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

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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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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Correlation of wing-leg identity in ectopic FGF-induced chimeric limbs with the differential expression of chick Tbx5 and Tbx4

Development ◽

10.1242/dev.125.1.51 ◽

1998 ◽

Vol 125 (1) ◽

pp. 51-60 ◽

Cited By ~ 5

Author(s):

H. Ohuchi ◽

J. Takeuchi ◽

H. Yoshioka ◽

Y. Ishimaru ◽

K. Ogura ◽

...

Keyword(s):

Growth Factor ◽

Fibroblast Growth Factor ◽

Expression Patterns ◽

Ectopic Expression ◽

Limb Bud ◽

Chick Embryos ◽

Fibroblast Growth ◽

Limb Buds ◽

Specific Position

It has been reported that members of the fibroblast growth factor (FGF) family can induce additional limb formation in the flank of chick embryos. The phenotype of the ectopic limb depends on the somite level at which it forms: limbs in the anterior flank resemble wings, whereas those in the posterior flank resemble legs. Ectopic limbs located in the mid-flank appear chimeric, possessing characteristics of both wings and legs; feather buds are present in the anterior halves with scales and claws in the posterior halves. To study the mechanisms underlying the chimerism of these additional limbs, we cloned chick Tbx5 and Tbx4 to use as forelimb and hindlimb markers and examined their expression patterns in FGF-induced limb buds. We found that Tbx5 and Tbx4 were differentially expressed in the anterior and posterior halves of additional limb buds in the mid-flank, respectively, consistent with the chimeric patterns of the integument. A boundary of Tbx5/Tbx4 exists in all ectopic limbs, indicating that the additional limbs are essentially chimeric, although the degree of chimerism is dependent on the position. The boundary of Tbx5/Tbx4 expression is not fixed at a specific position within the interlimb region, but dependent upon where FGF was applied. Since the ectopic expression patterns of Tbx5/Tbx4 in the additional limbs are closely correlated with the patterns of their chimeric phenotypes, it is likely that Tbx5 and Tbx4 expression in the limb bud is involved in determination of the forelimb and hindlimb identities, respectively, in vertebrates.

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The chick limbless mutation causes abnormalities in limb bud dorsal-ventral patterning: implications for the mechanism of apical ridge formation

Development ◽

10.1242/dev.122.12.3851 ◽

1996 ◽

Vol 122 (12) ◽

pp. 3851-3861 ◽

Cited By ~ 18

Author(s):

U. Grieshammer ◽

G. Minowada ◽

J.M. Pisenti ◽

U.K. Abbott ◽

G.R. Martin

Keyword(s):

Gene Expression ◽

Limb Development ◽

Positional Information ◽

Limb Bud ◽

Local Application ◽

Chick Embryos ◽

Limb Buds ◽

Early Stages ◽

Potential Link

In chick embryos homozygous for the limbless mutation, limb bud outgrowth is initiated, but a morphologically distinct apical ridge does not develop and limbs do not form. Here we report the results of an analysis of gene expression in limbless mutant limb buds. Fgf4, Fgf8, Bmp2 and Msx2, genes that are expressed in the apical ridge of normal limb buds, are not expressed in the mutant limb bud ectoderm, providing molecular support for the hypothesis that limb development fails in the limbless embryo because of the inability of the ectoderm to form a functional ridge. Moreover, Fgf8 expression is not detected in the ectoderm of the prospective limb territory or the early limb bud of limbless embryos. Since the early stages of limb bud outgrowth occur normally in the mutant embryos, this indicates that FGF8 is not required to promote initial limb bud outgrowth. In the absence of FGF8, Shh is also not expressed in the mutant limb buds, although its expression can be induced by application of FGF8-soaked beads. These observations support the hypothesis that Fgf8 is required for the induction of Shh expression during normal limb development. Bmp2 expression was also not detected in mutant limb mesoderm, consistent with the hypothesis that SHH induces its expression. In contrast, SHH is not required for the induction of Hoxd11 or Hoxd13 expression, since expression of both these genes was detected in the mutant limb buds. Thus, some aspects of mesoderm A-P patterning can occur in the absence of SHH and factors normally expressed in the apical ridge. Intriguingly, mutant limbs rescued by local application of FGF displayed a dorsalized feather pattern. Furthermore, the expression of Wnt7a, Lmx1 and En1, genes involved in limb D-V patterning, was found to be abnormal in mutant limb buds. These data suggest that D-V patterning and apical ridge formation are linked, since they show that the limbless mutation affects both processes. We present a model that explains the potential link between D-V positional information and apical ridge formation, and discuss the possible function of the limbless gene in terms of this model.

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Activation of Fgf-4 and HoxD gene expression by BMP-2 expressing cells in the developing chick limb

Development ◽

10.1242/dev.122.6.1821 ◽

1996 ◽

Vol 122 (6) ◽

pp. 1821-1828 ◽

Cited By ~ 17

Author(s):

D.M. Duprez ◽

K. Kostakopoulou ◽

P.H. Francis-West ◽

C. Tickle ◽

P.M. Brickell

Keyword(s):

Gene Expression ◽

Anterior Part ◽

Ectopic Expression ◽

Mirror Image ◽

Limb Bud ◽

Bone Morphogenetic Protein 2 ◽

Morphogenetic Protein ◽

Wing Bud ◽

Hoxd Gene

Bone morphogenetic protein-2 (BMP-2) has been implicated in the polarizing region signalling pathway, which specifies pattern across the antero-posterior of the developing vertebrate limb. Retinoic acid and Sonic Hedgehog (SHH) can act as polarizing signals; when applied anteriorly in the limb bud, they induce mirror-image digit duplications and ectopic Bmp-2 expression in anterior mesenchyme. In addition, the two signals can activate Fgf-4 expression in anterior ridge and HoxD expression in anterior mesenchyme. We tested the role of BMP-2 in this signalling cascade by ectopically expressing human BMP-2 (hBMP-2) at the anterior margin of the early wing bud using a replication defective retroviral vector, and found that ectopic expression of Fgf-4 was induced in the anterior part of the apical ectodermal ridge, followed later by ectopic expression of Hoxd-11 and Hoxd-13 in anterior mesenchyme. This suggests that BMP-2 is involved in regulating Fgf-4 and HoxD gene expression in the normal limb bud. Ectopically expressed hBMP-2 also induced duplication of digit 2 and bifurcation of digit 3, but could not produce the mirror-image digit duplications obtained with SHH-expressing cells. These results suggest that BMP-2 may be involved primarily in maintenance of the ridge, and in the link between patterning and outgrowth of the limb bud.

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Retroviral expression of FGF-2 (bFGF) affects patterning in chick limb bud

Development ◽

10.1242/dev.118.1.95 ◽

1993 ◽

Vol 118 (1) ◽

pp. 95-104 ◽

Cited By ~ 10

Author(s):

B.B. Riley ◽

M.P. Savage ◽

B.K. Simandl ◽

B.B. Olwin ◽

J.F. Fallon

Keyword(s):

Growth Factor ◽

Fibroblast Growth Factor ◽

Limb Development ◽

Limb Bud ◽

Fibroblast Growth Factor 2 ◽

Fibroblast Growth ◽

Donor Tissue ◽

Wing Bud

To investigate the role of fibroblast growth factor-2 (basic fibroblast growth factor) in chick limb development, we constructed a replication-defective spleen necrosis virus to ectopically express fibroblast growth factor-2 in stage 20–22 chick limb bud. Because infecting cells in vivo proved to be inefficient, limb bud cells were dissociated, infected in vitro, and then grafted back into host limbs. This procedure caused duplications of anterior skeletal elements, including proximal humerus, distal radius, and digits 2 and 3. Eighty-nine percent of host wings receiving infected grafts at their anterior borders had duplications of one or more of these elements. The frequency of duplication declined dramatically when infected cells were grafted to progressively more posterior sites of host limb buds, and grafting to the posterior border had no effect at all. Several techniques were used to determine the role of infected tissue in forming skeletal duplications. First, staining with an fibroblast growth factor-2 specific monoclonal antibody showed higher than endogenous levels of fibroblast growth factor-2 expression associated with extra elements. Second, the host/donor composition of duplicated elements was determined by simultaneously infecting donor cells with viruses encoding fibroblast growth factor-2 or beta-galactosidase; donor tissue was then visualized by X-gal staining. Patterns of ectopic fibroblast growth factor-2 expression and X-gal staining confirmed the presence of infected donor tissue near duplicated structures, but the duplicated skeletal elements themselves showed very little staining. Similar results were obtained in duplications caused by infected quail wing bud cells grafted to the chick wing bud. These observations suggest that fibroblast growth factor-2-expressing donor tissue induced host tissue to form normally patterned extra elements. In support of this conclusion, implanting beads containing fibroblast growth factor-2 caused partial duplications of digit 2. These data provide the first direct evidence that fibroblast growth factor-2 plays a role in patterning in the limb bud.

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Identification of Autosomal Regions Involved in Drosophila Raf Function

Genetics ◽

10.1093/genetics/156.2.763 ◽

2000 ◽

Vol 156 (2) ◽

pp. 763-774 ◽

Cited By ~ 2

Author(s):

Willis Li ◽

Elizabeth Noll ◽

Norbert Perrimon

Keyword(s):

Limb Development ◽

Target Gene ◽

Biological Function ◽

Genetic Interaction ◽

Ectopic Expression ◽

Tor Signaling ◽

Downstream Effector ◽

Early Embryos ◽

Genomic Regions

Abstract Raf is an essential downstream effector of activated p21Ras (Ras) in transducing proliferation or differentiation signals. Following binding to Ras, Raf is translocated to the plasma membrane, where it is activated by a yet unidentified “Raf activator.” In an attempt to identify the Raf activator or additional molecules involved in the Raf signaling pathway, we conducted a genetic screen to identify genomic regions that are required for the biological function of Drosophila Raf (Draf). We tested a collection of chromosomal deficiencies representing ∼70% of the autosomal euchromatic genomic regions for their abilities to enhance the lethality associated with a hypomorphic viable allele of Draf, DrafSu2. Of the 148 autosomal deficiencies tested, 23 behaved as dominant enhancers of Draf Su2, causing lethality in Draf Su2 hemizygous males. Four of these deficiencies identified genes known to be involved in the Drosophila Ras/Raf (Ras1/Draf) pathway: Ras1, rolled (rl, encoding a MAPK), 14-3-3ϵ, and bowel (bowl). Two additional deficiencies removed the Drosophila Tec and Src homologs, Tec29A and Src64B. We demonstrate that Src64B interacts genetically with Draf and that an activated form of Src64B, when overexpressed in early embryos, causes ectopic expression of the Torso (Tor) receptor tyrosine kinase-target gene tailless. In addition, we show that a mutation in Tec29A partially suppresses a gain-of-function mutation in tor. These results suggest that Tec29A and Src64B are involved in Tor signaling, raising the possibility that they function to activate Draf. Finally, we discovered a genetic interaction between Draf Su2 and Df(3L)vin5 that revealed a novel role of Draf in limb development. We find that loss of Draf activity causes limb defects, including pattern duplications, consistent with a role for Draf in regulation of engrailed (en) expression in imaginal discs.

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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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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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Evolution of limb development in cephalopod mollusks

10.1101/379735 ◽

2018 ◽

Author(s):

Oscar A. Tarazona ◽

Davys H. Lopez ◽

Leslie A. Slota ◽

Martin J. Cohn

Keyword(s):

Ventral Sucker ◽

Limb Development ◽

Common Ancestor ◽

Ectopic Expression ◽

Mirror Image ◽

Signaling Networks ◽

Limb Buds ◽

Dorsoventral Polarity ◽

Developmental Mechanisms ◽

Parallel Activation

AbstractCephalopod mollusks evolved numerous anatomical novelties, including arms and tentacles, but little is known about the developmental mechanisms underlying cephalopod limb evolution. Here we show that all three axes of cuttlefish limbs are patterned by the same signaling networks that act in vertebrates and arthropods, although they evolved limbs independently. In cuttlefish limb buds, Hedgehog is expressed anteriorly. Posterior transplantation of Hedgehog-expressing cells induced mirror-image limb duplications. Bmp and Wnt signals, which establish dorsoventral polarity in vertebrate and arthropod limbs, are similarly polarized in cuttlefish. Inhibition of Bmp2/4 dorsally caused ectopic expression of Notum, which marks the ventral sucker field, and ectopic sucker development. Cuttlefish also show proximodistal regionalization of Hth, Exd, Dll, Dac, Sp8/9, and Wnt expression, which delineates arm and tentacle sucker fields. These results suggest that cephalopod limbs evolved by parallel activation of a genetic program for appendage development that was present in the bilaterian common ancestor.

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