The limb field mesoderm determines initial limb bud anteroposterior asymmetry and budding independent of sonic hedgehog or apical ectodermal gene expressions

Development ◽  
1996 ◽  
Vol 122 (8) ◽  
pp. 2319-2330 ◽  
Author(s):  
M.A. Ros ◽  
A. Lopez-Martinez ◽  
B.K. Simandl ◽  
C. Rodriguez ◽  
J.C. Izpisua Belmonte ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sonic Hedgehog ◽  
Limb Bud ◽  
Gene Expressions ◽  
Limb Buds ◽  
Limb Initiation ◽  
Anteroposterior Polarity ◽  
Initial Pattern ◽  
Do So

We have analyzed the pattern of expression of several genes implicated in limb initiation and outgrowth using limbless chicken embryos. We demonstrate that the expressions of the apical ridge associated genes, Fgf-8, Fgf-4, Bmp-2 and Bmp-4, are undetectable in limbless limb bud ectoderm; however, FGF2 protein is present in the limb bud ectoderm. Shh expression is undetectable in limbless limb bud mesoderm. Nevertheless, limbless limb bud mesoderm shows polarization manifested by the asymmetric expression of Hoxd-11, −12 and −13, Wnt-5a and Bmp-4 genes. The posterior limbless limb bud mesoderm, although not actually expressing Shh, is competent to express it if supplied with exogenous FGF or transplanted to a normal apical ridge environment, providing further evidence of mesodermal asymmetry. Exogenous FGF applied to limbless limb buds permits further growth and determination of recognizable skeletal elements, without the development of an apical ridge. However, the cells competent to express Shh do so at reduced levels; nevertheless, Bmp-2 is then rapidly expressed in the posterior limbless mesoderm. limbless limb buds appear as bi-dorsal structures, as the entire limb bud ectoderm expresses Wnt-7a, a marker for dorsal limb bud ectoderm; the ectoderm fails to express En-1, a marker of ventral ectoderm. As expected, C-Lmx1, which is downstream of Wnt-7a, is expressed in the entire limbless limb bud mesoderm. We conclude that anteroposterior polarity is established in the initial limb bud prior to Shh expression, apical ridge gene expression or dorsal-ventral asymmetry. We propose that the initial pattern of gene expressions in the emergent limb bud is established by axial influences on the limb field. These permit the bud to emerge with asymmetric gene expression before Shh and the apical ridge appear. We report that expression of Fgf-8 by the limb ectoderm is not required for the initiation of the limb bud. The gene expressions in the pre-ridge limb bud mesoderm, as in the limb bud itself, are unstable without stimulation from the apical ridge and the polarizing region (Shh) after budding is initiated. We propose that the defect in limbless limb buds is the lack of a dorsal-ventral interface in the limb bud ectoderm where the apical ridge induction signal would be received and an apical ridge formed. These observations provide evidence for the hypothesis that the dorsal-ventral ectoderm interface is a precondition for apical ridge formation.

Determination of anteroposterior polarity in the axolotl forelimb by an interaction between limb and flank rudiments

Development ◽  
1977 ◽  
Vol 39 (1) ◽  
pp. 151-168
Author(s):  
J. M. W. Slack
Keyword(s):  
Anterior Margin ◽  
Morphological Character ◽  
Normal Development ◽  
Host Tissue ◽  
Limb Bud ◽  
Simple Rule ◽  
Classical Work ◽  
Anteroposterior Polarity ◽  
Do So

1. It is shown that the mesoderm in the prospective forelimb-bud of the axolotl embryo is thickened and divided into somatic and splanchnic layers, while that of the flank is thinner and undivided. The first sign of the limb-bud itself appears at stage 38. 2. A whole, a half or a third of a limb rudiment can develop into a normal or reduplicated limb when transplanted to the flank. 3. An anterior half of a limb rudiment fails to develop when transplanted to the head but will do so if accompanied by a small piece of flank tissue. 4. Small pieces of tissue from a wide area of the flank will cause reduplication of the forelimb if grafted to the anterior margin of the rudiment. It is shown that the whole of the reduplication is formed from host tissue and has the morphological character of the host. 5. Reduplications have posterior structures arranged symmetrically on both sides of the midline. Both muscles and cartilages are duplicated. 6. It is suggested that the same interaction between prospective flank and limb is responsible for the capacity for growth on the head, the induction of reduplications and the formation of the anteroposterior pattern of the limb in normal development. 7. A simple rule is proposed which explains the occurrence of reduplications in classical work on the amphibian limb.

Analysis of Hox gene expression in the chick limb bud

Development ◽  
1996 ◽  
Vol 122 (5) ◽  
pp. 1449-1466 ◽  
Author(s):  
C.E. Nelson ◽  
B.A. Morgan ◽  
A.C. Burke ◽  
E. Laufer ◽  
E. DiMambro ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Sonic Hedgehog ◽  
Hind Limb ◽  
Hox Genes ◽  
Expression Patterns ◽  
Limb Bud ◽  
Posterior Portion ◽  
Hox Gene ◽  
Hoxd Gene

The vertebrate Hox genes have been shown to be important for patterning the primary and secondary axes of the developing vertebrate embryo. The function of these genes along the primary axis of the embryo has been generally interpreted in the context of positional specification and homeotic transformation of axial structures. The way in which these genes are expressed and function during the development of the secondary axes, particularly the limb, is less clear. In order to provide a reference for understanding the role of the Hox genes in limb patterning, we isolated clones of 23 Hox genes expressed during limb development, characterized their expression patterns and analyzed their regulation by the signalling centers which pattern the limb. The expression patterns of the Abd-B-related Hoxa and Hoxd genes have previously been partially characterized; however, our study reveals that these genes are expressed in patterns more dynamic and complex than generally appreciated, only transiently approximating simple, concentric, nested domains. Detailed analysis of these patterns suggests that the expression of each of the Hoxa and Hoxd genes is regulated in up to three independent phases. Each of these phases appears to be associated with the specification and patterning of one of the proximodistal segments of the limb (upper arm, lower arm and hand). Interestingly, in the last of these phases, the expression of the Hoxd genes violates the general rule of spatial and temporal colinearity of Hox gene expression with gene order along the chromosome. In contrast to the Abd-B-related Hoxa and Hoxd genes, which are expressed in both the fore and hind limbs, different sets of Hoxc genes are expressed in the two limbs. There is a correlation between the relative position of these genes along the chromosome and the axial level of the limb bud in which they are expressed. The more 3′ genes are expressed in the fore limb bud while the 5′ genes are expressed in the hind limb bud; intermediate genes are transcribed in both limbs. However, there is no clear correlation between the relative position of the genes along the chromosome and their expression domains within the limb. With the exception of Hoxc-11, which is transcribed in a posterior portion of the hind limb, Hoxc gene expression is restricted to the anterior/proximal portion of the limb bud. Importantly, comparison of the distributions of Hoxc-6 RNA and protein products reveals posttranscriptional regulation of this gene, suggesting that caution must be exercised in interpreting the functional significance of the RNA distribution of any of the vertebrate Hox genes. To understand the genesis of the complex patterns of Hox gene expression in the limb bud, we examined the propagation of Hox gene expression relative to cell proliferation. We find that shifts in Hox gene expression cannot be attributed to passive expansion due to cell proliferation. Rather, phase-specific Hox gene expression patterns appear to result from a context-dependent response of the limb mesoderm to Sonic hedgehog. Sonic hedgehog (the patterning signal from the Zone of Polarizing Activity) is known to be able to activate Hoxd gene expression in the limb. Although we find that Sonic hedgehog is capable of initiating and polarizing Hoxd gene expression during both of the latter two phases of Hox gene expression, the specific patterns induced are not determined by the signal, but depend upon the temporal context of the mesoderm receiving the signal. Misexpression of Sonic hedgehog also reveals that Hoxb-9, which is normally excluded from the posterior mesenchyme of the leg, is negatively regulated by Sonic hedgehog and that Hoxc-11, which is expressed in the posterior portion of the leg, is not affected by Sonic hedgehog and hence is not required to pattern the skeletal elements of the lower leg.

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.

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.

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.

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.

scholarly journals Establishing the pattern of the vertebrate limb

Development ◽  
2020 ◽  
Vol 147 (17) ◽  
pp. dev177956 ◽  
Author(s):  
Caitlin McQueen ◽  
Matthew Towers
Keyword(s):  
Gene Expression ◽  
Pattern Formation ◽  
Limb Bud ◽  
Complex Pattern ◽  
Digit Number ◽  
Vertebrate Limb ◽  
Undifferentiated Cells

ABSTRACTThe vertebrate limb continues to serve as an influential model of growth, morphogenesis and pattern formation. With this Review, we aim to give an up-to-date picture of how a population of undifferentiated cells develops into the complex pattern of the limb. Focussing largely on mouse and chick studies, we concentrate on the positioning of the limbs, the formation of the limb bud, the establishment of the principal limb axes, the specification of pattern, the integration of pattern formation with growth and the determination of digit number. We also discuss the important, but little understood, topic of how gene expression is interpreted into morphology.

scholarly journals A role of polycomb group genes in the regulation of gap gene expression in Drosophila.

Genetics ◽  
1994 ◽  
Vol 136 (4) ◽  
pp. 1341-1353 ◽  
Author(s):  
F Pelegri ◽  
R Lehmann
Keyword(s):  
Gene Expression ◽  
Small Region ◽  
Early Embryo ◽  
Polycomb Group ◽  
Drosophila Embryo ◽  
Polycomb Group Genes ◽  
Gap Gene ◽  
Anteroposterior Polarity

Abstract Anteroposterior polarity of the Drosophila embryo is initiated by the localized activities of the maternal genes, bicoid and nanos, which establish a gradient of the hunchback (hb) morphogen. nanos determines the distribution of the maternal Hb protein by regulating its translation. To identify further components of this pathway we isolated suppressors of nanos. In the absence of nanos high levels of Hb protein repress the abdomen-specific genes knirps and giant. In suppressor-of-nanos mutants, knirps and giant are expressed in spite of high Hb levels. The suppressors are alleles of Enhancer of zeste (E(z)) a member of the Polycomb group (Pc-G) of genes. We show that E(z), and likely other Pc-G genes, are required for maintaining the expression domains of knirps and giant initiated by the maternal Hb protein gradient. We have identified a small region of the knirps promoter that mediates the regulation by E(z) and hb. Because Pc-G genes are thought to control gene expression by regulating chromatin, we propose that imprinting at the chromatin level underlies the determination of anteroposterior polarity in the early embryo.

scholarly journals Determination of Genetic Diversity Based on RAPD molecular Marker and ParARF3 Gene Expressions in some Apricot Genotypes in Iraq

2021 ◽  
Vol 34 (2) ◽  
pp. 60-74
Author(s):  
Ali S. A. Al-Janabi ◽  
Arshad N. Alhasnawi
Keyword(s):  
Gene Expression ◽  
Genetic Diversity ◽  
Genetic Distance ◽  
Genetic Similarity ◽  
Marine Alga ◽  
Gene Expressions ◽  
Negative Results ◽  
Minimum Genetic Distance ◽  
Relative Gene

Employing DNA markers allowed determining genetic diversity and relationships amongst five apricot genotypes. In this study, two relative gene expressions pertaining to ParARF3 gene, which could be distinguished from the genotypes that were exposed to various concentrations of marine alga treatments. As per our results, screening of seven primers with the DNA of 5 apricot genotypes was done, and six primers were generated while the primer OPN–16 gave negative results. The total quantity of fragments generated by 6 primers was 80 at an average of 13.33 fragments ̸primer. The highest unique percentage band depicted in U-17 touched 23%, and the total number of polymorphic bands touched 17 fragments with the average reaching 2.83 fragments ̸primer. The number of monomorphic lied in the range of 5 to 10, with a total of 47 monomorphic. Primer M 32 yielded the highest number of monomorphic bands reaching 10. Between Zaghenia and Zinni, a maximum genetic distance value of 0.8 was reached with less similarity value of 20%. A minimum genetic distance value of 0.44721 was noted between Kaisy and Baia while the high similarity value touched 55.3%. According to the cluster tree analysis, the genotypes were generally split into two key groups: A and B. The Zinni group, which included one apricot genotype, showed genetic similarity of 20% with the other genotypes present in B group. The B group was split into two sub-clusters B1 and B2 and the genetic similarity reached 44%. The ParARF3 relative gene expression pertaining to Zinni genotypes was second as well as convergent with that of gene expression for Zaghenia genotype results. Baia and Kaisy genotypes lied in between the lowest and highest ParARF3 value gene expression exposed to Marine Alga. These outcomes showed that RAPD markers offer an effectual alternative for the plant species genetic characterisation.

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