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Lee Niswander - One of the best experts on this subject based on the ideXlab platform.

  • BMP controls proximodistal outgrowth, via induction of the Apical Ectodermal Ridge, and dorsoventral patterning in the vertebrate limb.
    Development (Cambridge England), 2001
    Co-Authors: Sandrine Pizette, Cory Abate-shen, Lee Niswander
    Abstract:

    Dorsoventral (DV) patterning of the vertebrate limb requires the function of the transcription factor Engrailed 1 (EN1) in the ventral ectoderm. EN1 restricts, to the dorsal half of the limb, the expression of the two genes known to specify dorsal pattern. Limb growth along the proximodistal (PD) axis is controlled by the Apical Ectodermal Ridge (AER), a specialized epithelium that forms at the distal junction between dorsal and ventral ectoderm. Using retroviral-mediated misexpression of the bone morphogenetic protein (BMP) antagonist Noggin or an activated form of the BMP receptor in the chick limb, we demonstrate that BMP plays a key role in both DV patterning and AER induction. Thus, the DV and PD axes are linked by a common signal. Loss and gain of BMP function experiments show that BMP signaling is both necessary and sufficient to regulate EN1 expression, and consequently DV patterning. Our results also indicate that BMPs are required during induction of the AER. Manipulation of BMP signaling results in either disruptions in the endogenous AER, leading to absent or severely truncated limbs or the formation of ectopic AERs that can direct outgrowth. Moreover, BMP controls the expression of the MSX transcription factors, and our results suggest that MSX acts downstream of BMP in AER induction. We propose that the BMP signal bifurcates at the level of EN1 and MSX to mediate differentially DV patterning and AER induction, respectively.

  • BMPs negatively regulate structure and function of the limb Apical Ectodermal Ridge
    Development (Cambridge England), 1999
    Co-Authors: Sandrine Pizette, Lee Niswander
    Abstract:

    The Apical Ectodermal Ridge (AER), a transient specialized epithelium at the distal limb tip, is essential for vertebrate embryonic limb outgrowth along the proximodistal axis. Among all the molecules expressed in the AER, only the Fibroblast Growth Factors (FGFs) have been shown to substitute for its function in limb outgrowth. After specification of the skeletal progenitors is complete, the AER regresses, having fulfilled its function. However, the cellular processes underlying AER regression remain largely unclear, and the molecular ones, totally unknown. Members of the Bone Morphogenetic Protein (BMP) family are expressed in the AER throughout its life and in the mesenchyme. Our studies using misexpression of Noggin, a BMP inhibitor, reveal an unsuspected role for BMPs in the negative regulation of Fgf expression and AER function. We find that BMPs limit limb outgrowth by promoting AER regression, as BMP inhibition results in persistence of the AER, prolonged Fgf expression and excess soft-tissue growth. In addition, the Noggin misexpression studies uncover an earlier role for BMPs in repression of AER function. Noggin overexpression results in extension of the AER anteriorly and loss of AER asymmetry. We show that overall the AER becomes taller, and its anterior half becomes more similar to a normal posterior AER. In addition, Fgf4 transcripts, which are usually restricted to the posterior half of the AER, are now also expressed anteriorly. Moreover, ectopicFgf4 expression is induced independently of Sonic Hedgehog, contrary to current models of Fgf4 regulation in the limb. Our studies also provide insight into the activity of the hypothesized Apical Ectodermal maintenance factor (AEMF), which is thought to maintain the tall shape of the posterior part of the AER. Our work shows that the AER is negatively regulated by BMP.

  • Inhibition of NF-κB activity results in disruption of the Apical Ectodermal Ridge and aberrant limb morphogenesis
    Nature, 1998
    Co-Authors: Paul B. Bushdid, Lee Niswander, Dana M. Brantley, Fiona E. Yull, Gareth L. Blaeuer, Loren H. Hoffman, Lawrence D. Kerr
    Abstract:

    In Drosophila, the Dorsal protein establishes the embryonic dorso–ventral axis during development1. Here we show that the vertebrate homologue of Dorsal, nuclear factor-kappa B (NF-κB), is vital for the formation of the proximo–distal organizer of the developing limb bud, the Apical Ectodermal Ridge (AER). Transcription of the NF-κB proto-oncogene c-rel is regulated, in part, during morphogenesis of the limb bud by AER-derived signals such as fibroblast growth factors. Interruption of NF-κB activity using viral-mediated delivery of an inhibitor results in a highly dysmorphic AER, reduction in overall limb size, loss of distal elements and reversal in the direction of limb outgrowth. Furthermore, inhibition of NF-κB activity in limb mesenchyme leads to a reduction in expression of Sonic hedgehog and Twist but derepresses expression of the bone morphogenetic protein-4 gene. These results are the first evidence that vertebrate NF-κB proteins act to transmit growth factor signals between the ectoderm and the underlying mesenchyme during embryonic limb formation.

  • limb deformity proteins role in mesodermal induction of the Apical Ectodermal Ridge
    Development, 1997
    Co-Authors: Julie Kuhlman, Lee Niswander
    Abstract:

    During early limb development, distal tip ectoderm is induced by the underlying mesenchyme to form the Apical Ectodermal Ridge. Subsequent limb growth and patterning depend on reciprocal signaling between the mesenchyme and Ridge. Mice that are homozygous for mutations at the limb deformity (ld) locus do not form a proper Ridge and the anteroposterior axis of the limb is shortened. Skeletal analyses reveal shortened limbs that involve loss and fusion of distal bones and digits, defects in both anteroposterior and proximodistal patterning. Using molecular markers and mouse-chick chimeras we examined the Ridge-mesenchymal interactions to determine the origin of the ld patterning defects. In the ld Ridge, fibroblast growth factor 8 (Fgf8) RNA is decreased and Fgf4 RNA is not detected. In the ld mesenchyme, Sonic hedgehog (Shh), Evx1 and Wnt5a expression is decreased. In chimeras between ld ectoderm and wild-type mesenchyme, a Ridge of normal morphology and function is restored, Fgf8 and Shh are expressed normally, Fgf4 is induced and a normal skeletal pattern arises. These results suggest that the ld mesenchyme is unable to induce the formation of a completely functional Ridge. This primary defect causes a disruption of Ridge function and subsequently leads to the patterning defects observed in ld limbs. We propose a model in which Ridge induction requires at least two phases: an early competence phase, which includes induction of Fgf8 expression, and a later differentiation phase in which Fgf4 is induced and a morphological Ridge is formed. Ld proteins appear to act during the differentiation phase.

  • Effect of FGF on Gene Expression in Chick Limb Bud Cells in Vivo and in Vitro
    Developmental biology, 1995
    Co-Authors: Astrid Vogel, Desmond Roberts-clarke, Lee Niswander
    Abstract:

    Fibroblast growth factors (FGFs) are central to signaling in the developing limb. FGF-2 and FGF-4 can substitute for the Apical Ectodermal Ridge to maintain both limb bud outgrowth and polarizing region signaling. Here, we have repeated and extended previous studies and investigated local effects of the Apical Ectodermal Ridge on gene expression of Msx-1, 5' members of the HoxD complex, and Bmp-2 in the limb bud mesenchyme and tested whether members of the FGF family can substitute for the Ridge to maintain their expression patterns. We found that expression of Msx-1, Hoxd-13, and Bmp-2 in posterior limb bud mesenchyme is dependent on a local signal from the Apical Ectodermal Ridge. When the Apical Ectodermal Ridge of young chick wing buds is removed, or when posterior cells are taken from the bud and placed in culture, expression of Msx-1, Hoxd-13, and Bmp-2 is not detectable in posterior mesenchymal cells. Local application of FGF-soaked beads to posterior limb mesenchyme following Ridge removal or addition of FGF to cultured cells maintains expression of Msx-1, Hoxd-13, and Bmp-2. In contrast, expression of Hoxd-11 in posterior mesenchyme appears to be stable in the absence of either the Apical Ectodermal Ridge or FGF. Expression of Msx-1 in anterior and Apical cells is also locally maintained by the Apical Ectodermal Ridge and this effect can be reproduced by local application of FGF. Furthermore, the addition of FGF to cultured anterior limb bud cells maintains their ability to respond to positional cues when grafted back into limb buds.

Robert A. Kosher - One of the best experts on this subject based on the ideXlab platform.

  • Pleiotropic patterning response to activation of Shh signaling in the limb Apical Ectodermal Ridge.
    Developmental dynamics : an official publication of the American Association of Anatomists, 2011
    Co-Authors: Chi-kuang Leo Wang, Robert A. Kosher, William B. Upholt, Louis J. Pierro, Mizuyo H. Tsugane, Victoria Scranton, Caroline N. Dealy
    Abstract:

    Sonic hedgehog (Shh) signaling in the limb plays a central role in coordination of limb patterning and outgrowth. Shh expression in the limb is limited to the cells of the zone of polarizing activity (ZPA), located in posterior limb bud mesoderm. Shh is not expressed by limb ectoderm or Apical Ectodermal Ridge (AER), but recent studies suggest a role for AER–Shh signaling in limb patterning. Here, we have examined the effects of activation of Shh signaling in the AER. We find that targeted expression of Shh in the AER activates constitutive Shh signaling throughout the AER and subjacent limb mesoderm, and causes a range of limb patterning defects with progressive severity from mild polydactyly, to polysyndactyly with proximal defects, to severe oligodactyly with phocomelia and partial limb ventralization. Our studies emphasize the importance of control of the timing, level and location of Shh pathway signaling for limb anterior–posterior, proximal–distal, and dorsal–ventral patterning. Developmental Dynamics 240:1289–1302, 2011. © 2011 Wiley-Liss, Inc.

  • the expression pattern of the distal less homeo containing gene dlx 5 in the developing chick limb bud suggests its involvement in Apical Ectodermal Ridge activity pattern formation and cartilage differentiation
    Mechanisms of Development, 1995
    Co-Authors: Deborah Ferrari, Jennifer Gannon, William B. Upholt, Lauro Sumoy, Anthony M. C. Brown, Robert A. Kosher
    Abstract:

    Abstract Here we report the isolation from a chick limb bud cDNA library of a cDNA that contains the full coding sequence of chicken Dlx-5, a member of the Distal-less (Dlx) family of homeo☐-containing genes that encode homeodomains highly similar to that of the Drosophila Distal-less gene, a gene that is required for limb development in the Drosophila embryo. The expression pattern of Dlx-5 in the developing chick limb bud suggests that it may be involved in several aspects of limb morphogenesis. Dlx-5 is expressed in the Apical Ectodermal Ridge (AER) which directs the outgrowth and patterning of underlying limb mesoderm. During early limb development Dlx-5 is also expressed in the mesoderm at the anterior margin of the limb bud and in a discrete group of mesodermal cells at the mid-proximal posterior margin that corresponds to the posterior necrotic zone. These mesodermal domains of Dlx-5 expression roughly correspond to the anterior and posterior boundaries of the progress zone, the group of highly proliferating undifferentiated mesodermal cells underneath the AER that will give rise to the skeletal elements of the limb and associated structures. The AER and anterior and posterior mesodermal domains of Dlx-5 expression are regions in which the homeo☐-containing gene Msx-2 is also highly expressed, suggesting that Dlx-5 and Msx-2 might be involved in regulatory networks that control AER activity and demarcate the progress zone. In addition, Dlx-5 is expressed in high amounts by the differentiating cartilaginous skeletal elements of the limb, suggesting it may be involved in regulating the onset of limb cartilage differentiation.

  • Identification of a Spatially Specific Enhancer Element in the Chicken Msx-2 Gene That Regulates Its Expression in the Apical Ectodermal Ridge of the Developing Limb Buds of Transgenic Mice
    Developmental biology, 1995
    Co-Authors: Lauro Sumoy, Robert A. Kosher, Chi-kuang Leo Wang, Alexander C. Lichtler, Louis J. Pierro, William B. Upholt
    Abstract:

    Msx-2 is a member of the Msx family of homeobox-containing genes expressed in a variety of embryonic tissues involved in epithelial-mesenchymal interactions and pattern formation. In the developing chick limb bud, Msx-2 is expressed in the Apical Ectodermal Ridge, which plays a crucial role in directing the growth and patterning of limb mesoderm. In addition, Msx-2 is expressed in the anterior nonskeletal-forming mesoderm of the limb bud, in the posterior necrotic zone, and in the interdigital mesenchyme. Studies of the altered expression patterns of Msx-2 in amelic and polydactylous mutant chick limbs have suggested that the Apical Ectodermal Ridge and mesodermal domains of Msx-2 expression are independently regulated and that there might be separate cis-regulatory elements in the Msx-2 gene controlling its spatially distinct domains of expression. To test this hypothesis, we have isolated the chicken Msx-2 gene and have tested the ability of various regions of the gene to target expression of LacZ reporter gene to specific regions of the limbs of transgenic mice. A variety of these constructs are consistently expressed only in the Apical Ectodermal Ridge and the ectoderm of the genital tubercle and are not expressed in the mesoderm of the limb bud or in other regions of the embryo where the endogenous Msx-2 gene is expressed. These results suggest the presence of spatially specific cis-regulatory elements in the Msx-2 gene. We identified a 348-bp region in the 5' flanking region of the Msx-2 gene which can act as an Apical Ectodermal Ridge enhancer element when placed in reverse orientation in front of the reporter gene with transcription initiation directed by the minimal hsp68 promoter.

  • The expression pattern of the chicken homeobox-containing gene GHox-7 in developing polydactylous limb buds suggests its involvement in Apical Ectodermal Ridge-directed outgrowth of limb mesoderm and in programmed cell death
    Differentiation; research in biological diversity, 1993
    Co-Authors: Caroline N.d. Coelho, William B. Upholt, Robert A. Kosher
    Abstract:

    The limb buds of the polydactylous mutant embryos, talpid2 and diplopodia-5, possess expanded distal apexes surmounted by prolongated thickened Apical Ectodermal Ridges that promote the outgrowth and formation of digits from both the anterior and posterior mesoderm of the mutant limb buds. The chicken homeobox-containing gene GHox-7 exhibits an expanded domain of expression throughout the expanded subRidge mesoderm of the mutant limb buds, providing support for the hypothesis that GHox-7 expression by subRidge mesenchymal cells is involved in the outgrowth-promoting effect of the Apical Ectodermal Ridge. During normal limb development GHox-7 is also expressed by the mesoderm in the proximal anterior nonchondrogenic periphery of the limb bud, which includes, but is not limited to the anterior necrotic zone. GHox-7 is also expressed in the posterior necrotic zone at the mid-proximal posterior edge of the limb bud. In contrast, GHox-7 is not expressed in either the proximal anterior or posterior peripheral mesoderm of talpid2 and diplopodia-5 limb buds which lack proximal anterior and posterior necrotic zones. Furthermore, retinoic acid-coated bead implants, which diminish cell death in the anterior necrotic zone, elicit a local inhibition of GHox-7 expression in the proximal anterior peripheral mesoderm. These results support the suggestion that GHox-7 may be involved in defining regions of programmed cell death during limb development. Furthermore, these studies indicate that the distal subRidge and proximal anterior nonchondrogenic mesodermal domains of GHox-7 expression are independently regulated.

Hiroyuki Ide - One of the best experts on this subject based on the ideXlab platform.

  • fgf7 and fgf10 directly induce the Apical Ectodermal Ridge in chick embryos
    Developmental Biology, 1999
    Co-Authors: Hideyo Ohuchi, Koji Tamura, Hiroyuki Ide, Sayuri Yoneitamura, Tetsuya Endo, Hiroshi Yajima
    Abstract:

    During vertebrate limb development, the Apical Ectodermal Ridge (AER) plays a vital role in both limb initiation and distal outgrowth of the limb bud. In the early chick embryo the prelimb bud mesoderm induces the AER in the overlying ectoderm. However, the direct inducer of the AER remains unknown. Here we report that FGF7 and FGF10, members of the fibroblast growth factor family, are the best candidates for the direct inducer of the AER. FGF7 induces an ectopic AER in the flank ectoderm of the chick embryo in a different manner from FGF1, -2, and -4 and activates the expression of Fgf8, an AER marker gene, in a cultured flank ectoderm without the mesoderm. Remarkably, FGF7 and FGF10 applied in the back induced an ectopic AER in the dorsal median ectoderm. Our results suggest that FGF7 and FGF10 directly induce the AER in the ectoderm both of the flank and of the dorsal midline and that these two regions have the competence for AER induction. Formation of the AER of the dorsal median ectoderm in the chick embryo is likely to appear as a vestige of the dorsal fin of the ancestors.

  • Apical Ectodermal Ridge induction by the transplantation of En-1-overexpressing ectoderm in chick limb bud
    Development growth & differentiation, 1998
    Co-Authors: Mikiko Tanaka, Koji Tamura, Yasuyo Shigetani, Sayaka Sugiyama, Harukazu Nakamura, Hiroyuki Ide
    Abstract:

    In the early chick embryo, the dorsal ventral (DV) boundary organizes the Apical Ectodermal Ridge (AER) structure in the limb bud field. Here it is reported that Engrailed-1 (En-1), a homolog of the Drosophila segment polarity gene engrailed expressed in the ventral limb ectoderm, participates in AER formation at the DV boundary of the limb bud. Restricted ectopic expression of En-1 in the dorsal side of the limb bud by transplantation of En-1-overexpressing ectoderm induces ectopic AER at the boundary of En-1-positive and -negative cells. The results suggest that En-1 is involved in AER formation at the DV boundary of the limb bud.

  • MSX1 EXPRESSING MESODERM IS IMPORTANT FOR THE Apical Ectodermal Ridge (AER)-SIGNAL TRANSFER IN CHICK LIMB DEVELOPMENT
    Development growth & differentiation, 1997
    Co-Authors: Kenji Hara, Hiroyuki Ide
    Abstract:

    The Apical Ectodermal Ridge (AER) is a specialized thickening of the distal limb ectoderm, and its signals are known to support limb morphogenesis. The expression of a homeobox gene, Msx1, in the distal limb mesoderm depends on signals from the AER. In the present paper it is reported that Msx1 expression in the distal mesoderm is necessary for the transfer of AER signals in chick limb buds. Interruption of AER-mesoderm interaction by insertion of a thick filter led to the inhibition of pattern specification in the mesoderm just under the filter. In such cases, the expression of Msx1 disappeared in the mesoderm under the filter, suggesting that AER is able to signal over short ranges. In advanced limb buds, Msx1 is also expressed in the proximal mesoderm under the anterior ectoderm. However, it was found that a grafted antero-proximal mesoderm shows no inhibitory effects on pattern specification of the host mesoderm, as is the case with the distal mesoderm. On the other hand, grafted mesoderms without potent Msx1 re-expression, even underneath AER, disturbed normal limb development. In such cases, the expression of Msx1 disappeared in the mesoderm under the grafts, whereas Fgf-8 expression was maintained in the AER above the graft. These results indicate that the expression of Msx1 in the mesoderm is important for the transfer of AER signals.

  • Apical Ectodermal Ridge-DEPENDENT EXPRESSION OF THE CHICK 67 KDA LAMININ BINDING PROTEIN GENE (CLBP) IN DEVELOPING LIMB BUD
    Zoological science, 1997
    Co-Authors: Kenji Hara, Ken Satoh, Hiroyuki Ide
    Abstract:

    Apical Ectodermal Ridge (AER)-mesoderm interaction is important for morphogenesis in the developing chick limb bud. Genes whose expression is dependent upon the presence of AER, are likely to play important roles in the AER-mesoderm interaction. We report here the gene expression pattern of the chick homolog of the 67 kDa laminin binding protein (LBP), which is a non-integrin laminin receptor whose function relates to cell attachment, spreading, and polarization. Northern analysis showed that a single 1.4 kb transcript exists in stage 20 limb buds and which is dramatically reduced 24 hr after removal of AER. In situ hybridization analysis revealed that the chick 67 kDa laminin binding protein gene (cLbp) was expressed in the mesodermal region overlapping the Msx1-expressing domain and in the AER in early stage limb buds. Expression in the mesoderm was gradually restricted to the distal region underneath the AER as development proceeds. The expression in the limb mesoderm could be induced by local application of FGF-2 which could thus mimic the AER functions. These results indicated that the expression of cLbp depends on AER signals and that the 67 kDa non-integrin receptor binding to laminin plays a role in the AER-mesoderm interaction.

Cheryll Tickle - One of the best experts on this subject based on the ideXlab platform.

  • Dorso-ventral Ectodermal compartments and origin of Apical Ectodermal Ridge in developing chick limb
    Development (Cambridge England), 1997
    Co-Authors: Muriel Altabef, Jonathan D.w. Clarke, Cheryll Tickle
    Abstract:

    We wish to understand how limbs are positioned with respect to the dorso-ventral axis of the body in vertebrate embryos, and how different regions of limb bud ectoderm, i.e. dorsal ectoderm, Apical Ridge and ventral ectoderm, originate. Signals from dorsal and ventral ectoderm control dorso-ventral patterning while the Apical Ectodermal Ridge (AER) controls bud outgrowth and patterning along the proximo-distal axis. We show, using cell-fate tracers, the existence of two distinct Ectodermal compartments, dorsal versus ventral, in both presumptive limb and flank of early chick embryos. This organisation of limb ectoderm is the first direct evidence, in vertebrates, of compartments in non-neural ectoderm. Since the Apical Ridge appears to be confined to this compartment boundary, this positions the limb. The mesoderm, unlike the ectoderm, does not contain two separate dorsal and ventral cell lineages, suggesting that dorsal and ventral ectoderm compartments may be important to ensure appropriate control of mesodermal cell fate. Surprisingly, we also show that cells which form the Apical Ridge are initially scattered in a wide region of early ectoderm and that both dorsal and ventral ectoderm cells contribute to the Apical Ridge, intermingling to some extent within it.

  • FGF-4 replaces the Apical Ectodermal Ridge and directs outgrowth and patterning of the limb
    Cell, 1993
    Co-Authors: Lee Niswander, Cheryll Tickle, Astrid Vogel, Iain Booth, Gail R Martin
    Abstract:

    The Apical Ectodermal Ridge plays a key role in limb development. We show that recombinant FGF-4 can substitute for the Ridge to provide all the signals necessary for virtually complete outgrowth and patterning of the chick limb. FGF-4 stimulates proliferation of cells in the distal mesenchyme and maintains a signal from the posterior to the distal mesenchyme that appears to be required for elaboration of skeletal elements in the normal proximodistal sequence. Moreover, retinoic acid, which is capable of providing polarizing activity, can supply this signal. This suggests that polarizing activity plays a role in patterning along the proximodistal axis, in addition to its well-established role in anteroposterior patterning. Taken together, the data suggest a simple mechanism whereby FGF-4 links growth and pattern formation during limb development.

  • fgf 4 maintains polarizing activity of posterior limb bud cells in vivo and in vitro
    Development, 1993
    Co-Authors: Astrid Vogel, Cheryll Tickle
    Abstract:

    The polarizing region is a major signalling tissue involved in patterning the tissues of the vertebrate limb. The polarizing region is located at the posterior margin of the limb bud and can be recognized by its ability to induce additional digits when grafted to the anterior margin of a chick limb bud. The signal from the polarizing region operates at the tip of the bud in the progress zone, a zone of undifferentiated mesenchymal cells, maintained by interactions with the Apical Ectodermal Ridge. A number of observations have pointed to a link between the Apical Ectodermal Ridge and signalling by the polarizing region. To test this possibility, we removed the posterior Apical Ectodermal Ridge of chick wing buds and assayed posterior mesenchyme for polarizing activity. When the Apical Ectodermal Ridge is removed, there is a marked decrease in polarizing activity of posterior cells. The posterior Apical Ectodermal Ridge is known to express FGF-4 and we show that the decrease in polarizing activity of posterior cells of wing buds that normally follows Ridge removal can be prevented by implanting a FGF-4-soaked bead. Furthermore, we show that both ectoderm and FGF-4 maintain polarizing activity of limb bud cells in culture.

Tsutomu Nohno - One of the best experts on this subject based on the ideXlab platform.

  • A Wnt3a variant participates in chick Apical Ectodermal Ridge formation: distinct biological activities of Wnt3a splice variants in chick limb development.
    Development growth & differentiation, 2007
    Co-Authors: Tomohiro Narita, Shin Ichiro Nishimatsu, Naoyuki Wada, Tsutomu Nohno
    Abstract:

    Wnt/β-catenin signaling is involved in the formation of the Apical Ectodermal Ridge (AER) during vertebrate limb development. Although Wnt3a is a potent ligand for chick AER formation, whether chick Wnt3a can induce Fgf8 expression in chick embryos is unclear and the Wnt ligand involved in chick AER formation remains unknown. Here, we examined whether another Wnt3a isoform is expressed in the AER, and whether Wnt3 contributes to AER formation in chick as well as mouse embryos. We found that chick Wnt3 was not expressed in the presumptive limb ectoderm at the early stages of AER formation. Using 5′-rapid amplification of cDNA ends, we isolated another chick Wnt3a transcript. This novel variant, Wnt3a variant 2, induced Fgf8 in the limb ectoderm and activated the β-catenin pathway in vivo and in vitro. These data showed that Wnt3a variant 2 is an active form of chick Wnt3a that regulates chick AER formation.