the tissue loss affects to fully formed organs which play a transitory functional role in the developing individual. This is the case of the loss of the tail in the developing anura amphibia during metamorphosis. In other cases cell death appears closely linked to morphogenesis an appears to play an sculpturing role for the achievement of the final shape of a developing structure. This appears to be the case in “interdigital cell death” occurring during morphogenesis of the digits of the amniote embryos.

Fine structural study of interdigital membranes during formation of the digits of the duck foot reveals that the interdigital necrosis is accompanied by a high deposition of collagenous material in the epithelio-mesenchymal interface, rupture of the basal lamina and detachment of ectodermal cells into the amniotic sac. These changes are similar to those observed in the regressing interdigital membrane of the chick although their intensity and temporal extension are less pronounced in the duck. It is suggested that these changes account for the disappearance of the marginal zone of the duck interdigital membranes. The possible causal relationship between the different structural changes are discussed.

In an attempt to clarify the mechanism of production of the syndactyly induced by Janus Green B (JGB) we have studied the morphology and structural modifications of the chick embryo leg bud after JGB administration by means of (1) neutral red vital staining, (2) whole-mount cartilage staining and β) light microscopy and transmission and scanning electron microscopy. The results show that the well-known inhibition of interdigital cell death is accompanied by a precocious alteration of the epithelial tissue and especially of the epithelial-mesenchymal interface. 24 h after JGB administration the cells of the AER reduce the number of junctions and the basal ectodermal cells are detached into the mesenchymal tissue in zones in which the basal lamina undergoes disruption. In addition the interdigital mesenchymal cells diverted from the dying program are able to undergo a rapid differentiation into cartilage. It is proposed that the mechanism of production of JGB-induced syndactyly might be due to an alteration of the normal epithelial-mesenchymal interactions rather than to a direct inhibitory effect of the JGB on the dying program.

With rapid progress in understanding the genes that control limb development and patterning interest is becoming focused on the factors that permit the emergence of the limb bud. The current hypothesis is that FGF-8 from the mesonephros induces limb initiation. To test this, the inductive interaction between the Wolffian duct and intermediate mesoderm was blocked rostral to the limb field, preventing mesonephric differentiation while maintaining the integrity of the limb field. The experimental outcome was monitored by following expression of cSim1 and Lmx1, molecular markers for the duct and the mesonephros, respectively. Evidence is presented that the intermediate mesoderm undergoes apoptosis when the inductive interaction with the Wolffian duct is blocked. fgf-8 expression was undetectable in the mesonephric area of embryos with confirmed absence of mesonephros; nevertheless, limb buds formed and limb development was normal. The mesonephros in general, and specifically its fgf-8 expression, was shown to be unnecessary for limb initiation and development; the hypothesis linking the mesonephros and limb development is not supported. Further studies of axial influences on limb initiation should now concentrate on medial structures such as Hensen's node and paraxial mesoderm; the alternative that no axial influences are required should also be examined.

The ventricular fiber architecture has been studied anatomically and histologically in 25 human hearts. Special attention has been paid to the form of insertion of the myocardial fibers and to the existence or nonexistence of an independent system of fibers for each ventricle. Three different myocardial layers--superficial (subepicardial), middle, and deep (subendocardial)--have been distinguished according to the fiber direction through the ventricular mass. All the fibers are common for both ventricles with the exception of a thick middle layer peculiar to the left ventricle. The tendinous cords, the central fibrous body, and the arterial fibrous rings are the major zones of insertion of myocardial fibers. On the basis of our results we have attempted to correlate the ventricular architecture with the valvular dynamics.

The anatomy of the human aortic valve was studied by SEM in 36 subjects without cardiac pathology who had died of various accidental causes. Villous and lamellar tissue excrescences were observed at the node of Arantius and at the limit between the lunules and the load-bearing portion of the leaflets. The morphology of these structures suggests that they represent areas in which valve tissue becomes detached into the bloodstream. Fenestrations were present in the lunules of 14 specimens, with a higher incidence in specimens from subjects who were middle-aged or older. Our observations suggest that fenestrations appear initially as small perforations which then coalesce to form larger apertures. Two main types of endothelial cells, elongated and polygonal were detected on the endothelial surface of the leaflets. Both types of cells display a constant mode of arrangement on the different segments of the leaflets (lunules, node of Arantius and load-bearing portion of the leaflet). The possible relationships between endothelial cell morphology and the pattern of mechanical stress to which the leaflets are subjected is discussed.

In this work we have attempted to characterize the programmed cell death process in the chick embryonic interdigital tissue. Interdigital cell death is a prominent phenomenon during limb development and has the role of sculpturing the digits. Morphological changes in the regressing interdigital tissue studied by light, transmission and scanning electron microscopy were correlated with the occurrence of internucleosomal DNA fragmentation, evaluated using agarose gels. Programming of the cell death process was also analyzed by testing the chondrogenic potential of the interdigital mesenchyme, in high density cultures. Our results reveal a progressive loss of the chondrogenic potential of the interdigital mesenchyme, detectable 36 hours before the onset of the degenerative process. Internucleosomal DNA fragmentation was only detected concomitant with the appearance of cells dying with the morphology of apoptosis, but unspecific DNA fragmentation was also present at the same time. This unspecific DNA fragmentation was explained by a precocious activation of the phagocytic removal of the dying cells, confirmed in the tissue sections. From our observations it is suggested that programming of cell death involves changes before endonuclease activation. Further, cell surface changes involved in the phagocytic uptake of the dying cells appear to be as precocious as endonuclease activation.

The pattern and structure of the blood vessels of the interdigital spaces of the leg bud have been studied by means of Indian ink injections and transmission electron microscopy in the chick and duck embryos. The results show that in the chick the interdigital necrotic process responsible for the freeing of the digits is followed by regression of the blood vessels. In the webbed foot of the duck, the interdigital necrotic processes are not followed by vascular regression. Transmission electron microscopic studies show that both in the chick and in the duck, interdigital blood vessels are immature structures lacking basal lamina. Dead cells of presumably endothelial origin were detected in the lumen of the regressing blood vessels of the chick but not in the duck. However, the intensity of this cell death process does not appear to be high enough to account by itself for the disappearance of the interdigital blood vessels. The possible relationships between interdigital mesenchymal cell death and vascular regression are discussed.

Proteoglycans are ubiquitous extracellular matrix molecules whose role in development remains poorly understood. In the developing chick limb, the nature and possible roles of a number of extracellular matrix proteins is well documented. Much less is known of the biochemical nature, and more importantly, the roles of proteoglycans. Using a panel of monoclonal antibodies (Mabs) which recognise specific epitopes on the constituent chondroitin/dermatan sulphate chains, we show that distinct sub-populations of proteoglycans are dynamically expressed within the limb ectoderm, the ectodermal basement membrane and the limb mesenchyme. In particular, prior to chondrogenesis, chondroitin-6-sulphate-rich proteoglycans containing over-sulphated domains residue predominantly within the mesenchymal extracellular matrix ECM, whilst chondroitin-4-sulphate (C-4-S) is associated with the ectodermal basement membrane and subjacent mesenchymal ECM. At stage 24, C-4-S is also localized in the prechondrogenic condensation. Concomitantly with overt chondrogenesis, the epitopes recognized by the Mabs become restricted to the chondrifying skeletal elements and the undifferentiated distal mesenchyme. The significance of these findings has yet to be elucidated.