Mutations in mouse Aristaless-like4 cause Strong's luxoid polydactyly

Development ◽  
1998 ◽  
Vol 125 (14) ◽  
pp. 2711-2721 ◽  
Author(s):  
S. Qu ◽  
S.C. Tucker ◽  
J.S. Ehrlich ◽  
J.M. Levorse ◽  
L.A. Flaherty ◽  
...  
Keyword(s):  
Limb Development ◽  
Evolutionary Biology ◽  
Biochemical Characterization ◽  
Limb Bud ◽  
Critical Threshold ◽  
Homeodomain Protein ◽  
Loss Of Function ◽  
Posterior Aspect ◽  
Vertebrate Limb Development ◽  
Zone Of Polarizing Activity

Mutations that affect vertebrate limb development provide insight into pattern formation, evolutionary biology and human birth defects. Patterning of the limb axes depends on several interacting signaling centers; one of these, the zone of polarizing activity (ZPA), comprises a group of mesenchymal cells along the posterior aspect of the limb bud that express sonic hedgehog (Shh) and plays a key role in patterning the anterior-posterior (AP) axis. The mechanisms by which the ZPA and Shh expression are confined to the posterior aspect of the limb bud mesenchyme are not well understood. The polydactylous mouse mutant Strong's luxoid (lst) exhibits an ectopic anterior ZPA and expression of Shh that results in the formation of extra anterior digits. Here we describe a new chlorambucil-induced deletion allele, lstAlb, that uncovers the lst locus. Integration of the lst genetic and physical maps suggested the mouse Aristaless-like4 (Alx4) gene, which encodes a paired-type homeodomain protein that plays a role in limb patterning, as a strong molecular candidate for the Strong's luxoid gene. In genetic crosses, the three lst mutant alleles fail to complement an Alx4 gene-targeted allele. Molecular and biochemical characterization of the three lst alleles reveal mutations of the Alx4 gene that result in loss of function. Alx4 haploinsufficiency and the importance of strain-specific modifiers leading to polydactyly are indicative of a critical threshold requirement for Alx4 in a genetic program operating to restrict polarizing activity and Shh expression in the anterior mesenchyme of the limb bud, and suggest that mutations in Alx4 may also underlie human polydactyly.

The role of Alx-4 in the establishment of anteroposterior polarity during vertebrate limb development

Development ◽  
1998 ◽  
Vol 125 (22) ◽  
pp. 4417-4425 ◽  
Author(s):  
M. Takahashi ◽  
K. Tamura ◽  
D. Buscher ◽  
H. Masuya ◽  
S. Yonei-Tamura ◽  
...  
Keyword(s):  
Negative Feedback ◽  
Limb Development ◽  
Feedback Loop ◽  
Limb Bud ◽  
Negative Regulator ◽  
Negative Feedback Loop ◽  
Vertebrate Limb ◽  
Limb Outgrowth ◽  
Vertebrate Limb Development

We have determined that Strong's Luxoid (lstJ) [corrected] mice have a 16 bp deletion in the homeobox region of the Alx-4 gene. This deletion, which leads to a frame shift and a truncation of the Alx-4 protein, could cause the polydactyly phenotype observed in lstJ [corrected] mice. We have cloned the chick homologue of Alx-4 and investigated its expression during limb outgrowth. Chick Alx-4 displays an expression pattern complementary to that of shh, a mediator of polarizing activity in the limb bud. Local application of Sonic hedgehog (Shh) and Fibroblast Growth Factor (FGF), in addition to ectodermal apical ridge removal experiments suggest the existence of a negative feedback loop between Alx-4 and Shh during limb outgrowth. Analysis of polydactylous mutants indicate that the interaction between Alx-4 and Shh is independent of Gli3, a negative regulator of Shh in the limb. Our data suggest the existence of a negative feedback loop between Alx-4 and Shh during vertebrate limb outgrowth.

Retinoic acid signaling is required during early chick limb development

Development ◽  
1996 ◽  
Vol 122 (5) ◽  
pp. 1385-1394 ◽  
Author(s):  
J.A. Helms ◽  
C.H. Kim ◽  
G. Eichele ◽  
C. Thaller
Keyword(s):  
Retinoic Acid ◽  
Limb Development ◽  
Acid Synthesis ◽  
Apical Ectodermal Ridge ◽  
Limb Bud ◽  
Retinoid Receptor ◽  
Limb Morphogenesis ◽  
Wing Bud ◽  
Zone Of Polarizing Activity

In the chick limb bud, the zone of polarizing activity controls limb patterning along the anteroposterior and proximodistal axes. Since retinoic acid can induce ectopic polarizing activity, we examined whether this molecule plays a role in the establishment of the endogenous zone of polarizing activity. Grafts of wing bud mesenchyme treated with physiologic doses of retinoic acid had weak polarizing activity but inclusion of a retinoic acid-exposed apical ectodermal ridge or of prospective wing bud ectoderm evoked strong polarizing activity. Likewise, polarizing activity of prospective wing mesenchyme was markedly enhanced by co-grafting either a retinoic acid-exposed apical ectodermal ridge or ectoderm from the wing region. This equivalence of ectoderm-mesenchyme interactions required for the establishment of polarizing activity in retinoic acid-treated wing buds and in prospective wing tissue, suggests a role of retinoic acid in the establishment of the zone of polarizing activity. We found that prospective wing bud tissue is a high-point of retinoic acid synthesis. Furthermore, retinoid receptor-specific antagonists blocked limb morphogenesis and down-regulated a polarizing signal, sonic hedgehog. Limb agenesis was reversed when antagonist-exposed wing buds were treated with retinoic acid. Our results demonstrate a role of retinoic acid in the establishment of the endogenous zone of polarizing activity.

scholarly journals Wdpcp Regulates Proliferation and Differentiation in the Developing Limb via Hedgehog Signaling

2021 ◽  
Author(s):  
Mark Langhans ◽  
Rocky Tuan ◽  
Peter Alexander ◽  
Jingtao Gao ◽  
Bing Wang ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Limb Development ◽  
Hedgehog Signaling ◽  
Proliferative Response ◽  
Limb Bud ◽  
In Vitro Assays ◽  
Loss Of Function ◽  
Growth Plates ◽  
Skeletal Phenotype

Abstract Background: Mice with a loss of function mutation in Wdpcp were described previously to display severe birth defects in the developing heart, neural tube, and limb buds. Further characterization of the skeletal phenotype of Wdpcp null mice was limited by perinatal lethality. Results: We utilized Prx1-Cre mice to generate limb bud mesenchyme specific deletion of Wdpcp. These mice recapitulated the appendicular skeletal phenotype of the Wdpcp null mice including polydactyl and limb bud signaling defects. Examination of later stages of limb development demonstrated decreased size of cartilage anlagen, delayed calcification, and abnormal growth plates. Utilizing in vitro assays, we demonstrated that loss of Wdpcp in skeletal progenitors lead to loss of hedgehog signaling responsiveness and associated proliferative response. In vitro chondrogenesis assays showed this loss of hedgehog and proliferative response was associated with decreased expression of early chondrogenic marker N-Cadherin. E14.5 forelimbs demonstrated delayed ossification and expression of osteoblast markers Runx2 and Sp7. P0 growth plates demonstrated loss of hedgehog signaling markers and expansion of the hypertrophic zones of the growth plate. In vitro osteogenesis assays demonstrated decreased osteogenic differentiation of Wdpcp null mesenchymal progenitors in response to hedgehog stimulation. Conclusions: These findings demonstrate how Wdpcp and associated regulation of the hedgehog signaling pathway plays an important role at multiple stages of skeletal development. Wdpcp is necessary for positive regulation of hedgehog signaling and associated proliferation is key to the initiation of chondrogenesis. At later stages, Wdpcp facilitates the robust hedgehog response necessary for chondrocyte hypertrophy and osteogenic differentiation.

Slug, a zinc finger gene previously implicated in the early patterning of the mesoderm and the neural crest, is also involved in chick limb development

Development ◽  
1997 ◽  
Vol 124 (9) ◽  
pp. 1821-1829
Author(s):  
M.A. Ros ◽  
M. Sefton ◽  
M.A. Nieto
Keyword(s):  
Zinc Finger ◽  
Sonic Hedgehog ◽  
Limb Development ◽  
Limb Bud ◽  
Mesenchymal Phenotype ◽  
Signalling Molecules ◽  
Slug Expression ◽  
Snail Family ◽  
Zone Of Polarizing Activity ◽  
Signalling Systems

The great advances made over the last few years in the identification of signalling molecules that pattern the limb bud along the three axes make the limb an excellent model system with which to study developmental mechanisms in vertebrates. The understanding of the signalling networks and their mutual interactions during limb development requires the characterisation of the corresponding downstream genes. In this study we report the expression pattern of Slug, a zinc-finger-containing gene of the snail family, during the development of the limb, and its regulation by distinct axial signalling systems. Slug expression is highly dynamic, and at different stages of limb development can be correlated with the zone of polarizing activity, the progress zone and the interdigital areas. We show that the maintenance of its expression is dependent on signals from the apical ectodermal ridge and independent of Sonic Hedgehog. We also report that, in the interdigit, apoptotic cells lie outside of the domains of Slug expression. The correlation of Slug expression with areas of undifferentiated mesenchyme at stages of tissue differentiation is consistent with its role in early development, in maintaining the mesenchymal phenotype and repressing differentiation processes. We suggest that Slug is involved in the epithelial-mesenchymal interactions that lead to the maintenance of the progress zone.

Expression of chicken fibroblast growth factor homologous factor (FHF)-1 and of differentially spliced isoforms of FHF-2 during development and involvement of FHF-2 in chicken limb development

Development ◽  
1999 ◽  
Vol 126 (2) ◽  
pp. 409-421
Author(s):  
I. Munoz-Sanjuan ◽  
B.K. Simandl ◽  
J.F. Fallon ◽  
J. Nathans
Keyword(s):  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Neural Tube ◽  
Limb Development ◽  
Limb Bud ◽  
Fibroblast Growth ◽  
Limb Buds ◽  
Adjacent Structures ◽  
Morphological Defects ◽  
Zone Of Polarizing Activity

Members of the fibroblast growth factor (FGF) family have been identified as signaling molecules in a variety of developmental processes, including important roles in limb bud initiation, growth and patterning. This paper reports the cloning and characterization of the chicken orthologues of fibroblast growth factor homologous factors-1 and −2 (cFHF-1/cFGF-12 and cFHF-2/cFGF-13, respectively). We also describe the identification of a novel, conserved isoform of FHF-2 in chickens and mammals. This isoform arises by alternative splicing of the first exon of the FHF-2 gene and is predicted to encode a polypeptide with a distinct amino-terminus. Whole-mount in situ hybridization reveals restricted domains of expression of cFHF-1 and cFHF-2 in the developing neural tube, peripheral sensory ganglia and limb buds, and shows that the two cFHF-2 transcript isoforms are present in non-overlapping spatial distributions in the neural tube and adjacent structures. In the developing limbs, cFHF-1 is confined to the posterior mesoderm in an area that encompasses the zone of polarizing activity and cFHF-2 is confined to the distal anterior mesoderm in a region that largely overlaps the progress zone. Ectopic cFHF-2 expression is induced adjacent to grafts of cells expressing Sonic Hedgehog and the zone of cFHF-2 expression is expanded in talpid2 embryos. In the absence of the apical ectodermal ridge or in wingless or limbless mutant embryos, expression of cFHF-1 and cFHF-2 is lost from the limb bud. A role for cFHF-2 in the patterning and growth of skeletal elements is implied by the observation that engraftment of developing limb buds with QT6 cells expressing a cFHF-2 isoform that is normally expressed in the limb leads to a variety of morphological defects. Finally, we show that a secreted version of cFHF-2 activates the expression of HoxD13, HoxD11, Fgf-4 and BMP-2 ectopically, consistent with cFHF-2 playing a role in anterior-posterior patterning of the limb.

The bHLH transcription factor dHAND controls Sonic hedgehog expression and establishment of the zone of polarizing activity during limb development

Development ◽  
2000 ◽  
Vol 127 (11) ◽  
pp. 2461-2470 ◽  
Author(s):  
J. Charite ◽  
D.G. McFadden ◽  
E.N. Olson
Keyword(s):  
Transcription Factor ◽  
Sonic Hedgehog ◽  
Limb Development ◽  
Target Genes ◽  
Ectopic Expression ◽  
Tissue Growth ◽  
Limb Bud ◽  
Bhlh Transcription Factor ◽  
Expression Domain ◽  
Zone Of Polarizing Activity

Limb outgrowth and patterning of skeletal elements are dependent on complex tissue interactions involving the zone of polarizing activity (ZPA) in the posterior region of the limb bud and the apical ectodermal ridge. The peptide morphogen Sonic hedgehog (SHH) is expressed specifically in the ZPA and, when expressed ectopically, is sufficient to mimic its functions, inducing tissue growth and formation of posterior skeletal elements. We show that the basic helix-loop-helix transcription factor dHAND is expressed posteriorly in the developing limb prior to Shh and subsequently occupies a broad domain that encompasses the Shh expression domain. In mouse embryos homozygous for a dHAND null allele, limb buds are severely underdeveloped and Shh is not expressed. Conversely, misexpression of dHAND in the anterior region of the limb bud of transgenic mice results in formation of an additional ZPA, revealed by ectopic expression of Shh and its target genes, and resulting limb abnormalities that include preaxial polydactyly with duplication of posterior skeletal elements. Analysis of mouse mutants in which Hedgehog expression is altered also revealed a feedback mechanism in which Hedgehog signaling is required to maintain the full dHAND expression domain in the developing limb. Together, these findings identify dHAND as an upstream activator of Shh expression and important transcriptional regulator of limb development.

Experiments on developing limb buds of the axolotl Ambystoma mexicanum

Development ◽  
1980 ◽  
Vol 57 (1) ◽  
pp. 177-187
Author(s):  
M. Maden ◽  
B. C. Goodwin
Keyword(s):  
Limb Development ◽  
Ambystoma Mexicanum ◽  
Limb Bud ◽  
Limb Buds ◽  
Vertebrate Limb ◽  
Two Systems ◽  
Vertebrate Limb Development ◽  
Supernumerary Limbs

Various experiments were performed on the limb buds of axolotls to compare the behaviour of amphibian limbs with that previously reported for chick limbs. Following removalof the tip or whole limb bud, extensive powers of regulation were observed since complete limbs always formed. Similarly after distal to proximal grafts intercalary regulation occurred to produce perfect limbs and after proximal to distal grafts serial repetitionsresulted. Transplantation and rotation of limb buds to reverse either the dorso-ventral,antero-posterior or both axes resulted in the induction of supernumerary limbs in a large proportion of cases. Such regulatory behaviour of axolotl limb buds is in contrast to the mosaic nature of chick limbs and as a result, theories such as the progress-zone theory which have been formulated on the basis of data from chick limbs are not relevant togeneral principles of vertebrate limb development. Possible reasons for the diverse behaviour between the two systems are discussed.

Experiments on Anuran limb buds and their significance for principles of vertebrate limb development

Development ◽  
1981 ◽  
Vol 63 (1) ◽  
pp. 243-265
Author(s):  
M. Maden
Keyword(s):  
Limb Development ◽  
Rana Temporaria ◽  
Limb Bud ◽  
Limb Buds ◽  
Dorsoventral Axis ◽  
Vertebrate Limb ◽  
Vertebrate Limb Development ◽  
Supernumerary Limbs ◽  
Standard Set ◽  
And Inversion

A standard set of six experiments performed on the limb buds of two species of Anurans - Rana temporaria and Xenopus laevis -are described. The experiments are limb-bud amputation, distal to proximal shifts, proximal to distal shifts, inversion of the dorsoventral axis inversion of the anteroposterior axis and inversion of both axes. The results are compared to those previously reported for Urodeles and chicks to determine whether any principles of vertebrate limb development can be formulated. It appears that the proximodistal axis becomes increasingly mosaic from the Urodeles through Anurans to chicks. In the transverse axes however, there is much more uniformity of behaviour in the production of supernumerary limbs. The relation between the type of limb development (regulative or mosaic) and the subsequent regenerative powers of the adult limb is discussed.

Evidence that members of the Cut/Cux/CDP family may be involved in AER positioning and polarizing activity during chick limb development

Development ◽  
2000 ◽  
Vol 127 (23) ◽  
pp. 5133-5144
Author(s):  
A.T. Tavares ◽  
T. Tsukui ◽  
J.C. Izpisua Belmonte
Keyword(s):  
Limb Development ◽  
Limb Bud ◽  
Wing Margin ◽  
Lateral Plate ◽  
Posterior Limb ◽  
Downstream Target ◽  
Notch Pathways ◽  
Specialized Region ◽  
Dorsoventral Boundary ◽  
Cycle Regulation

In vertebrates, the apical ectodermal ridge (AER) is a specialized epithelium localized at the dorsoventral boundary of the limb bud that regulates limb outgrowth. In Drosophila, the wing margin is also a specialized region located at the dorsoventral frontier of the wing imaginal disc. The wingless and Notch pathways have been implicated in positioning both the wing margin and the AER. One of the nuclear effectors of the Notch signal in the wing margin is the transcription factor cut. Here we report the identification of two chick homologues of the Cut/Cux/CDP family that are expressed in the developing limb bud. Chick cux1 is expressed in the ectoderm outside the AER, as well as around ridge-like structures induced by (β)-catenin, a downstream target of the Wnt pathway. cux1 overexpression in the chick limb results in scalloping of the AER and limb truncations, suggesting that Cux1 may have a role in limiting the position of the AER by preventing the ectodermal cells around it from differentiating into AER cells. The second molecule of the Cut family identified in this study, cux2, is expressed in the pre-limb lateral plate mesoderm, posterior limb bud and flank mesenchyme, a pattern reminiscent of the distribution of polarizing activity. The polarizing activity is determined by the ability of a certain region to induce digit duplications when grafted into the anterior margin of a host limb bud. Several manipulations of the chick limb bud show that cux2 expression is regulated by retinoic acid, Sonic hedgehog and the posterior AER. These results suggest that Cux2 may have a role in generating or mediating polarizing activity. Taking into account the probable involvement of Cut/Cux/CDP molecules in cell cycle regulation and differentiation, our results raise the hypothesis that chick Cux1 and Cux2 may act by modulating proliferation versus differentiation in the limb ectoderm and polarizing activity regions, respectively.

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