Vertebrate limb development and possible clues to diversity in limb form

Chick embryos are good models for vertebrate development. The principles that underlie chick wing development have been discovered and there is increasing knowledge about the molecules involved. The importance of identifying molecules is that this provides a direct link to understanding the genetic basis of diversity in form. Chick wing development will be compared with limb development in other vertebrates. Possible mechanisms that could lead to variations in form, including limb reductions and limblessness, differences between fore- and hindlimbs, limb proportions, and interdigital webbing can be suggested.     

Involvement of Wnt-5a in chondrogenic pattern formation in the chick limb bud

Members of the Wnt family are known to play diverse roles in the organogenesis of vertebrates. The full-coding sequences of chicken Wnt-5a were identified and the role it plays in limb development was examined by comparing its expression pattern with that of two other Wnt members, Wnt-4 and Wnt-11, and by misexpressing it with a retrovirus vector in the limb bud. Wnt-5a expression is detected in the limb-forming region at stage 14, and in the apical ectodermal ridge and distal mesenchyme of the limb bud. The signal was graded along the proximal-distal axis at stages 20-28 and also along the anterior-posterior axis during early stages. It disappeared in the cartilage-forming region after stage 26, and was restricted to the region surrounding the phalanges at stage 34. Wnt-4 and Wnt-11, other members of the Wnt-5a-subclass, were expressed with a distinct spatiotemporal pattern during the later phase. Wnt-4 was expressed in the articular structure and Wnt-11 was expressed in the dorsal and ventral mesenchyme adjacent to the ectoderm. Wnt-5a expression was partially reduced after apical ectodermal ridge removal, whereas Wnt-11 expression was down-regulated by dorsal ectoderm removal. Therefore, expression of these Wnt was differentially regulated by the ectodermal signal. Misexpression of Wnt-5a in the limb bud with the retrovirus resulted in truncation of long bones predominantly in the zeugopod because of retarded chondrogenic differentiation. Distal elements, such as the phalanges and metacarpals, were not significantly reduced in size. These results suggest that Wnt-5a is involved in pattern formation along the proximal-distal axis by regulation of chondrogenic differentiation.     

Evolution and development of the primate limb skeleton

The order Primates is composed of many closely related lineages, each having a relatively well established phylogeny supported by both the fossil record and molecular data. 1 Primate evolution is characterized by a series of adaptive radiations beginning early in the Cenozoic era. Studies of these radiations have uncovered two major trends. One is that substantial amounts of morphological diversity have been produced over short periods of evolutionary time. 2 The other is that consistent and repeated patterns (variational tendencies 3 ) are detected. Taxa within clades, such as the strepsirrhines of Madagascar and the platyrrhines of the Neotropics, have diversified in body size, substrate preference, and diet. 2 , 4 - 6 The diversification of adaptive strategies within such clades is accompanied by repeated patterns of change in cheiridial proportions 7 , 8 (Fig. 1) and tooth‐cusp morphology. 9 There are obvious adaptive, natural‐selection based explanations for these patterns. The hands and feet are in direct contact with a substrate, so their form would be expected to reflect substrate preference, whereas tooth shape is related directly to the functional demands of masticating foods having different mechanical properties. What remains unclear, however, is the role of developmental and genetic processes that underlie the evolutionary diversity of the primate body plan. Are variational tendencies a signature of constraints in developmental pathways? What is the genetic basis for similar morphological transformations among closely related species? These are a sampling of the types of questions we believe can be addressed by future research integrating evidence from paleontology, comparative morphology, and developmental genetics.     

Dual requirement of ectodermal Smad4 during AER formation and termination of feedback signaling in mouse limb buds

BMP signaling is pivotal for normal limb bud development in vertebrate embryos and genetic analysis of receptors and ligands in the mouse revealed their requirement in both mesenchymal and ectodermal limb bud compartments. In this study, we genetically assessed the potential essential functions of SMAD4, a mediator of canonical BMP/TGFß signal transduction, in the mouse limb bud ectoderm. Msx2‐Cre was used to conditionally inactivate Smad4 in the ectoderm of fore‐ and hindlimb buds. In hindlimb buds, the Smad4 inactivation disrupts the establishment and signaling by the apical ectodermal ridge (AER) from early limb bud stages onwards, which results in severe hypoplasia and/or aplasia of zeugo‐ and autopodal skeletal elements. In contrast, the developmentally later inactivation of Smad4 in forelimb buds does not alter AER formation and signaling, but prolongs epithelial‐mesenchymal feedback signaling in advanced limb buds. The late termination of SHH and AER‐FGF signaling delays distal progression of digit ray formation and inhibits interdigit apoptosis. In summary, our genetic analysis reveals the temporally and functionally distinct dual requirement of ectodermal Smad4 during initiation and termination of AER signaling. genesis 51:660–666. © 2013 Wiley Periodicals, Inc.     

The effect of vitamin A (retinoids) on pattern formation implies a uniformity of developmental mechanisms throughout the animal kingdom

Retinoids are low molecular weight, lipophilic derivatives of vitamin A which have a profound effect upon the development of a diverse array of animals. Here, I review these effects on Invertebrates: a colonial hydroid, a colonial ascidian, and Vertebrates: the regenerating amphibian limb, the developing chick limb bud, the regenerating amphibian tail, the anteroposterior axis of the early embryo, the developing chick embryo skin. There is a striking uniformity of effect of retinoids on pattern formation when applied to these diverse organisms. The majority react by being posteriorized in their development, although additional effects can also be seen. Several hypotheses which can explain these results are discussed along with the deduction that they lead to: retinoids may be components of a universal developmental mechanism or they may simply act in a similar way to alter a universal developmental mechanism. In either case the experimental analysis of retinoid effects on development has important implications for the evolution of developmental mechanisms.     

A single-cell census of mouse limb development identifies complex spatiotemporal dynamics of skeleton formation

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Existence of gradient in cell adhesiveness along the developing Xenopus hind limb bud, shown by a cellular sorting-out experiment in vitro

To examine the possibility of a difference in cell adhesiveness along the developing Xenopus hind limb bud axes, single mesenchymal cells from developing hind limb buds were cultured, allowing them to form an aggregate in a gyratory culture system. By observing the distribution of cells within aggregates, it was found that sorting-out occurred between cells from different positions and different stages. Cells derived from more distal positions tended to be situated interiorly in the aggregates. According to Steinberg's differential adhesion hypothesis, these results support the idea that there is a graded difference in cell adhesiveness along the proximo-distal axis of the developing limb, with adhesiveness increasing distally. Although similar sorting-out was observed between anterior and posterior cell populations, it could not be determined which cell populations were definitely more cohesive. These properties may be correlated with the experimentally demonstrated 'positional value', which should be different among cells located at different positions along the axes of the developing vertebrate limb bud.     

Early steps of paired fin development in zebrafish compared with tetrapod limb development

The development of zebrafish paired fins and tetrapod forelimbs and hindlimbs show striking similarities at the molecular level. In recent years, the zebrafish, Danio rerio has become a valuable model for the study of the development of vertebrate paired appendages and several large-scale mutagenesis screens have identified novel fin mutants. This review summarizes recent advances in research into zebrafish paired fin development and highlights features that are shared with and distinct from limb development in other main animal models.     

Ectodermal FGFs induce perinodular inhibition of limb chondrogenesis in vitro and in vivo via FGF receptor 2

The formation of cartilage elements in the developing vertebrate limb, where they serve as primordia for the appendicular skeleton, is preceded by the appearance of discrete cellular condensations. Control of the size and spacing of these condensations is a key aspect of skeletal pattern formation. Limb bud cell cultures grown in the absence of ectoderm formed continuous sheet-like masses of cartilage. With the inclusion of ectoderm, these cultures produced one or more cartilage nodules surrounded by zones of noncartilaginous mesenchyme. Ectodermal fibroblast growth factors (FGF2 and FGF8), but not a mesodermal FGF (FGF7), substituted for ectoderm in inhibiting chondrogenic gene expression, with some combinations of the two ectodermal factors leading to well-spaced cartilage nodules of relatively uniform size. Treatment of cultures with SU5402, an inhibitor FGF receptor tyrosine kinase activity, rendered FGFs ineffective in inducing perinodular inhibition. Inhibition of production of FGF receptor 2 (FGFR2) by transfection of wing and leg cell cultures with antisense oligodeoxynucleotides blocked appearance of ectoderm- or FGF-induced zones of perinodular inhibition of chondrogenesis and, when introduced into the limb buds of developing embryos, led to shorter, thicker, and fused cartilage elements. Because FGFR2 is expressed mainly at sites of precartilage condensation during limb development in vivo and in vitro, these results suggest that activation of FGFR2 by FGFs during development elicits a lateral inhibitor of chondrogenesis that limits the expansion of developing skeletal elements.     

Fibroblast growth factor-induced gene expression and cartilage pattern formation in chick limb bud recombinants

To clarify the roles of fibroblast growth factors (FGF) in limb cartilage pattern formation, the effects of various FGF on recombinant limbs that were composed of dissociated and reaggregated mesoderm and ectodermal jackets were examined. Fibroblast growth factor-soaked beads were inserted just under the apical ectodermal ridge (AER) of recombinant limbs and the recombinant limbs were grafted and allowed to develop. Control recombinant limbs without FGF beads formed one or two cartilage elements. Recombinants with FGF-4 beads formed up to five cartilage elements, which were aligned along the anteroposterior (AP) axis. Each cartilage element showed digit-like segmentation. In contrast, recombinants with FGF-2 beads showed formation of multiple thick and unsegmented cartilage rods, which elongated inside and outside the AP plane from the distal end of the recombinants. Recombinants with FGF-8 beads formed a truncated cartilage pattern and recombinants with FGF-10 beads formed a cartilage pattern similar to that of the control recombinants. The expression of the Fgf-8, Msx-1 and Hoxa-13 genes in the developing recombinant limbs were examined. FGF-4 induced extension of the length of the Fgf-8-positive epidermis, or AER, along the AP axis 5 days after grafting, at which time the digits are specified. FGF-2 induced expansion of the Msx-1-positive area, first in the proximal direction and then along the dorsoventral axis. The functions of these FGF in recombinant and normal limb patterning are discussed in this paper.     

The Wnt antagonist Frzb-1 regulates chondrocyte maturation and long bone development during limb skeletogenesis

The Wnt antagonist Frzb-1 is expressed during limb skeletogenesis, but its roles in this complex multistep process are not fully understood. To address this issue, we determined Frzb-1 gene expression patterns during chick long bone development and carried out gain- and loss-of-function studies by misexpression of Frzb-1, Wnt-8 (a known Frzb-1 target), or different forms of the intracellular Wnt mediator LEF-1 in developing limbs and cultured chondrocytes. Frzb-1 expression was quite strong in mesenchymal prechondrogenic condensations and then characterized epiphyseal articular chondrocytes and prehypertrophic chondrocytes in growth plates. Virally driven Frzb-1 misexpression caused shortening of skeletal elements, joint fusion, and delayed chondrocyte maturation, with consequent inhibition of matrix mineralization, metalloprotease expression, and marrow/bone formation. In good agreement, misexpression of Frzb-1 or a dominant-negative form of LEF-1 in cultured chondrocytes maintained the cells at an immature stage. Instead, misexpression of Wnt-8 or a constitutively active LEF-1 strongly promoted chondrocyte maturation, hypertrophy, and calcification. Immunostaining revealed that the distribution of endogenous Wnt mediator beta-catenin changes dramatically in vivo and in vitro, from largely cytoplasmic in immature proliferating and prehypertrophic chondrocytes to nuclear in hypertrophic mineralizing chondrocytes. Misexpression of Frzb-1 prevented beta-catenin nuclear relocalization in chondrocytes in vivo or in vitro. The data demonstrate that Frzb-1 exerts a strong influence on limb skeletogenesis and is a powerful and direct modulator of chondrocyte maturation, phenotype, and function. Phases of skeletogenesis, such as terminal chondrocyte maturation and joint formation, appear to be particularly dependent on Wnt signaling and thus very sensitive to Frzb-1 antagonistic action.     

Gene expression and pattern formation during early embryonic development in amphibians

Temporal and spatial gene expression and inductive interactions control the establishment of the body plan during embryogenesis in invertebrates and vertebrates. The best-studied vertebrate model system is the amphibian embryo. Seventy-five years after the famous organizer experiment of Hans Spemann and Hilde Mangold in 1924 our knowledge of the molecular mechanisms of the multi-step formation of embryonic axis has substantially improved. Although in the 30s and 40s the interest of many laboratories was focussed on neural induction (determination of the central nervous system), only crude factors from so-called heterogeneous inducers (liver, bone marrow, etc.,) could be isolated by the traditional biochemical techniques available at this time. An important breakthrough was the characterization and purification of a mesoderm inducing factor, the so-called vegetalizing factor (homologous to Activin) in highly purified from chicken embryos. Much later after the introduction of molecular techniques Vgl and Activin (both belonging to the TGF-β family) and FGFs could be identified as important factors for mesoderm formation. It was in the 90s that secreted neuralizing factors (chordin, noggin, follistatin and cerberus) could be detected, which are expressed at the dorsal side of the early embryo including the Spemann organizer. In contrast to the classical view, these proteins act as antagonists to factors like BMP-4 localized on the ventral side. Of special interest was the fact that inDrosophila sog, homologous to chordin, determines the ventral side, whiledpp, homologous toBMP-4, participates in the formation of the dorsal side. These data of evolutionary conserved genes in both invertebrates and vertebrates support the view that they are descendents of common ancestors, the urbilateralia, living around 300 million years ago. The expression of those genes coding for secreted proteins is closely related to inductive interactions between cells and germ layers. Recently it was shown that planar signals are not sufficient to generate a specific anterior/posterior pattern during the primary steps of neural induction, i.e., formation of the central nervous system in amphibians.     

Initiation of limb regeneration: the critical steps for regenerative capacity

While urodele amphibians (newts and salamanders) can regenerate limbs as adults, other tetrapods (reptiles, birds and mammals) cannot and just undergo wound healing. In adult mammals such as mice and humans, the wound heals and a scar is formed after injury, while wound healing is completed without scarring in an embryonic mouse. Completion of regeneration and wound healing takes a long time in regenerative and non-regenerative limbs, respectively. However, it is the early steps that are critical for determining the extent of regenerative response after limb amputation, ranging from wound healing with scar formation, scar-free wound healing, hypomorphic limb regeneration to complete limb regeneration. In addition to the accumulation of information on gene expression during limb regeneration, functional analysis of signaling molecules has recently shown important roles of fibroblast growth factor (FGF), Wnt/beta-catenin and bone morphogenic protein (BMP)/Msx signaling. Here, the routine steps of wound healing/limb regeneration and signaling molecules specifically involved in limb regeneration are summarized. Regeneration of embryonic mouse digit tips and anuran amphibian (Xenopus) limbs shows intermediate regenerative responses between the two extremes, those of adult mammals (least regenerative) and urodele amphibians (more regenerative), providing a range of models to study the various abilities of limbs to regenerate.     

Signals regulating muscle formation in the limb during embryonic development

Classically, somites have been the preparation of choice for the study of muscle development, while the limb bud is the preferred model of axis formation. Nevertheless, the limb bud offers some experimental advantages for muscle studies. This review describes the successive events involved in limb muscle formation during embryonic development, the properties of the key marker molecules and resumes our current knowledge of the signalling pathways involved.