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

Development ◽  
1996 ◽  
Vol 122 (12) ◽  
pp. 3851-3861 ◽  
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.

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 ◽  
1992 ◽  
Vol 115 (2) ◽  
pp. 629-637 ◽  
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)

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

Development ◽  
1998 ◽  
Vol 125 (3) ◽  
pp. 351-357 ◽  
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.

scholarly journals Retinoids reprogramme pre-bud mesenchyme to give changes in limb pattern

Development ◽  
1987 ◽  
Vol 100 (4) ◽  
pp. 723-733 ◽  
Author(s):  
S.M. Wilde ◽  
S.E. Wedden ◽  
C. Tickle
Keyword(s):  
Retinoic Acid ◽  
Effective Treatment ◽  
Limb Development ◽  
Apical Ectodermal Ridge ◽  
Limb Bud ◽  
Chick Embryos ◽  
Early Stages ◽  
Limb Pattern

Retinoic acid was locally applied to presumptive limb regions of chick embryos to find out the earliest time at which the limb pattern can be reprogrammed. When beads soaked in retinoic acid were placed in the appropriate positions in embryos at stage 10 or older, duplicated or reduced leg patterns resulted. To pin point the time at which the cells in the limb rudiment respond to the retinoid, beads were removed at various times and the lengths of exposure required to reprogramme limb development found. The early limb rudiments require longer exposures to give duplications than late rudiments. The effective treatment periods last at least until stage 17 when the limb bud and apical ectodermal ridge develop. In contrast, the length of exposure to reduce the limb is constant at early stages. Retinoids first start acting to produce duplicated structures between stages 10 and 13. Therefore, retinoids appear to begin to reprogramme the cells as soon as they are determined to give rise to a limb.

scholarly journals The effect of local application of retinoic acid to the anterior margin of the developing chick limb

Development ◽  
1983 ◽  
Vol 78 (1) ◽  
pp. 269-289
Author(s):  
Dennis Summerbell
Keyword(s):  
Retinoic Acid ◽  
Limb Development ◽  
Anterior Margin ◽  
Mirror Image ◽  
Limb Bud ◽  
Local Application ◽  
Low Doses ◽  
Full Dose ◽  
Early Stages ◽  
High Doses

Local application of retinoic acid to the chick limb bud produces effects that are dose and/or stage dependent. Low doses and/or old stages tend to give normal limbs or perhaps one or two supernumerary digits of a more anterior character. Medium doses and/or intermediate stages tend to give full mirror-image supernumeraries with two or even three extra digits including particularly digits of a posterior character. High doses and/or early stages give limbs in which supernumerary digits fail to form or are lost, and in which even host skeletal elements are missing or reduced. The effects are graded over the full dose and/or stage range. Various explanations are discussed in the context of the current hypotheses of limb development. We conclude that one should not necessarily interpret the results as evidence that retinoids normally play a part in the control of development or regeneration.

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

Development ◽  
1999 ◽  
Vol 126 (21) ◽  
pp. 4729-4736
Author(s):  
L. Lettice ◽  
J. Hecksher-Sorensen ◽  
R.E. Hill
Keyword(s):  
Gene Expression ◽  
Pattern Formation ◽  
Limb Development ◽  
Limb Bud ◽  
Mouse Mutation ◽  
Number Of Factors ◽  
Limb Outgrowth ◽  
Gene Functions ◽  
Regulatory Loop ◽  
Limb Initiation

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.

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 ◽  
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.

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

Development ◽  
1998 ◽  
Vol 125 (1) ◽  
pp. 51-60 ◽  
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.

scholarly journals SMAD4 target genes are part of a transcriptional network that integrates the response to BMP and SHH signaling during early limb bud patterning

Development ◽  
2021 ◽  
Author(s):  
Julie Gamart ◽  
Iros Barozzi ◽  
Frédéric Laurent ◽  
Robert Reinhardt ◽  
Laurène Ramos Martins ◽  
...  
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Target Genes ◽  
Cholesterol Biosynthesis ◽  
Limb Bud ◽  
Limb Buds ◽  
Shh Signaling ◽  
Mesenchymal Progenitors ◽  
Repressive Effect ◽  
Mouse Limb

SMAD4 regulates gene expression in response to BMP and TGFβ signal transduction and is required for diverse morphogenetic processes, but its target genes have remained largely elusive. Here, we identify the SMAD4 target genes in mouse limb buds using an epitope-tagged Smad4 allele for ChIP-seq analysis in combination with transcription profiling. This analysis shows that SMAD4 predominantly mediates BMP signal-transduction during early limb bud development. Unexpectedly, the expression of cholesterol biosynthesis enzymes is precociously down-regulated and intracellular cholesterol levels are reduced in Smad4-deficient limb bud mesenchymal progenitors. Most importantly, our analysis reveals a predominant function of SMAD4 in up-regulating target genes in the anterior limb bud mesenchyme. Analysis of differentially expressed genes shared between Smad4- and Shh-deficient limb buds corroborates this function of SMAD4 and also reveals the repressive effect of SMAD4 on posterior genes that are up-regulated in response to SHH signaling. This analysis uncovers opposing trans-regulatory inputs from SHH and SMAD4-mediated BMP signal transduction on anterior and posterior gene expression during the digit patterning and outgrowth in early limb buds.

Hox-4 gene expression in mouse/chicken heterospecific grafts of signalling regions to limb buds reveals similarities in patterning mechanisms

Development ◽  
1992 ◽  
Vol 115 (2) ◽  
pp. 553-560 ◽  
Author(s):  
J.C. Izpisua-Belmonte ◽  
J.M. Brown ◽  
A. Crawley ◽  
D. Duboule ◽  
C. Tickle
Keyword(s):  
Gene Expression ◽  
Gene Network ◽  
Primitive Streak ◽  
Mouse Embryos ◽  
Limb Bud ◽  
Chick Embryos ◽  
Mouse Limb ◽  
Mouse Cells ◽  
Species Specific ◽  
Genital Tubercle

The products of Hox-4 genes appear to encode position in developing vertebrate limbs. In chick embryos, a number of different signalling regions when grafted to wing buds lead to duplicated digit patterns. We grafted tissue from the equivalent regions in mouse embryos to chick wing buds and assayed expression of Hox-4 genes in both the mouse cells in the grafts and in the chick cells in the responding limb bud using species specific probes. Tissue from the mouse limb polarizing region and anterior primitive streak respecify anterior chick limb bud cells to give posterior structures and lead to activation of all the genes in the complex. Mouse neural tube and genital tubercle grafts, which give much less extensive changes in pattern, do not activate 5′-located Hox-4 genes. Analysis of expression of Hox-4 genes in mouse cells in the grafted signalling regions reveals no relationship between expression of these genes and strength of their signalling activity. Endogenous signals in the chick limb bud activate Hox-4 genes in grafts of mouse anterior limb cells when placed posteriorly and in grafts of mouse anterior primitive streak tissue. The activation of the same gene network by different signalling regions points to a similarity in patterning mechanisms along the axes of the vertebrate body.

Bone morphogenetic proteins and a signalling pathway that controls patterning in the developing chick limb

Development ◽  
1994 ◽  
Vol 120 (1) ◽  
pp. 209-218 ◽  
Author(s):  
P.H. Francis ◽  
M.K. Richardson ◽  
P.M. Brickell ◽  
C. Tickle
Keyword(s):  
Gene Expression ◽  
Retinoic Acid ◽  
Bone Morphogenetic Proteins ◽  
Limb Development ◽  
Posterior Part ◽  
Signalling Pathway ◽  
Limb Bud ◽  
Bone Morphogenetic Protein 2 ◽  
Close Relationship ◽  
Wing Bud

We show here that bone morphogenetic protein 2 (BMP-2) is involved in patterning the developing chick limb. During early stages of limb development, mesenchymal expression of the Bmp-2 gene is restricted to the posterior part of the bud, in a domain that colocalizes with the polarizing region. The polarizing region is a group of cells at the posterior margin of the limb bud that can respecify the anteroposterior axis of the limb when grafted anteriorly and can activate expression of genes of the HoxD complex. We dissect possible roles of BMP-2 in the polarizing region signalling pathway by manipulating the developing wing bud. Retinoic acid application, which mimics the effects of polarizing region grafts, activates Bmp-2 gene expression in anterior cells. This shows that changes in anteroposterior pattern are correlated with changes in Bmp-2 expression. When polarizing region grafts are placed at the anterior margin of the wing bud, the grafts continue to express the Bmp-2 gene and also activate Bmp-2 expression in the adjacent anterior host mesenchyme. These data suggest that BMP-2 is part of the response pathway to the polarizing signal, rather than being the signal itself. In support of this, BMP-2 protein does not appear to have any detectable polarizing activity when applied to the wing bud. The pattern of Bmp-4 gene expression in the developing wing bud raises the possibility that BMP-2 and BMP-4 could act in concert. There is a close relationship, both temporal and spatial, between the activation of the Bmp-2 and Hoxd-13 genes in response to retinoic acid and polarizing region grafts, suggesting that expression of the two genes might be linked.

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