Wnt signaling in limb organogenesis

Secreted signaling molecules of the Wnt family have been found to play a central role in controlling embryonic development of a wide range of taxa from Hydra to humans. The most extensively studied Wnt signaling pathway is the canonical Wnt pathway, which controls gene expression by stabilizing β-catenin, and regulates a multitude of developmental processes. More recently, noncanonical Wnt pathways, which are β-catenin-independent, have been found to be important developmental regulators. Understanding the mechanisms of Wnt signaling is essential for the development of novel preventive and therapeutic approaches of human diseases. Limb development is a paradigm to study the principles of Wnt signaling in various developmental contexts. In the developing vertebrate limb, Wnt signaling has been shown to have important functions during limb bud initiation, limb outgrowth, early limb patterning, and later limb morphogenesis events. This review provides a brief overview on the diversity of Wnt-dependent signaling events during embryonic development of the vertebrate limb.     

Wnt signaling in limb organogenesis

Secreted signaling molecules of the Wnt family have been found to play a central role in controlling embryonic development of a wide range of taxa from Hydra to humans. The most extensively studied Wnt signaling pathway is the canonical Wnt pathway, which controls gene expression by stabilizing β-catenin, and regulates a multitude of developmental processes. More recently, noncanonical Wnt pathways, which are β-catenin-independent, have been found to be important developmental regulators. Understanding the mechanisms of Wnt signaling is essential for the development of novel preventive and therapeutic approaches of human diseases. Limb development is a paradigm to study the principles of Wnt signaling in various developmental contexts. In the developing vertebrate limb, Wnt signaling has been shown to have important functions during limb bud initiation, limb outgrowth, early limb patterning, and later limb morphogenesis events. This review provides a brief overview on the diversity of Wnt-dependent signaling events during embryonic development of the vertebrate limb.Key words: Wnts, limb initiation, outgrowth, patterning, morphogenesis     

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 dominant hemimelia mutation uncouples epithelial-mesenchymal interactions and disrupts anterior mesenchyme formation in mouse hindlimbs.

Epithelial-mesenchymal interactions are essential for both limb outgrowth and pattern formation in the limb. Molecules capable of communication between these two tissues are known and include the signaling molecules SHH and FGF4, FGF8 and FGF10. Evidence suggests that the pattern and maintenance of expression of these genes are dependent on a number of factors including regulatory loops between genes expressed in the AER and those in the underlying mesenchyme. We show here that the mouse mutation dominant hemimelia (Dh) alters the pattern of gene expression in the AER such that Fgf4, which is normally expressed in a posterior domain, and Fgf8, which is expressed throughout are expressed in anterior patterns. We show that maintenance of Shh expression in the posterior mesenchyme is not dependent on either expression of Fgf4 or normal levels of Fgf8 in the overlying AER. Conversely, AER expression of Fgf4 is not directly dependent on Shh expression. Also the reciprocal regulatory loop proposed for Fgf8 in the AER and Fgf10 in the underlying mesenchyme is also uncoupled by this mutation. Early during the process of limb initiation, Dh is involved in regulating the width of the limb bud, the mutation resulting in selective loss of anterior mesenchyme. The Dh gene functions in the initial stages of limb development and we suggest that these initial roles are linked to mechanisms that pattern gene expression in the AER.     

Tbx2 Terminates Shh/Fgf Signaling in the Developing Mouse Limb Bud by Direct Repression of Gremlin1

Vertebrate limb outgrowth is driven by a positive feedback loop that involves Sonic hedgehog (Shh) and Gremlin1 (Grem1) in the posterior limb bud mesenchyme and Fibroblast growth factors (Fgfs) in the overlying epithelium. Proper spatio-temporal control of these signaling activities is required to avoid limb malformations such as polydactyly. Here we show that, in Tbx2-deficient hindlimbs, Shh/Fgf4 signaling is prolonged, resulting in increased limb bud size and duplication of digit 4. In turn, limb-specific Tbx2 overexpression leads to premature termination of this signaling loop with smaller limbs and reduced digit number as phenotypic manifestation. We show that Tbx2 directly represses Grem1 in distal regions of the posterior limb mesenchyme allowing Bone morphogenetic protein (Bmp) signaling to abrogate Fgf4/9/17 expression in the overlying epithelium. Since Tbx2 itself is a target of Bmp signaling, our data identify a growth-inhibiting positive feedback loop (Bmp/Tbx2/Grem1). We propose that proliferative expansion of Tbx2-expressing cells mediates self-termination of limb bud outgrowth due to their refractoriness to Grem1 induction.     

Ectodermal Wnt-6 promotes Myf5-dependent avian limb myogenesis

Limb muscles of vertebrates are derived from precursor cells that migrate from the lateral edge of the dermomyotome into the limb bud. Although several signaling molecules have been reported to be involved in the process of limb myogenesis, none of their activities has led to a consolidate idea about the limb myogenic pathway. Particularly, the role of ectodermal signals in limb myogenesis is still obscure. Here, we investigated the role of the ectoderm and ectodermal Wnt-6 during limb muscle development. We found that ectopic expression of Wnt-6 in the limb bud specifically extends the expression domains of Pax3, Paraxis, Myf5, Myogenin, Desmin and Myosin heavy chain (MyHC) but inhibits MyoD expression. Ectoderm removal results in a loss of expression of all of these myogenic markers. We show that Wnt-6 can compensate the absence of the ectoderm by rescuing the expression of Pax3, Paraxis, Myf5, Myogenin, Desmin and MyHC but not MyoD. These results show that, in chick, at least two signals from the limb ectoderm are necessary for muscle development. One of the signals is Wnt-6, which plays a unique role in promoting limb myogenesis via Pax3/Paraxis-Myf5, whereas the other putative signaling pathway involving MyoD expression is negatively regulated by Wnt-6 signaling.     

Developmental origins of precocial forelimbs in marsupial neonates

Marsupial mammals are born in an embryonic state, as compared with their eutherian counterparts, yet certain features are accelerated. The most conspicuous of these features are the precocial forelimbs, which the newborns use to climb unaided from the opening of the birth canal to the teat. The developmental mechanisms that produce this acceleration are unknown. Here we show that heterochronic and heterotopic changes early in limb development contribute to forelimb acceleration. Using Tbx5 and Tbx4 as fore- and hindlimb field markers, respectively, we have found that, compared with mouse, both limb fields arise notably early during opossum development. Patterning of the forelimb buds is also accelerated, as Shh expression appears early relative to the outgrowth of the bud itself. In addition, the forelimb fields and forelimb myocyte allocation are increased in size and number, respectively, and migration of the spinal nerves into the forelimb bud has been modified. This shift in the extent of the forelimb field is accompanied by shifts in Hox gene expression along the anterior-posterior axis. Furthermore, we found that both fore- and hindlimb fields arise gradually during gastrulation and extension of the embryonic axis, in contrast to the appearance of the limb fields in their entirety in all other known cases. Our results show a surprising evolutionary flexibility in the early limb development program of amniotes and rule out the induction of the limb fields by mature structures such as the somites or mesonephros.     

Gremlin-mediated BMP antagonism induces the epithelial-mesenchymal feedback signaling controlling metanephric kidney and limb organogenesis

Epithelial-mesenchymal feedback signaling is the key to diverse organogenetic processes such as limb bud development and branching morphogenesis in kidney and lung rudiments. This study establishes that the BMP antagonist gremlin (Grem1) is essential to initiate these epithelial-mesenchymal signaling interactions during limb and metanephric kidney organogenesis. A Grem1 null mutation in the mouse generated by gene targeting causes neonatal lethality because of the lack of kidneys and lung septation defects. In early limb buds, mesenchymal Grem1 is required to establish a functional apical ectodermal ridge and the epithelial-mesenchymal feedback signaling that propagates the sonic hedgehog morphogen. Furthermore, Grem1-mediated BMP antagonism is essential to induce metanephric kidney development as initiation of ureter growth, branching and establishment of RET/GDNF feedback signaling are disrupted in Grem1-deficient embryos. As a consequence, the metanephric mesenchyme is eliminated by apoptosis, in the same way as the core mesenchymal cells of the limb bud.     

Retention of lateral motor column neurons during the phase of rapid cell loss after limb amputation in Rana pipiens tadpoles

Target tissue regulation of naturally occurring neuronal death during development has often been studied by removing a limb bud and then analyzing spinal motor neuron number later in development. The present study focuses on the necessity for limb presence in the initiation of the most rapid phase of cell loss from the lateral motor column (LMC) and in the control of neuron number during this restricted developmental period. Unilateral hindlimb amputation in larval Rana pipiens at the time of onset of rapid LMC cell loss resulted in an unequal, bilateral retention of excess motor neurons (i.e., less cell loss than normally occurs) during this phase. Limb traumatization, with axotomy, also resulted in reduced bilateral LMC cell loss, although to a lesser extent than did amputation. Absence of the limb or axonal transection presumably prevents the communication of motor neurons with their differentiating targets and thus interferes with the flow of information required for the selective events of neuronal loss and survival. The presence of the limb with intact axons is essential at the time that LMC cell loss normally ensues for both the initiation and progression of the phase of greatest cell loss.     

The recombinant limb as a model for the study of limb patterning, and its application to muscle development

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. Received: 31 August 1998 / Accepted: 29 September 1998     

Evidence that the limb bud ectoderm is required for survival of the underlying mesoderm

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.     

The Osr1 and Osr2 genes act in the pronephric anlage downstream of retinoic acid signaling and upstream of Wnt2b to maintain pectoral fin development

Vertebrate odd-skipped related genes (Osr) have an essential function during the formation of the intermediate mesoderm (IM) and the kidney structures derived from it. Here, we show that these genes are also crucial for limb bud formation in the adjacent lateral plate mesoderm (LPM). Reduction of zebrafish Osr function impairs fin development by the failure of tbx5a maintenance in the developing pectoral fin bud. Osr morphant embryos show reduced wnt2b expression, and increasing Wnt signaling in Osr morphant embryos partially rescues tbx5a expression. Thus, Osr genes control limb bud development in a non-cell-autonomous manner, probably through the activation of Wnt2b. Finally, we demonstrate that Osr genes are downstream targets of retinoic acid (RA) signaling. Therefore, Osr genes act as a relay within the genetic cascade of fin bud formation: by controlling the expression of the signaling molecule Wnt2ba in the IM they play an essential function transmitting the RA signaling originated in the somites to the LPM.     

Cell populations synthesizing cartilage proteoglycan core protein in the early chick limb bud.

Cartilage specific macromolecules are known to be synthesized in the mesenchyme of the embryonic chick limb bud, especially in areas of prechondrogenic condensations (Shinomura et al, 1984). Even though the mesenchyme seems homogeneous according to histological criteria, studies in the past have suggested the presence of different cell populations with different chondrogenic potential (Solursh et al, 1982; Swalla et al, 1984). In this study we have investigated by means of flow cytometry, the synthesis of proteoglycan core protein during early development of the chick limb bud in order to identify the different chondrocyte progenitor cells. We were able to identify by virtue of different size and density a cell population which synthesizes core protein extensively at stage 24 and stage 25 of development. This cell population synthesizes core protein predominantly at the proximal half of the limb bud at stage 24. However at stage 25 the same population synthesizes core protein predominantly at the distal half of the limb bud. These observations indicate that the distal half of stage 25 limb bud is mostly homogeneous with prechondrogenic cells and is in agreement with in vitro experiments that show high chondrogenic potential of the mesenchymal cells from this stage.