A novel family of T-box genes in urodele amphibian limb development and regeneration: candidate genes involved in vertebrate forelimb/hindlimb patterning

In certain urodeles, a lost appendage, including hand and foot, can be completely replaced through epimorphic regeneration. The regeneration process involves cellular activities similar to those described for embryogenesis. Working on the assumption that the morphological pattern specific for a forelimb or a hindlimb is controlled by different gene activities in the two limbs, we employed a mRNA differential display screen for the detection of candidate limb identity genes. Using this approach, we have isolated a newt gene which in regenerating and developing limbs reveals properties expected of a gene having a role in controlling limb morphology: (1) it is exclusively expressed in the forelimbs, but not hindlimbs, (2) during embryonic development its expression is co-incident with forelimb bud formation, (3) it has an elevated message level throughout the undifferentiated limb bud and the blastema, respectively, and (4) it is expressed only in mesenchymal, but not in epidermal tissues. This novel newt gene shares a conserved DNA-binding domain, the T-box, with putative transcription factors including the Brachyury (T) gene product. In a following PCR-based screen, we used the evolutionarily conserved T-box motif and amplified a family of related genes in the newt; their different expression patterns in normal and regenerating forelimbs, hindlimbs and tail suggest, in general, an important role of T-domain proteins in vertebrate pattern formation.

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Role of the chicken homeobox-containing genes GHox-4.6 and GHox-8 in the specification of positional identities during the development of normal and polydactylous chick limb buds

Development ◽

10.1242/dev.115.2.629 ◽

1992 ◽

Vol 115 (2) ◽

pp. 629-637 ◽

Cited By ~ 3

Author(s):

C.N. Coelho ◽

W.B. Upholt ◽

R.A. Kosher

Keyword(s):

Limb Development ◽

Ectopic Expression ◽

Limb Bud ◽

Chick Embryos ◽

Limb Buds ◽

Hox Gene ◽

Wing Bud ◽

Coated Bead ◽

Experimentally Induced

During early stages of normal chick limb development, the homeobox-containing (HOX) gene GHox-4.6 is expressed throughout the posterior mesoderm of the wing bud from which most of the skeletal elements including the digits will develop, whereas GHox-8 is expressed in the anterior limb bud mesoderm which will not give rise to skeletal elements. In the present study, we have examined the expression of GHox-4.6 and GHox-8 in the wing buds of two polydactylous mutant chick embryos, diplopodia-5 and talpid2, from which supernumerary digits develop from anterior limb mesoderm, and have also examined the expression of these genes in response to polarizing zone grafts and retinoic acid-coated bead implants which induce the formation of supernumerary digits from anterior limb mesoderm. We have found that the formation of supernumerary digits from the anterior mesoderm in mutant and experimentally induced polydactylous limb buds is preceded by the ectopic expression of GHox-4.6 in the anterior mesoderm and the coincident suppression of GHox-8 expression in the anterior mesoderm. These observations suggest that the anterior mesoderm of the polydactylous limb buds is “posteriorized” and support the suggestion that GHox-8 and GHox-4.6, respectively, are involved in specifying the anterior non-skeletal and posterior digit-forming regions of the limb bud. Although the anterior mesodermal domain of GHox-8 expression is severely impaired in the mutant and experimentally induced polydactylous limb buds, this gene is expressed by the prolonged, thickened apical ectodermal ridges of the polydactylous limb buds that extend along the distal anterior as well as the distal posterior mesoderm.(ABSTRACT TRUNCATED AT 250 WORDS)

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

Development ◽

10.1242/dev.125.22.4417 ◽

1998 ◽

Vol 125 (22) ◽

pp. 4417-4425 ◽

Cited By ~ 6

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.

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Retinoic acid signaling is required during early chick limb development

Development ◽

10.1242/dev.122.5.1385 ◽

1996 ◽

Vol 122 (5) ◽

pp. 1385-1394 ◽

Cited By ~ 10

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.

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Sonic hedgehog is not required for polarising activity in the Doublefoot mutant mouse limb bud

Development ◽

10.1242/dev.125.3.351 ◽

1998 ◽

Vol 125 (3) ◽

pp. 351-357 ◽

Cited By ~ 2

Author(s):

C. Hayes ◽

J.M. Brown ◽

M.F. Lyon ◽

G.M. Morriss-Kay

Keyword(s):

Gene Expression ◽

Limb Development ◽

Target Genes ◽

Anterior Part ◽

Expression Patterns ◽

Ectopic Expression ◽

Mutant Gene ◽

Limb Bud ◽

Active Constituent ◽

Limb Buds

The mouse mutant Doublefoot (Dbf) shows preaxial polydactyly of all four limbs. We have analysed limb development in this mutant with respect to morphogenesis, gene expression patterns and ectopic polarising activity. The results reveal a gain-of-function mutation at a locus that mediates pattern formation in the developing limb. Shh expression is identical with that of wild-type embryos, i.e. there is no ectopic expression. However, mesenchyme from the anterior aspects of Dbf/+ mutant limb buds, when transplanted to the anterior side of chick wing buds, induces duplication of the distal skeletal elements. Mid-distal mesenchymal transplants from early, but not later, Dbf/+ limb buds are also able to induce duplication. This demonstration of polarising activity in the absence of Shh expression identifies the gene at the Dbf locus as a new genetic component of the Shh signalling pathway, which (at least in its mutated form) is able to activate signal transduction independently of Shh. The mutant gene product is sufficient to fulfil the signalling properties of Shh including upregulation of the direct Shh target genes Ptc and Gli, and induction of the downstream target genes Bmp2, Fgf4 and Hoxd13. The expression domains of all these genes extend from their normal posterior domains into the anterior part of the limb bud without being focused on a discrete ectopic site. These observations dissociate polarising activity from Shh gene expression in the Dbf/+ limb bud. We suggest that the product of the normal Dbf gene is a key active constituent of the polarising region, possibly acting in the extracellular compartment.

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Etudes expérimentales sur le rôle des somites au cours des premiers stades du développement du membre antérieur chez l'embryon du Chélonien Emys orbicularis L.

Development ◽

10.1242/dev.32.2.417 ◽

1974 ◽

Vol 32 (2) ◽

pp. 417-430

Author(s):

Par J. Vasse

Keyword(s):

Experimental Study ◽

Developmental Stage ◽

Limb Development ◽

Limb Bud ◽

Stages Of Development ◽

Emys Orbicularis ◽

Bud Development ◽

Early Stages

Experimental study on the role of the somites during the early stages of development of the front limbs of the embryo of the chelonian Emys orbicularis L. Ablation of postotic somites 6–13 on one side in embryos of Emys orbicularis L. at the developmental stage when 20–23 somite pairs were present, led to arrest of forelimb-bud development in the somatopleure adjacent to the ablated somites on the operated side. Limb development in the somatopleure adjacent to intact somites on the operated side was unaffected, attaining the same stage as on the non-operated side. Ablation at later stages (25–33 somite pairs) did not prevent development of the limb adjacent to the ablated somites. When a part of the prospective somatopleure was injured, the remaining part formed a small limb-bud. When an obstacle was placed between the somatopleural mesoderm and the adjacent somite, development of the somatopleure stopped at this level. These results corroborate those obtained from previous studies in various reptilian embryos concerning the role of the ventral somite extensions as activators of proliferation in the somatopleural mesoderm. Injury to the ventral extension alone led to serious disturbances in the somatopleural mesoderm adjacent to this somite.

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The role of Engrailed in establishing the dorsoventral axis of the chick limb

Development ◽

10.1242/dev.124.12.2317 ◽

1997 ◽

Vol 124 (12) ◽

pp. 2317-2324 ◽

Cited By ~ 8

Author(s):

C. Logan ◽

A. Hornbruch ◽

I. Campbell ◽

A. Lumsden

Keyword(s):

Expression Patterns ◽

Limb Bud ◽

Dorsoventral Patterning ◽

Signalling Molecules ◽

Endogenous Expression ◽

Signalling Molecule ◽

Normal Expression ◽

Dorsal Ectoderm ◽

Mutation Analyses

Expression and mutation analyses in mice suggest that the homeobox-containing gene Engrailed (En) plays a role in dorsoventral patterning of the limb. During the initial stages of limb bud outgrowth, En-1 mRNA and protein are uniformly distributed throughout the ventral limb bud ectoderm. Limbs of En-1(−/−) mice display a double dorsal phenotype suggesting that normal expression of En-1 in the ventral ectoderm is required to establish and/or maintain ventral limb characteristics. Loss of En-1 function also results in ventral expansion of the apical ectodermal ridge (AER), suggesting that En-1 is also required for proper formation of the AER. To further investigate the role En plays in dorsoventral patterning and AER formation, we have used the replication competent retroviral vector, RCAS, to mis-express mouse En-1 in the early chick limb bud. We show that ectopic En-1 expression in dorsal ectoderm is sufficient to repress the endogenous expression of the dorsal ectodermal marker Wnt7a, with a resultant decrease in Lmx1 expression in underlying dorsal mesenchyme. Furthermore, the AER is disrupted morphologically and the expression patterns of the AER signalling molecules Fgf-8 and Fgf-4 are altered. Consistent with recent evidence that there is a reciprocal interaction between signalling molecules in the dorsal ectoderm, AER, and zone of polarising activity (ZPA), loss of Wnt7a, Fgf-8 and Fgf-4 expression leads to a decrease in expression of the signalling molecule Shh in the ZPA. These results strongly support the idea that, in its normal domain of expression, En-1 represses Wnt7a-mediated dorsal differentiation by limiting the expression of Wnt7a to the dorsal ectoderm. Furthermore, our results provide additional evidence that En-1 is involved in AER formation and suggest that En-1 may act to define ventral ectodermal identity.

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Why we have (only) five fingers per hand: hox genes and the evolution of paired limbs

Development ◽

10.1242/dev.116.2.289 ◽

1992 ◽

Vol 116 (2) ◽

pp. 289-296 ◽

Cited By ~ 4

Author(s):

C.J. Tabin

Keyword(s):

Limb Development ◽

Hox Genes ◽

Expression Patterns ◽

Limb Bud ◽

Developmental Constraint ◽

Limb Morphogenesis ◽

Different Types ◽

Embryonic Limb ◽

Limb Pattern ◽

Anterior Posterior

Limb development has long been a model system for studying vertebrate pattern formation. The advent of molecular biology has allowed the identification of some of the key genes that regulate limb morphogenesis. One important class of such genes are the homeobox-containing, or Hox genes. Understanding of the roles these genes play in development additionally provides insights into the evolution of limb pattern. Hox gene expression patterns divide the embryonic limb bud into five sectors along the anterior/posterior axis. The expression of specific Hox genes in each domain specifies the developmental fate of that region. Because there are only five distinct Hox-encoded domains across the limb bud there is a developmental constraint prohibiting the evolution of more than five different types of digits. The expression patterns of Hox genes in modern embryonic limb buds also gives clues to the shape of the ancestral fin field from which the limb evolved, hence elucidating the evolution of the tetrapod limb.

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Involvement of T-box genes Tbx2-Tbx5 in vertebrate limb specification and development

Development ◽

10.1242/dev.125.13.2499 ◽

1998 ◽

Vol 125 (13) ◽

pp. 2499-2509 ◽

Cited By ~ 14

Author(s):

J.J. Gibson-Brown ◽

S.I. Agulnik ◽

L.M. Silver ◽

L. Niswander ◽

V.E. Papaioannou

Keyword(s):

Gene Expression ◽

Limb Development ◽

T Box ◽

Cell Pellet ◽

Wing Bud ◽

Zone Of Polarizing Activity ◽

The Relationship ◽

Tbx2 Expression ◽

Limb Specification

We have recently shown in mice that four members of the T-box family of transcription factors (Tbx2-Tbx5) are expressed in developing limb buds, and that expression of two of these genes, Tbx4 and Tbx5, is primarily restricted to the developing hindlimbs and forelimbs, respectively. In this report, we investigate the role of these genes in limb specification and development, using the chick as a model system. We induced the formation of ectopic limbs in the flank of chick embryos to examine the relationship between the identity of the limb-specific T-box genes being expressed and the identity of limb structures that subsequently develop. We found that, whereas bud regions expressing Tbx4 developed characteristic leg structures, regions expressing Tbx5 developed characteristic wing features. In addition, heterotopic grafts of limb mesenchyme (wing bud into leg bud, and vice versa), which are known to retain the identity of the donor tissue after transplantation, retained autonomous expression of the appropriate, limb-specific T-box gene, with no evidence of regulation by the host bud. Thus there is a direct relationship between the identity of the structures that develop in normal, ectopic and recombinant limbs, and the identity of the T-box gene(s) being expressed. To investigate the regulation of T-box gene expression during limb development, we employed several other embryological manipulations. By surgically removing the apical ectodermal ridge (AER) from either wing or leg buds, we found that, in contrast to all other genes implicated in the patterning of developing appendages, maintenance of T-box gene expression is not dependent on the continued provision of signals from the AER or the zone of polarizing activity (ZPA). By generating an ectopic ZPA, by grafting a sonic hedgehog (SHH)-expressing cell pellet under the anterior AER, we found that Tbx2 expression can lie downstream of SHH. Finally, by grafting a SHH-expressing cell pellet to the anterior margin of a bud from which the AER had been removed, we found that Tbx2 may be a direct, short-range target of SHH. Our findings suggest that these genes are intimately involved in limb development and the specification of limb identity, and a new model for the evolution of vertebrate appendages is proposed.

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hrT is required for cardiovascular development in zebrafish

Development ◽

10.1242/dev.129.21.5093 ◽

2002 ◽

Vol 129 (21) ◽

pp. 5093-5101 ◽

Cited By ~ 1

Author(s):

Daniel P. Szeto ◽

Kevin J. P. Griffin ◽

David Kimelman

Keyword(s):

Late Onset ◽

Cardiovascular Development ◽

Lateral Plate ◽

T Box ◽

Vascular Morphogenesis ◽

Developing Heart ◽

Evolutionarily Conserved ◽

Heart Formation ◽

Cardiovascular Cells

The recently identified zebrafish T-box gene hrT is expressed in the developing heart and in the endothelial cells forming the dorsal aorta. Orthologs of hrT are expressed in cardiovascular cells fromDrosophila to mouse, suggesting that the function of hrT is evolutionarily conserved. The role of hrT in cardiovascular development, however, has not thus far been determined in any animal model. Using morpholino antisense oligonucleotides, we show that zebrafish embryos lacking hrT function have dysmorphic hearts and an absence of blood circulation. Although the early events in heart formation were normal inhrT morphant embryos, subsequently the hearts failed to undergo looping, and late onset defects in chamber morphology and gene expression were observed. In particular, we found that the loss of hrT function led to a dramatic upregulation of tbx5, a gene required for normal heart morphogenesis. Conversely, we show that overexpression of hrT causes a significant downregulation of tbx5, indicating that one key role ofhrT is to regulate the levels of tbx5. Secondly, we found that HrT is required to inhibit the expression of the blood lineage markersgata1 and gata2 in the most posterior lateral plate mesoderm. Finally, we show that HrT is required for vasculogenesis in the trunk, leading to similar vascular defects to those observed in midline mutants such as floating head. hrT expression in the vascular progenitors depends upon midline mesoderm, indicating that this expression is one important component of the response to a midline-derived signal during vascular morphogenesis.

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Pigeon foot feathering reveals conserved limb identity networks

10.1101/602987 ◽

2019 ◽

Author(s):

Elena F. Boer ◽

Hannah F. Van Hollebeke ◽

Sungdae Park ◽

Carlos R. Infante ◽

Douglas B. Menke ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Limb Development ◽

Limb Bud ◽

Differentially Expressed ◽

Evolutionarily Conserved ◽

Genes Encoding ◽

Embryonic Limb ◽

Summary Statement ◽

Limb Identity

AbstractThe tetrapod limb is a stunning example of evolutionary diversity, with dramatic variation not only among distantly related species, but also between the serially homologous forelimbs (FLs) and hindlimbs (HLs) within species. Despite this variation, highly conserved genetic and developmental programs underlie limb development and identity in all tetrapods, raising the question of how limb diversification is generated from a conserved toolkit. In some breeds of domestic pigeon, shifts in the expression of two conserved limb identity transcription factors,PITX1andTBX5, are associated with the formation of feathered HLs with partial FL identity. To determine how modulation ofPITX1andTBX5expression affects downstream gene expression, we compared the transcriptomes of embryonic limb buds from pigeons with scaled and feathered HLs. We identified a set of differentially expressed genes enriched for genes encoding transcription factors, extracellular matrix proteins, and components of developmental signaling pathways with important roles in limb development. A subset of the genes that distinguish scaled and feathered HLs are also differentially expressed between FL and scaled HL buds in pigeons, pinpointing a set of gene expression changes downstream ofPITX1andTBX5in the partial transformation from HL to FL identity. We extended our analyses by comparing pigeon limb bud transcriptomes to chicken, anole lizard, and mammalian datasets to identify deeply conservedPITX1- andTBX5-regulated components of the limb identity program. Our analyses reveal a suite of predominantly low-level gene expression changes that are conserved across amniotes to regulate the identity of morphologically distinct limbs.Summary statementIn feather-footed pigeons, mutant alleles ofPITX1andTBX5drive the partial redeployment of an evolutionarily conserved forelimb genetic program in the hindlimb.

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