Journal articles on the topic 'Limb bud'
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1
Lee, Christopher T., Luoping Li, Norio Takamoto, James F. Martin, Francesco J. DeMayo, Ming-Jer Tsai, and Sophia Y. Tsai. "The Nuclear Orphan Receptor COUP-TFII Is Required for Limb and Skeletal Muscle Development." Molecular and Cellular Biology 24, no. 24 (December 15, 2004): 10835–43. http://dx.doi.org/10.1128/mcb.24.24.10835-10843.2004.
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ABSTRACT The nuclear orphan receptor COUP-TFII is widely expressed in multiple tissues and organs throughout embryonic development, suggesting that COUP-TFII is involved in multiple aspects of embryogenesis. Because of the early embryonic lethality of COUP-TFII knockout mice, the role of COUP-TFII during limb development has not been determined. COUP-TFII is expressed in lateral plate mesoderm of the early embryo prior to limb bud formation. In addition, COUP-TFII is also expressed in the somites and skeletal muscle precursors of the limbs. Therefore, in order to study the potential role of COUP-TFII in limb and skeletal muscle development, we bypassed the early embryonic lethality of the COUP-TFII mutant by using two methods. First, embryonic chimera analysis has revealed an obligatory role for COUP-TFII in limb bud outgrowth since mutant cells are unable to contribute to the distally growing limb mesenchyme. Second, we used a conditional-knockout approach to ablate COUP-TFII specifically in the limbs. Loss of COUP-TFII in the limbs leads to hypoplastic skeletal muscle development, as well as shorter limbs. Taken together, our results demonstrate that COUP-TFII plays an early role in limb bud outgrowth but not limb bud initiation. Also, COUP-TFII is required for appropriate development of the skeletal musculature of developing limbs.
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Carrington, J. L., and J. F. Fallon. "Initial limb budding is independent of apical ectodermal ridge activity; evidence from a limbless mutant." Development 104, no. 3 (November 1, 1988): 361–67. http://dx.doi.org/10.1242/dev.104.3.361.
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Outgrowth of normal chick limb bud mesoderm is dependent on the presence of a specialized epithelium called the apical ectodermal ridge. This ectodermal ridge is induced by the mesoderm at about the time of limb bud formation. The limbless mutation in the chick affects apical ectodermal ridge formation in the limb buds of homozygotes. The initial formation of the limb bud appears to be unaffected by the mutation but no ridge develops and further outgrowth, which is normally dependent on the ridge, does not take place. As a result, limbless chicks develop without limbs. In the present study, which utilized a pre-limb-bud recombinant technique, limbless mesoderm induced an apical ectodermal ridge in grafted normal flank ectoderm. However, at stages when normal flank ectoderm is capable of responding to ridge induction, limbless flank ectoderm did not form a ridge or promote outgrowth of a limb in response to normal presumptive wing bud mesoderm. We conclude from this that the limbless mutation affects the ability of the ectoderm to form a ridge. In addition, because the limbless ectoderm has no morphological ridge and no apparent ridge activity (i.e. it does not stabilize limb elements in stage-18 limb bud mesoderm), the limbless mutant demonstrates that the initial formation of the limb bud is independent of apical ectodermal ridge activity.
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Zuniga, A., and R. Zeller. "Gli3 (Xt) and formin (ld) participate in the positioning of the polarising region and control of posterior limb-bud identity." Development 126, no. 1 (January 1, 1999): 13–21. http://dx.doi.org/10.1242/dev.126.1.13.
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During initiation of limb-bud outgrowth in vertebrate embryos, the polarising region (limb-bud organizer) is established upon activation of the Sonic Hedgehog (SHH) signaling molecule at the posterior limb-bud margin. Another hallmark of establishing anteroposterior limb-bud identities is the colinear activation of HoxD genes located at the 5′ end of the cluster (5′HoxD genes). The unique and shared functions of Gli3 and formin in these determinative events were genetically analyzed using single and double homozygous Extra-toes (Xt; disrupting Gli3) and limb deformity (ld; disrupting formin) mouse embryos. Analysis of the limb skeletal phenotypes reveals genetic interaction of the two genes. In addition to loss of digit identity and varying degrees of polydactyly, proximal skeletal elements are severely shortened in Xt;ld double homozygous limbs. The underlying molecular defects affect both establishment of the polarising region and posterior limb-bud identity. In particular, the synergism between Gli3- and formin-mediated mesenchyme-AER interactions positions the SHH signaling center at the posterior limb-bud margin. The present study shows that establishment and positioning of the polarising region is regulated both by restriction of Shh through Gli3 and its positive feedback regulation through formin. Concurrently, Gli3 functions independently of formin during initial posterior nesting of 5′HoxD domains, whereas their subsequent distal restriction and anterior expansion depends on genetic interaction of Gli3 and formin.
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Ros, M. A., A. Lopez-Martinez, B. K. Simandl, C. Rodriguez, J. C. Izpisua Belmonte, R. Dahn, and J. F. Fallon. "The limb field mesoderm determines initial limb bud anteroposterior asymmetry and budding independent of sonic hedgehog or apical ectodermal gene expressions." Development 122, no. 8 (August 1, 1996): 2319–30. http://dx.doi.org/10.1242/dev.122.8.2319.
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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.
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Grieshammer, U., G. Minowada, J. M. Pisenti, U. K. Abbott, and G. R. Martin. "The chick limbless mutation causes abnormalities in limb bud dorsal-ventral patterning: implications for the mechanism of apical ridge formation." Development 122, no. 12 (December 1, 1996): 3851–61. http://dx.doi.org/10.1242/dev.122.12.3851.
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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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Scaal, M., A. Bonafede, V. Dathe, M. Sachs, G. Cann, B. Christ, and B. Brand-Saberi. "SF/HGF is a mediator between limb patterning and muscle development." Development 126, no. 21 (November 1, 1999): 4885–93. http://dx.doi.org/10.1242/dev.126.21.4885.
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Scatter factor/hepatocyte growth factor (SF/HGF) is known to be involved in the detachment of myogenic precursor cells from the lateral dermomyotomes and their subsequent migration into the newly formed limb buds. As yet, however, nothing has been known about the role of the persistent expression of SF/HGF in the limb bud mesenchyme during later stages of limb bud development. To test for a potential role of SF/HGF in early limb muscle patterning, we examined the regulation of SF/HGF expression in the limb bud as well as the influence of SF/HGF on direction control of myogenic precursor cells in limb bud mesenchyme. We demonstrate that SF/HGF expression is controlled by signals involved in limb bud patterning. In the absence of an apical ectodermal ridge (AER), no expression of SF/HGF in the limb bud is observed. However, FGF-2 application can rescue SF/HGF expression. Excision of the zone of polarizing activity (ZPA) results in ectopic and enhanced SF/HGF expression in the posterior limb bud mesenchyme. We could identify BMP-2 as a potential inhibitor of SF/HGF expression in the posterior limb bud mesenchyme. We further demonstrate that ZPA excision results in a shift of Pax-3-positive cells towards the posterior limb bud mesenchyme, indicating a role of the ZPA in positioning of the premuscle masses. Moreover, we present evidence that, in the limb bud mesenchyme, SF/HGF increases the motility of myogenic precursor cells and has a role in maintaining their undifferentiated state during migration. We present a model for a crucial role of SF/HGF during migration and early patterning of muscle precursor cells in the vertebrate limb.
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Chan, D. C., A. Wynshaw-Boris, and P. Leder. "Formin isoforms are differentially expressed in the mouse embryo and are required for normal expression of fgf-4 and shh in the limb bud." Development 121, no. 10 (October 1, 1995): 3151–62. http://dx.doi.org/10.1242/dev.121.10.3151.
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Mice homozygous for the recessive limb deformity (ld) mutation display both limb and renal defects. The limb defects, oligodactyly and syndactyly, have been traced to improper differentiation of the apical ectodermal ridge (AER) and shortening of the anteroposterior limb axis. The renal defects, usually aplasia, are thought to result from failure of ureteric bud outgrowth. Since the ld locus gives rise to multiple RNA isoforms encoding several different proteins (termed formins), we wished to understand their role in the formation of these organs. Therefore, we first examined the embryonic expression patterns of the four major ld mRNA isoforms. Isoforms I, II and III (all containing a basic amino terminus) are expressed in dorsal root ganglia, cranial ganglia and the developing kidney including the ureteric bud. Isoform IV (containing an acidic amino terminus) is expressed in the notochord, the somites, the apical ectodermal ridge (AER) of the limb bud and the developing kidney including the ureteric bud. Using a lacZ reporter assay in transgenic mice, we show that this differential expression of isoform IV results from distinct regulatory sequences upstream of its first exon. These expression patterns suggest that all four isoforms may be involved in ureteric bud outgrowth, while isoform IV may be involved in AER differentiation. To define further the developmental consequences of the ld limb defect, we analyzed the expression of a number of genes thought to play a role in limb development. Most significantly, we find that although the AERs of ld limb buds express several AER markers, they do not express detectable levels of fibroblast growth factor 4 (fgf-4), which has been proposed to be the AER signal to the mesoderm. Thus we conclude that one or more formins are necessary to initiate and/or maintain fgf-4 production in the distal limb. Since ld limbs form distal structures such as digits, we further conclude that while fgf-4 is capable of supporting distal limb outgrowth in manipulated limbs, it is not essential for distal outgrowth in normal limb development. In addition, ld limbs show a severe decrease in the expression of several mesodermal markers, including sonic hedgehog (shh), a marker for the polarizing region and Hoxd-12, a marker for posterior mesoderm. We propose that incomplete differentiation of the AER in ld limb buds leads to reduction of polarizing activity and defects along the anteroposterior axis.
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Thaller, C., and G. Eichele. "Characterization of retinoid metabolism in the developing chick limb bud." Development 103, no. 3 (July 1, 1988): 473–83. http://dx.doi.org/10.1242/dev.103.3.473.
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Retinoids (vitamin A derivatives) have been shown to have striking effects on developing and regenerating vertebrate limbs. In the developing chick limb, retinoic acid is a candidate morphogen that may coordinate the pattern of cellular differentiation along the anteroposterior limb axis. We describe a series of investigations of the metabolic pathway of retinoids in the chick limb bud system. To study retinoid metabolism in the bud, all-trans-[3H]retinol, all-trans-[3H]retinal and all-trans-[3H]retinoic acid were released into the posterior region of the limb anlage, the area that contains the zone of polarizing activity, a tissue possibly involved in limb pattern formation. We found that the locally applied [3H]retinol is primarily converted to [3H]retinal, [3H]retinoic acid and a yet unidentified metabolite. When [3H]retinal is locally applied, it is either oxidized to [3H]retinoic acid or reduced to [3H]retinol. In contrast, local delivery of retinoic acid to the bud yields neither retinal nor retinol nor the unknown metabolite. This flow of metabolites agrees with the biochemical pathway of retinoids that has previously been elucidated in a number of other animal systems. To find out whether metabolism takes place directly in the treated limb bud, we have compared the amount of [3H]retinoid present in each of the four limb anlagen following local treatment of the right wing bud. The data suggest that retinoid metabolism takes place mostly in the treated limb bud. This local metabolism could provide a simple mechanism to generate in a controlled fashion the biologically active all-trans-retinoic acid from its abundant biosynthetic precursor retinol. In addition, local metabolism supports the hypothesis that retinoids are local chemical mediators involved in pattern formation.
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Al-Musawi, Duha, and Hayder Mubarak. "MESENCHYMAL CELL DEATH IN MOUSE LIMB BUD AFTER THE ONSET OF PRIMARY MYOGENESIS." Iraqi Journal of Medical Sciences 16, no. 1 (March 31, 2018): 41–50. http://dx.doi.org/10.22578/ijms.16.1.7.
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Background: The vertebrate limb bud develops as an outgrowth of mesoderm, which forms all their elements (muscles, nerves, vessels, bone, cartilage, and tendon). Myogenic precursor cells are seen at E11.5 mouse embryo, when the first nerve fascicles begin to enter the limb. The first signs of musculature masses are seen at E12.5 in both fore and hind limb buds. Apoptosis or programmed cell death is essential in the development of the limbs. In vertebrate, the developing limb morphogenesis depends on the appropriate spatial and temporal balance between cell death and cell proliferation. Objective: To perform comprehensive analysis of the proximo-distal pattern of cell death, evaluated by (TUNEL test) in cross sections of mouse limbs during prenatal development after onset of primary myogenesis. Methods: Fifteen pregnant female mice (Musmusculus) were divided into three groups according to the days of pregnancy into day (14, 16 and 19), only two embryos were taken from each mouse. All the limb buds were involved in this study. Paraffin embedded histological cross-sections of the limb buds were prepared, histological staining (using H&E stain) and TUNEL test labeling were done. Assessment of the number of apoptotic cells in the limb bud mesenchyme was done by counting these cells. Results: The H&E stained sections of the limb buds showed less amounts of mesenchymal tissues in older embryos (day 19). The TUNEL stain showed active apoptotic changes at proximal parts of the limb buds at gestational day 19, while the distal parts of the limbs buds showed active apoptotic changes at the early days (day 14). The evaluation of TUNEL test reaction in the proximal regions showed statistical significant increase of apoptotic cells in day 19 compared to day 14 (p = 0.001 for both). The mean number of apoptotic cells in the proximal regions were statistically significant (p = 0.001) between day 16 and day 19. While the mean number of apoptotic cells of distal regions of the limb buds was higher at day 14 compared to that of day 16 and day 19. These differences between day 14 and day 16 were statistically significant and between day 16 and day 19 while statistically non-significant between day 14 and day 19. Comparison of mean number of apoptotic cells between proximal and distal regions in all the three groups showed a statistically significant higher mean number of apoptotic cells in the distal regions compared to proximal region (p = 0.001). The mean number of the apoptotic cells in both regions (proximal and distal) of the limb buds revealed statistically significant differences between day 16 and day 19 (p = 0.001). Conclusion: Apoptosis was higher in all parts of the developing limbs during day 19, and that could be associated with degenerative changes occurring at the apical ectodermal ridge. Moreover, apoptosis was higher in the distal part of the limb bud and this may be due to more differentiation of the distal parts than in the proximal part of the limb bud. Keywords: Development, limb bud, mouse, embryo, TUNEL, apoptosis, mesenchyme Citation: Al-Musawi DML, Mubarak HJ. Mesenchymal Cell death in mouse limb bud after the onset of primary myogenesis. Iraqi JMS. 2018; Vol. 16(1): 41-50. doi: 10.22578/IJMS.16.1.7
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Gibson-Brown, J. J., S. I. Agulnik, L. M. Silver, L. Niswander, and V. E. Papaioannou. "Involvement of T-box genes Tbx2-Tbx5 in vertebrate limb specification and development." Development 125, no. 13 (July 1, 1998): 2499–509. http://dx.doi.org/10.1242/dev.125.13.2499.
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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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Maden, M., and D. Summerbell. "Retinoic acid-binding protein in the chick limb bud: identification at developmental stages and binding affinities of various retinoids." Development 97, no. 1 (September 1, 1986): 239–50. http://dx.doi.org/10.1242/dev.97.1.239.
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The application of retinoic acid (RA) to the developing chick limb bud causes 6-digit double posterior limbs to form instead of the normal 3-digit limb. As an attempt to begin a molecular analysis of this phenomenon we have identified and characterized a soluble cytoplasmic receptor for RA, namely cytoplasmic retinoic acid-binding protein (CRABP), from the cells of the chick limb bud. It is present from stages 20–35 at similar levels and has an apparent Kd of 140–280 nM. In competition experiments with other retinoids Ro 13-7410 was found to be the most effective at competing for sites on CRABP followed by all-trans-RA, 13-cis-RA, Ro 10-1670 and retinal. Retinol, retinyl palmitate, retinyl acetate, etretinate and arotinoid showed low or no affinity for CRABP. Specificity for binding was thus demonstrated since analogues with an acid end group competed effectively, the aldehyde competed less effectively and the ester or alcohol groups did not compete. At the concentration of RA that needs to be administered to cause duplications in the pattern of the limb bud, we estimate that 4% of the CRABP present in the limb bud has RA bound. The similarities between steroid receptors in the mediation of steroid hormone action and CRABP in the mediation of RA action is discussed. In this regard we note that while there are 104 steroid receptors per cell in other cell types we estimate that there are about 105 RA receptors per cell in the chick limb bud.
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Bee, J. A., and K. von der Mark. "An analysis of chick limb bud intercellular adhesion underlying the establishment of cartilage aggregates in suspension culture." Journal of Cell Science 96, no. 3 (July 1, 1990): 527–36. http://dx.doi.org/10.1242/jcs.96.3.527.
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To examine the mechanism of intercellular adhesion in the establishment of limb skeletal elements we have investigated the process of limb bud cell aggregation in vitro. Limb bud cells are aggregation-competent immediately after their trypsin:collagenase dissociation in the absence of calcium. This aggregation is largely Ca2(+)-independent (CI) and is completely and reversibly inhibited by cycloheximide. In contrast, when limb bud cells are first allowed to recover from Ca2(+)-free trypsin:collagenase dissociation, aggregation of the surviving population is exclusively Ca2(+)-dependent (CD) and completely and reversibly inhibited by cycloheximide. The presence of exogenous calcium during initial cell dissociation retains a functional CD aggregation mechanism. However, incubation of such cells with EGTA releases the CD component and converts the cells to a predominantly CI aggregation. Rabbits were immunized with limb bud cells exhibiting the recovered CD aggregation mechanism and the resulting immune sera were screened for their effect on cell aggregation. Relative to pre-immune sera, intact immune IgG agglutinated dissociated limb bud cells whilst immune Fab fragments inhibited their aggregation. The aggregation-inhibiting antiserum recognizes five major limb bud cell surface components with apparent molecular weights of 72K, 50K, 23K, 14.5K and 8.5K (K = 10(3) Mr), respectively. Limb bud cell surface plasma membranes were isolated by sucrose gradient density centrifugation and detergent-solubilized proteins coupled to Sepharose 4B with cyanogen bromide. Equivalent cell surface plasma membrane proteins were 125I-iodinated and applied to the affinity column. Limb bud cell surface protein affinity chromatography in the presence of exogenous calcium yields a single protein with an apparent molecular weight of approximately 8.5 K. This protein molecule elutes at 0.6 M NaCl, indicating a high affinity, is recognized by the aggregation-inhibiting antiserum, and is itself capable of inhibiting CD limb bud cell aggregation. Fab fragments prepared from rabbit antisera specifically directed against the affinity-purified material also inhibit CD limb bud cell aggregation and this inhibition is neutralized by the 8.5 K protein. Our data thus demonstrate that CD limb bud cell aggregation is not mediated by fibronectin and/or collagen type I and indicate that this process is governed by a novel 8.5 K cell adhesion molecule.
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Chang, W. Y., F. KhosrowShahian, M. Wolanski, R. Marshall, W. McCormick, S. Perry, and M. J. Crawford. "Conservation of Pitx1 expression during amphibian limb morphogenesis." Biochemistry and Cell Biology 84, no. 2 (April 1, 2006): 257–62. http://dx.doi.org/10.1139/o06-036.
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In contrast to the pattern of limb emergence in mammals, chicks, and the newt N. viridescens, embryos such as Xenopus laevis and Eleutherodactylus coqui initiate pelvic limb buds before they develop pectoral ones. We studied the expression of Pitx1 in X. laevis and E. coqui to determine if this paired-like homeodomain transcription factor directs differentiation specifically of the hindlimb, or if it directs the second pair of limbs to form, namely the forelimbs. We also undertook to determine if embryonic expression patterns were recapitulated during the regeneration of an amputated limb bud. Pitx1 is expressed in hindlimbs in both X. laevis and E. coqui, and expression is similar in both developing and regenerating limb buds. Expression in hindlimbs is restricted to regions of proliferating mesenchyme.Key words: regeneration, Xenopus laevis, limb bud, Pitx1 protein, specification.
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Maden, Malcolm. "The limb bud — part two." Nature 371, no. 6498 (October 1994): 560–61. http://dx.doi.org/10.1038/371560a0.
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Ohuchi, H., T. Nakagawa, A. Yamamoto, A. Araga, T. Ohata, Y. Ishimaru, H. Yoshioka, et al. "The mesenchymal factor, FGF10, initiates and maintains the outgrowth of the chick limb bud through interaction with FGF8, an apical ectodermal factor." Development 124, no. 11 (June 1, 1997): 2235–44. http://dx.doi.org/10.1242/dev.124.11.2235.
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Vertebrate limb formation has been known to be initiated by a factor(s) secreted from the lateral plate mesoderm. In this report, we provide evidence that a member of the fibroblast growth factor (FGF) family, FGF10, emanates from the prospective limb mesoderm to serve as an endogenous initiator for limb bud formation. Fgf10 expression in the prospective limb mesenchyme precedes Fgf8 expression in the nascent apical ectoderm. Ectopic application of FGF10 to the chick embryonic flank can induce Fgf8 expression in the adjacent ectoderm, resulting in the formation of an additional complete limb. Expression of Fgf10 persists in the mesenchyme of the established limb bud and appears to interact with Fgf8 in the apical ectoderm and Sonic hedgehog in the zone of polarizing activity. These results suggest that FGF10 is a key mesenchymal factor involved in the initial budding as well as the continuous outgrowth of vertebrate limbs.
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Fernandez-Teran, M., M. E. Piedra, I. S. Kathiriya, D. Srivastava, J. C. Rodriguez-Rey, and M. A. Ros. "Role of dHAND in the anterior-posterior polarization of the limb bud: implications for the Sonic hedgehog pathway." Development 127, no. 10 (May 15, 2000): 2133–42. http://dx.doi.org/10.1242/dev.127.10.2133.
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dHAND is a basic helix-loop-helix (bHLH) transcription factor essential for cardiovascular development. Here we analyze its pattern of expression and functional role during chick limb development. dHAND expression was observed in the lateral plate mesoderm prior to emergence of the limb buds. Coincident with limb initiation, expression of dHAND became restricted to the posterior half of the limb bud. Experimental procedures that caused mirror-image duplications of the limb resulted in mirror-image duplications of the pattern of dHAND expression along the anterior-posterior axis. Retroviral overexpression of dHAND in the limb bud produced preaxial polydactyly, corresponding to mild polarizing activity at the anterior border. At the molecular level, misexpression of dHAND caused ectopic activation of members of the Sonic hedgehog (Shh) pathway, including Gli and Patched, in the anterior limb bud. A subset of infected embryos displayed ectopic anterior activation of Shh. Other factors implicated in anterior-posterior polarization of the bud such as the most 5′ Hoxd genes and Bmp2 were also ectopically activated at the anterior border. Our results indicate a role for dHAND in the establishment of anterior-posterior polarization of the limb bud.
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Qu, S., K. D. Niswender, Q. Ji, R. van der Meer, D. Keeney, M. A. Magnuson, and R. Wisdom. "Polydactyly and ectopic ZPA formation in Alx-4 mutant mice." Development 124, no. 20 (October 15, 1997): 3999–4008. http://dx.doi.org/10.1242/dev.124.20.3999.
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Correct development of the limb is dependent on coordination between three distinct signaling centers. Recently, fibroblast growth factor-4 has been identified as a crucial determinant of AER function, which directs limb bud outgrowth, and Sonic hedgehog has been identified as a signaling molecule that mediates ZPA function, which specifies anterior-posterior patterning in the developing limb bud. In addition, Shh and FGF-4 reciprocally reinforce each other's expression via a positive feedback loop, providing a molecular basis for the coordination of limb bud outgrowth and anterior-posterior patterning. The mechanisms by which these signaling centers come to occupy their normal positions in the posterior limb bud during development are not understood. Here we identify and characterize Alx-4, a gene that encodes a paired-type homeodomain protein. Alx-4 is expressed in several populations of mesenchymal cells, including mesenchymal cells in the anterior limb bud, and mice homozygous for targeted disruption of the Alx-4 gene have multiple abnormalities, including preaxial polydactyly. The polydactyly is associated with the formation of an ectopic anterior ZPA, as indicated by anterior expression of Sonic hedgehog, HoxD13 and fibroblast growth factor-4. The expression of other candidate regulators of anterior-posterior positional information in the limb bud, including HoxB8 and Gli3, is not altered in Alx-4 mutant embryos. By chromosomal mapping experiments, Alx-4 is tightly linked to Strong's luxoid, a polydactylous mouse mutant. The results identify Alx-4 as a determinant of anterior-posterior positional identity in the limb and a component of a regulatory program that restricts ZPA formation to the posterior limb bud mesenchyme.
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Duboc, Veronique, and Malcolm P. O. Logan. "Regulation of limb bud initiation and limb-type morphology." Developmental Dynamics 240, no. 5 (February 28, 2011): 1017–27. http://dx.doi.org/10.1002/dvdy.22582.
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Nelson, C. E., B. A. Morgan, A. C. Burke, E. Laufer, E. DiMambro, L. C. Murtaugh, E. Gonzales, L. Tessarollo, L. F. Parada, and C. Tabin. "Analysis of Hox gene expression in the chick limb bud." Development 122, no. 5 (May 1, 1996): 1449–66. http://dx.doi.org/10.1242/dev.122.5.1449.
Full textAbstract:
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.
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Izpisua-Belmonte, J. C., J. M. Brown, A. Crawley, D. Duboule, and C. Tickle. "Hox-4 gene expression in mouse/chicken heterospecific grafts of signalling regions to limb buds reveals similarities in patterning mechanisms." Development 115, no. 2 (June 1, 1992): 553–60. http://dx.doi.org/10.1242/dev.115.2.553.
Full textAbstract:
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.
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Coelho, C. N., K. M. Krabbenhoft, W. B. Upholt, J. F. Fallon, and R. A. Kosher. "Altered expression of the chicken homeobox-containing genes GHox-7 and GHox-8 in the limb buds of limbless mutant chick embryos." Development 113, no. 4 (December 1, 1991): 1487–93. http://dx.doi.org/10.1242/dev.113.4.1487.
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It has been suggested that the reciprocal expression of the chicken homeobox-containing genes GHox-8 and GHox-7 by the apical ectodermal ridge and subjacent limb mesoderm might be involved in regulating the proximodistal outgrowth of the developing chick limb bud. In the present study the expression of GHox-7 and GHox-8 has been examined by in situ and dot blot hybridization in the developing limb buds of limbless mutant chick embryos. The limb buds of homozygous mutant limbless embryos form at the proper time in development (stage 17/18), but never develop an apical ectodermal ridge, fail to undergo normal elongation, and eventually degenerate. At stage 18, which is shortly following the formation of the limb bud, the expression of GHox-7 is considerably reduced (about 3-fold lower) in the mesoderm of limbless mutant limb buds compared to normal limb bud mesoderm. By stages 20 and 21, as the limb buds of limbless embryos cease outgrowth, GHox-7 expression in limbless mesoderm declines to very low levels, whereas GHox-7 expression increases in the mesoderm of normal limb buds which are undergoing outgrowth. In contrast to GHox-7, expression of GHox-8 in limbless mesoderm at stage 18 is quantitatively similar to its expression in normal limb bud mesoderm, and in limbless and normal mesoderm GHox-8 expression is highly localized in the anterior mesoderm of the limb bud. In normal limb buds, GHox-8 is also expressed in high amounts by the apical ectodermal ridge.(ABSTRACT TRUNCATED AT 250 WORDS)
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Cheng, T. C., T. A. Hanley, J. Mudd, J. P. Merlie, and E. N. Olson. "Mapping of myogenin transcription during embryogenesis using transgenes linked to the myogenin control region." Journal of Cell Biology 119, no. 6 (December 15, 1992): 1649–56. http://dx.doi.org/10.1083/jcb.119.6.1649.
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During vertebrate embryogenesis, the muscle-specific helix-loop-helix protein myogenin is expressed in muscle cell precursors in the developing somite myotome and limb bud before muscle fiber formation and is further upregulated during myogenesis. We show that cis-acting DNA sequences within the 5' flanking region of the mouse myogenin gene are sufficient to direct appropriate temporal, spatial, and tissue-specific transcription of myogenin during mouse embryogenesis. Myogenin-lacZ transgenes trace the fate of embryonic cells that activate myogenin transcription and suggest that myogenic precursor cells that migrate from the somite myotome to the limb bud are committed to a myogenic fate in the absence of myogenin transcription. Activation of a myogenin-lacZ transgene can occur in limb bud explants in culture, indicating that signals required for activation of myogenin transcription are intrinsic to the limb bud and independent of other parts of the embryo. These results reveal multiple populations of myogenic precursor cells during development and suggest the existence of regulators other than myogenic helix-loop-helix proteins that maintain cells in the early limb bud in the myogenic lineage.
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Todt, W. L., and J. F. Fallon. "Posterior apical ectodermal ridge removal in the chick wing bud triggers a series of events resulting in defective anterior pattern formation." Development 101, no. 3 (November 1, 1987): 501–15. http://dx.doi.org/10.1242/dev.101.3.501.
Full textAbstract:
The ability of the anterior apical ectodermal ridge to promote outgrowth in the chick wing bud when disconnected from posterior apical ridge was examined by rotating the posterior portion of the stage-19/20 to stage-21 wing bud around its anteroposterior axis. This permitted contact between the anterior and posterior mesoderm, without removing wing bud tissue. In a small but significant number of cases (10/54), anterior structures (digit 2) formed spatially isolated from posterior structures (digits 3 and 4). Thus, continuity with posterior ridge is not a prerequisite for anterior-ridge function in the wing bud. Nevertheless, posterior-ridge removal does result in anterior limb truncation. To investigate events leading to anterior truncation, we examined cell death patterns in the wing bud following posterior-ridge removal. We observed an abnormal area of necrosis along the posterior border of the wing bud at 6–12 h following posterior-ridge removal. This was followed by necrosis in the distal, anterior mesoderm at 48 h postoperatively and subsequent anterior truncation. Clearly, healthy posterior limb bud mesoderm is needed for anterior limb bud survival and development. We propose that anterior truncation is the direct result of anterior mesodermal cell death and that this may not be related to positional specification of anterior cells. In our view, cell death of anterior mesoderm, after posterior mesoderm removal, should not be used as evidence for a role in position specification by the polarizing zone during the limb bud stages of development. We suggest that the posterior mesoderm that maintains the anterior mesoderm need not be restricted to the mapped polarizing zone, but is more extensively distributed in the limb bud.
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Tickle, C. "Retinoic acid and chick limb bud development." Development 113, Supplement_1 (January 1, 1991): 113–21. http://dx.doi.org/10.1242/dev.113.supplement_1.113.
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The chick limb bud is a powerful experimental system in which to study pattern formation in vertebrate embryos. Exogenously applied retinoic acid, a vitamin A derivative, can bring about changes in pattern and, on several grounds, is a good candidate for an endogenous morphogen. As such, the local concentration of retinoic acid might provide cells with information about their position in relation to one axis of the limb. Alternatively, retinoic acid may be part of a more complex signalling system. Homeobox genes are possible target genes for regulation by retinoic acid in the limb. In particular, one homeobox gene, XlHbox 1 is expressed locally in the mesenchyme of vertebrate forelimbs and might code for an anterior position. When the pattern of the chick wing is changed by retinoic acid or by grafts of signalling tissue such that anterior cells now form posterior structures, the domain of XlHbox 1 expression expands rather than contracts. The expansion of XlHbox 1 expression correlates with shoulder girdle abnormalities. Retinoic acid application leads to visible changes in bud shape and this allows dissection of the way in which patterning is co-ordinated with morphogenesis. Results of recombination experiments and studies of changes in the apical ridge and proliferation in the mesenchyme suggest the following scheme: retinoic acid is involved in specification of position of mesenchyme cells; this specification determines their local interaction with the ridge that controls ridge morphology; the thickened apical ridge permits local proliferation in the underlying mesenchyme. The recent advances in molecular biology that permit analysis of the expression of various interesting genes in developing limbs hold out the promise that further investigation may soon allow a complete account of the patterning process in one part of the vertebrate embryo.
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Onda, H., D. J. Goldhamer, and R. A. Tassava. "An extracellular matrix molecule of newt and axolotl regenerating limb blastemas and embryonic limb buds: immunological relationship of MT1 antigen with tenascin." Development 108, no. 4 (April 1, 1990): 657–68. http://dx.doi.org/10.1242/dev.108.4.657.
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Several well-characterized extracellular matrix (ECM) components have been localized to the amphibian limb regenerate, but the identification and characterization of novel ECM molecules have received little attention. Here we describe, using mAb MT1 and immunocytochemistry, an ECM molecule expressed during limb regeneration and limb development. In limb stumps, mAb MT1 reactivity was restricted to tendons, myotendinous junctions, granules in the basal layers of epidermis, periosteum (newts) and perichondrium (axolotls). In regenerating limbs, reactivity in the distal limb stump was first detected 5 days and 1 day after amputation of newt and axolotl limbs, respectively. In both species, mAb MT1 recognized what appeared to be an abundant blastema matrix antigen, localized in both thin and thick cords between and sometimes closely associated with blastema cells. Reactivity was generally uniform throughout the blastema except for a particularly thick layer that was present immediately beneath the wound epithelium. During redifferentiation stages, mAb MT1 reactivity persisted among blastema cells and redifferentiating cartilage but was lost proximally in areas of muscle and connective tissue differentiation. During the entire period of embryonic limb development, mAb MT1 reactivity was seen in the ECM of the mesenchyme and in a layer beneath the limb bud ectoderm, similar to its distribution during regeneration. Considerable mAb MT1 reactivity was also associated with the developing somites. The reactivity of mAb MT1 in blastema and limb bud was similar if not identical to that of a polyclonal Ab against tenascin (pAbTN), a large, extracellular matrix glycoprotein implicated in growth control, inductive interactions, and other developmental events. This pAbTN effectively competed against mAb MT1 binding on blastema sections. In immunoblots, both mAb MT1 and pAbTN recognized a very high molecular weight (approximately Mr 1000 × 10(3)) protein in blastema extracts of both newts and axolotls. mAb MT1 immunoprecipitated a protein of Mr 1000K size which reacted to both mAb MT1 and pAbTN in immunoblots. These data show that tenascin is in the matrix of the urodele blastema and limb bud, and suggest that mAb MT1 identifies urodele tenascin.
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Daston, G., E. Lamar, M. Olivier, and M. Goulding. "Pax-3 is necessary for migration but not differentiation of limb muscle precursors in the mouse." Development 122, no. 3 (March 1, 1996): 1017–27. http://dx.doi.org/10.1242/dev.122.3.1017.
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The limb muscles of vertebrates are derived from precursor cells that migrate from the lateral edge of the dermomyotome into the limb bud. Previous studies have shown that the paired domain-containing transcription factor Pax-3 is expressed in the limb in cells that are precursors for limb muscles (Williams, B. and Ordahl, C.P. (1994) Development 120, 785–796). In splotch (Pax-3-) embryos, the limb muscles fail to develop and cells expressing Pax-3 are no longer found in the limb. In this paper we have analyzed the role of Pax-3 in the migration and subsequent differentiation of limb muscle precursors. By labeling somites adjacent to the prospective forelimb with the lipophilic dye DiI, we have shown that cells derived from these somites do not migrate into the limbs of splotch mice. The failure of limb muscle precursors to invade the limb in splotch mice is associated with the absence of c-met expression in premigratory cells, together with a change in the morphology of the ventral dermomyotome. In addition, we have shown the lateral half of somites derived from day E9.25 splotch embryos can undergo muscle differentiation when grafted into the limb bud stage 20 chick host embryos. Our results indicate that Pax-3 regulates the migration of limb muscle precursors into the limb and is not required for cells in the lateral somite to differentiate into muscle.
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Bell, K. M., and J. C. McLachlan. "Stimulation of division in mouse 3T3 cells by coculture with embryonic chick limb tissue." Development 86, no. 1 (April 1, 1985): 219–26. http://dx.doi.org/10.1242/dev.86.1.219.
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Two regions of the chick limb bud — the apical ectodermal ridge and the zone of polarizing activity — have been shown to influence cell division and pattern formation during normal development and following surgical manipulation. In this study, using a simple coculture system, together with autoradiography, we have shown that these morphogenetically active regions of the limb bud can stimulate quiescent 3T3 cells to initiate DNA synthesis to a significantly greater degree than comparable but morphogenetically inactive regions of the limb bud.
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Sermeus, Yvenn, Jef Vangheel, Liesbet Geris, Bart Smeets, and Przemko Tylzanowski. "Mechanical Regulation of Limb Bud Formation." Cells 11, no. 3 (January 26, 2022): 420. http://dx.doi.org/10.3390/cells11030420.
Full textAbstract:
Early limb bud development has been of considerable interest for the study of embryological development and especially morphogenesis. The focus has long been on biochemical signalling and less on cell biomechanics and mechanobiology. However, their importance cannot be understated since tissue shape changes are ultimately controlled by active forces and bulk tissue rheological properties that in turn depend on cell–cell interactions as well as extracellular matrix composition. Moreover, the feedback between gene regulation and the biomechanical environment is still poorly understood. In recent years, novel experimental techniques and computational models have reinvigorated research on this biomechanical and mechanobiological side of embryological development. In this review, we consider three stages of early limb development, namely: outgrowth, elongation, and condensation. For each of these stages, we summarize basic biological regulation and examine the role of cellular and tissue mechanics in the morphogenetic process.
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King, Mary, Jelena S. Arnold, Alan Shanske, and Bernice E. Morrow. "T-genes and limb bud development." American Journal of Medical Genetics Part A 140A, no. 13 (2006): 1407–13. http://dx.doi.org/10.1002/ajmg.a.31250.
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Logan, M., H. G. Simon, and C. Tabin. "Differential regulation of T-box and homeobox transcription factors suggests roles in controlling chick limb-type identity." Development 125, no. 15 (August 1, 1998): 2825–35. http://dx.doi.org/10.1242/dev.125.15.2825.
Full textAbstract:
The wing and the leg of the chick, although homologous structures, have characteristic patterns of skeletal elements, muscles, tendons, featherbuds and scales. Despite recent advances in understanding the common genetic pathways patterning the wing and leg, the molecular nature of the specification of limb-type identity has remained elusive. Embryological experiments have indicated the existence of limb-specific territories in the flank. In the newt, deviation of nerves from the limb into the flank can induce ectopic limbs to form from this tissue. In the chick, Fibroblast growth factor (FGF)-soaked beads applied to the flank can induce ectopic formation of limbs from the surrounding tissue. In both cases, the type of limb that forms, either a wing/forelimb or leg/hindlimb, is dependent upon the location to which the limb-inducing signal is applied. We have isolated and characterised three candidate genes for controlling limb identity in the chick. Two T-box transcription factors, cTbx4 and cTbx5, are expressed in a restricted manner in the leg bud and wing buds, respectively. cPtx1, a member of the Otx-related subclass of paired-type homeodomain proteins, is expressed exclusively in the leg bud. Using FGF to induce ectopic limb buds of wing, leg and intermediate identity, we show that early expression of cTbx5, cTbx4 and cPtx1 in the induced limb buds correlates with later wing- or leg-type identity of ectopic limbs. We observe a general correlation between the location of an ectopic outgrowth induced by FGF and the identity of the resulting limb but, significantly, we report that there is no definitive rostral-caudal level that divides the ectopic wing and leg territories.
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Yang, X. M., K. Vogan, P. Gros, and M. Park. "Expression of the met receptor tyrosine kinase in muscle progenitor cells in somites and limbs is absent in Splotch mice." Development 122, no. 7 (July 1, 1996): 2163–71. http://dx.doi.org/10.1242/dev.122.7.2163.
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Hepatocyte growth factor/scatter factor (HGF/SF) stimulates proliferation, dissociation, migration and morphogenesis of cells in culture. To investigate a possible role for HGF/SF and its receptor, the Met tyrosine kinase, in embryonic development, we have analyzed their expression in mouse embryos from day 7.5 of gestation by whole-mount in situ hybridization. Met expression is first detected in the ventral portion of somites at day 9.25 of gestation (22 somite embryo) at the level of fore limb buds. As somites mature, met expression is detected in caudal somites, and is confined to the lateral and media] tips of the dermomyotome and dermomyotome/myotome respectively. In contrast, HGF/SF is expressed exclusively in the mesodermal core of the limb bud. As the dermomyotome elongates ventrolaterally, the met-expressing cells at the lateral tip appear to detach from the somite, invade the limb bud and localize at the dorsal and ventral limb sides in close proximity to HGF/SF-expressing cells. At later stages, both met- and HGF/SF-expressing cells appear to migrate distally and localize to the digit forming area of the developing hand plate. Met expression in the lateral dermomyotome and limb bud coincides with expression of Pax-3, a marker for migrating muscle precursor cells in the somite and limb. Splotch-2H and Splotch-delayed mice, which harbor mutations in Pax-3, show major disruptions in early limb muscle development. Significantly, no met-expressing cells were observed in the limbs of homozygous Splotch-2H and Splotch-delayed animals, whereas HGF/SF expression was not affected. The restricted expression of met to a sub-population of Pax-3-expressing cells in the lateral tip of the dermomyotome, demonstrates that met represents a unique molecular marker for this migratory cell population. From these observations, together with the biological activities of HGF/SF, we propose that in homozygous Splotch embryos the failure of muscle precursors to migrate into and populate the limb bud results from a loss of met expression in the cells at the ventrolateral edge of the somitic dermomyotome.
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Ivanenko, Viacheslav N., Ekaterina A. Antonenko, Mikhail S. Gelfand, Jill Yager, and Frank D. Ferrari. "Changes in segmentation and setation along the anterior/posterior axis of the homonomous trunk limbs of a remipede (Crustacea, Arthropoda)." PeerJ 4 (August 10, 2016): e2305. http://dx.doi.org/10.7717/peerj.2305.
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This study describes the segmentation and setation at different developmental stages of the homonomous trunk limbs of the remipedeSpeleonectes tulumensisYager, 1987 collected in anchialine caves of the Yucatan Peninsula. Most homonomous trunk limbs originate ventrolaterally and are composed of two protopodal segments, three exopodal segments and four endopodal segments; contralateral limb pairs are united by a sternal bar. However, the last few posterior limbs originate ventrally, are smaller sized, and have regressively fewer segments, suggesting that limb development passes through several intermediate steps beginning with a limb bud. A terminal stage of development is proposed for specimens on which the posterior somite bears a simple bilobate limb bud, and the adjacent somite bears a limb with a protopod comprised of a coxapod and basipod, and with three exopodal and four endopodal segments. On each trunk limb there are 20 serially homologous groups of setae, and the numbers of setae on different limbs usually varies. These groups of setae are arranged linearly and are identified based on the morphology of the setae and their position on the segments. The number of setae in these groups increases gradually from the anterior homonomous limb to a maximum between limbs 8–12; the number then decreases sharply on the more posterior limbs. Changes in the number of setae, which reach a maximum between trunk limbs 8–12, differ from changes in segmentation which vary only over the last few posterior trunk limbs. Following a vector analysis that identified a spatial pattern for these 20 groups of setae among the different homonomous limbs, the hypothesis was confirmed that the number of setae in any given group and any given limb is correlated with the group, with the position of the somite along the body axis, and with the number of somites present on the specimens. This is the first vector analysis used to analyze a pattern of developmental changes in serially homologs of an arthropod. Development of remipede limbs are compared and contrasted with similar copepod limbs. Architecture, particularly the sternal bar uniting contralateral limb pairs, proposed as homologous, and development of trunk limb segmentation of the remipede is generally similar to that of copepods, but the remipede limb differs in several ways including an additional endopodal segment, the proximal, that appears simultaneously with the protopod during development.
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Riley, B. B., M. P. Savage, B. K. Simandl, B. B. Olwin, and J. F. Fallon. "Retroviral expression of FGF-2 (bFGF) affects patterning in chick limb bud." Development 118, no. 1 (May 1, 1993): 95–104. http://dx.doi.org/10.1242/dev.118.1.95.
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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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Ochiya, T., H. Sakamoto, M. Tsukamoto, T. Sugimura, and M. Terada. "Hst-1 (FGF-4) antisense oligonucleotides block murine limb development." Journal of Cell Biology 130, no. 4 (August 15, 1995): 997–1003. http://dx.doi.org/10.1083/jcb.130.4.997.
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The initiation of limb development depends on the site specific proliferation of the mesenchyme by the signals from the apical ectodermal ridge (AER) in embryonic mouse. We have previously reported that the local expression of Hst-1/Fgf-4 transcripts in AER of the mouse limb bud is developmentally regulated, expressed at 11 and 12 days post coitus (p.c.) embryo. In an effort to further understand the role of Hst-1/FGF-4 in mouse limb development, an antisense oligodeoxynucleotides (ODNs) study was performed. We first established a novel organ culture system to study mouse limb development in vitro. This system allows mouse limb bud at 9.5-10-d p.c. embryo, when placed on a sheet of extracellular matrix in a defined medium, to differentiate into a limb at 12.5-d p.c. embryo within 4.5 d. Using this organ culture system, we have shown that exposure of 9.5-10-d p.c. embryonal limb bud explants to antisense ODNs of Hst-1/FGF-4 blocks limb development. In contrast, sense and scrambled ODNs have no inhibitory effect on limb outgrowth, suggesting that Hst-1/FGF-4 may work as a potent inducing factor for mouse limb development.
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Muneoka, K., and E. H. Murad. "Intercalation and the cellular origin of supernumerary limbs in Xenopus." Development 99, no. 4 (April 1, 1987): 521–26. http://dx.doi.org/10.1242/dev.99.4.521.
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The hypothesis that a specialized polarizing zone controls the pattern of the anterior-posterior axis during limb development in Xenopus has been tested by analysing the cellular contribution to supernumerary limbs. Supernumerary limbs were generated by grafting hindlimb buds contralaterally between X. borealis and X. laevis to appose anterior and posterior limb tissues. Cells derived from these two species of Xenopus are readily identified by staining with quinacrine. The analysis of cellular contribution showed that supernumerary limbs consist of approximately half anterior-derived (57%) and half posterior-derived (43%) cells. These data are not consistent with the polarizing zone theory but are consistent with the hypothesis that both supernumerary limbs and normally developing limbs arise from intercalary interactions between limb bud cells with different positional values.
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Yamamoto, M., Y. Gotoh, K. Tamura, M. Tanaka, A. Kawakami, H. Ide, and A. Kuroiwa. "Coordinated expression of Hoxa-11 and Hoxa-13 during limb muscle patterning." Development 125, no. 7 (April 1, 1998): 1325–35. http://dx.doi.org/10.1242/dev.125.7.1325.
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The limb muscle precursor cells migrate from the somites and congregate into the dorsal and ventral muscle masses in the limb bud. Complex muscle patterns are formed by successive splitting of the muscle masses and subsequent growth and differentiation in a region-specific manner. Hox genes, known as key regulator genes of cartilage pattern formation in the limb bud, were found to be expressed in the limb muscle precursor cells. We found that HOXA-11 protein was expressed in the premyoblasts in the limb bud, but not in the somitic cells or migrating premyogenic cells in the trunk at stage 18. By stage 24, HOXA-11 expression began to decrease from the posterior halves of the muscle masses. HOXA-13 was expressed strongly in the myoblasts of the posterior part in the dorsal/ventral muscle masses and weakly in a few myoblasts of the anterior part of the dorsal muscle mass. Transplantation of the lateral plate of the presumptive wing bud to the flank induced migration of premyoblasts from somites to the graft. Under these conditions, HOXA-11 expression was induced in the migrating premyoblasts in the ectopic limb buds. Application of retinoic acid at the anterior margin of the limb bud causes duplication of the autopodal cartilage and transformation of the radius to the ulna, and at the same time induces duplication of the muscle pattern along the anteroposterior axis. Under these conditions, HOXA-13 was also induced in the anterior region of the ventral muscles in the zeugopod. These results suggest that Hoxa-11 and Hoxa-13 expression in the migrating premyoblasts is under the control of the limb mesenchyme and the polarizing signal(s). In addition, these results indicate that these Hox genes are involved in muscle patterning in the limb buds.
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Schotthoefer, Anna M., Anson V. Koehler, Carol U. Meteyer, and Rebecca A. Cole. "Influence of Ribeiroia ondatrae (Trematoda: Digenea) infection on limb development and survival of northern leopard frogs (Rana pipiens): effects of host stage and parasite-exposure level." Canadian Journal of Zoology 81, no. 7 (July 1, 2003): 1144–53. http://dx.doi.org/10.1139/z03-099.
Full textAbstract:
Recent evidence suggests that infection by larvae of the trematode Ribeiroia ondatrae accounts for a significant proportion of limb malformations currently observed in amphibian populations of North America. However, the effects of R. ondatrae infection on northern leopard frogs (Rana pipiens), one of the species most frequently reported with malformations, have not been adequately explored. Moreover, the risk factors associated with R. ondatrae-induced malformations have not been clearly identified. We examined the effects of timing of infection on tadpole survival and limb development. Rana pipiens tadpoles were individually exposed to R. ondatrae cercariae at the pre-limb-bud (Gosner stages 24 and 25), limb-bud (Gosner stages 27 and 28), or paddle (Gosner stages 3133) stages of development and monitored through metamorphosis. The effects of infection were stage-specific. Infections acquired at the pre-limb-bud stage resulted in a high mortality rate (47.597.5%), whereas tadpoles infected at the limb-bud stage displayed a high malformation rate (16% overall), and the magnitude of effects increased with the level of exposure to cercariae. In contrast, infections acquired at the paddle stage had no effect on limb development or tadpole survival, which suggests that the timing of R. ondatrae infection in relation to the stage structure of tadpole populations in the wild is an important determinant of the degree to which populations are affected by R. ondatrae.
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Tavares, A. T., T. Tsukui, and J. C. Izpisua Belmonte. "Evidence that members of the Cut/Cux/CDP family may be involved in AER positioning and polarizing activity during chick limb development." Development 127, no. 23 (December 1, 2000): 5133–44. http://dx.doi.org/10.1242/dev.127.23.5133.
Full textAbstract:
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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Wehrle-Haller, B., M. Koch, S. Baumgartner, J. Spring, and M. Chiquet. "Nerve-dependent and -independent tenascin expression in the developing chick limb bud." Development 112, no. 2 (June 1, 1991): 627–37. http://dx.doi.org/10.1242/dev.112.2.627.
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The extracellular matrix protein, tenascin, appears in a restricted pattern during organ morphogenesis. Tenascin accumulates along developing peripheral nerves as they leave the spinal cord and enter the limb mesenchyme (Wehrle and Chiquet, Development 110, 401–415, 1990). Here we found that most but not all tenascin deposited along growing nerves is of glial origin. By in situ hybridization with a tenascin cDNA probe, we determined the site of tenascin mRNA accumulation both in normal and nerve-free limbs. In normal wing buds, tenascin mRNA was first detected within the developing limb nerves. Vinculin-positive glial precursor cells, which comigrate with the axons, are the likely source of this tenascin message. In nerveless wing grafts, tenascin was first expressed in tendon primordia in the absence, and thus independently, from innervation. In contrast to normal limbs, grafted wing buds neither contained vinculin-positive glial precursor cells, nor expressed tenascin in regions proximal to tendon primordia. In normal wing buds, tenascin deposited by tendon primordia transiently parallels and surrounds certain developing nerves. After the major nerve pattern is established, tenascin mRNA disappears from nerves in the upper limb, but is expressed in perichondrium and tendons. We propose that glial tenascin facilitates the penetration of axons into the limb bud and is important for nerve fasciculation. In some places, early tendon primordia might help to guide the migration of axons and glial precursor cells towards their target.
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Bell, K. M. "The preliminary characterization of mitogens secreted by embryonic chick wing bud tissues in vitro." Development 93, no. 1 (April 1, 1986): 257–65. http://dx.doi.org/10.1242/dev.93.1.257.
Full textAbstract:
Embryonic chick wing bud tissues secrete diffusible mitogens when cultured in vitro (Bell & McLachlan, 1985). These molecules may play an important role in limb development since media conditioned by morphogenetically active regions of the wing bud possess greater mitogenic activity than media conditioned by non-morphogenetic regions. These studies show that while the chick-derived growth factors were mitogenic for mouse-derived NIH 3T3,10T1/2 and NR6 cells and chick limb bud cells, they did not stimulate DNA synthesis in 3B11, PC13 END, normal rat kidney or bovine endothelial cells. Furthermore, the effects of the chick-derived mitogens were synergistically enhanced by insulin and PGF2α but remained unaffected by ECDGF, EGF, FGF and MSA. These findings indicate that embryonic chick limb bud cells synthesize and secrete growth factors which resemble in function other well-characterized growth factors and in particular PDGF.
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McQueen, Caitlin, and Matthew Towers. "Establishing the pattern of the vertebrate limb." Development 147, no. 17 (September 1, 2020): dev177956. http://dx.doi.org/10.1242/dev.177956.
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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.
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Yokouchi, Y., K. Ohsugi, H. Sasaki, and A. Kuroiwa. "Chicken homeobox gene Msx-1: structure, expression in limb buds and effect of retinoic acid." Development 113, no. 2 (October 1, 1991): 431–44. http://dx.doi.org/10.1242/dev.113.2.431.
Full textAbstract:
A chicken gene carrying a homeobox highly homologous to the Drosophila muscle segment homeobox (msh) gene was isolated and designated as Msx-1. Conceptual translation from the longest ORF gave a protein of 259 amino acids lacking the conserved hexapeptide. Northern analysis detected a single 2.6 kb transcript. As early as day 2 of incubation, the transcript was detected but was not found in adult tissue. In situ hybridization analysis revealed that Msx-1 expression is closely related to a particular mesenchymal cell lineage during limb bud formation. In early stage embryos, Msx-1 was expressed in the somatopleure. When primordial mesenchyme cells for limb bud were generated from the Wolffian ridge of the somatopleure, Msx-1 expression began to diminish in the posterior half of the limb bud then in the presumptive cartilage-forming mesenchyme. In developing limb buds, remarkable expression was seen in the apical ectodermal ridge (AER), which is responsible for the sustained outgrowth and development of the limb. The Msx-1 transcripts were found in the limb mesenchymal cells in the region covering the necrotic zone and ectodermal cells overlying such mesenchymal cells. Both ectodermal and mesenchymal expression in limb bud were rapidly suppressed by local treatment of retinoic acid which can generate mirror-image duplication of digits. This indicates that retinoic acid alters the marginal presumptive non-cartilage forming mesenchyme cell lineage through suppression of Msx-1 expression.
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Lee, Hyunji, and Steven A. Vokes. "A limb bud morphogen bites the dust." Developmental Cell 57, no. 17 (September 2022): 2041–42. http://dx.doi.org/10.1016/j.devcel.2022.08.007.
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Hopyan, Sevan. "Biophysical regulation of early limb bud morphogenesis." Developmental Biology 429, no. 2 (September 2017): 429–33. http://dx.doi.org/10.1016/j.ydbio.2017.06.034.
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Morishita, Yoshihiro, and Yoh Iwasa. "Growth Based Morphogenesis of Vertebrate Limb Bud." Bulletin of Mathematical Biology 70, no. 7 (July 31, 2008): 1957–78. http://dx.doi.org/10.1007/s11538-008-9334-1.
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Fekete, D. M., and J. P. Brockes. "Evidence that the nerve controls molecular identity of progenitor cells for limb regeneration." Development 103, no. 3 (July 1, 1988): 567–73. http://dx.doi.org/10.1242/dev.103.3.567.
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Adult urodele amphibians can regenerate their limbs after amputation by a process that requires the presence of axons at the amputation plane. Paradoxically, if the limb develops in the near absence of nerves (the ‘aneurogenic’ limb) it can subsequently regenerate in a nerve-independent fashion. The growth zone (blastema) of regenerating limbs normally contains progenitor cells whose division is nerve-dependent. A monoclonal antibody that marks these nerve-dependent cells in the normal blastema does not stain the mesenchymal cells of developing limb buds and only stains the amputated limb bud when axons have reached the plane of amputation. This report shows that the blastemal cells of the regenerating aneurogenic limb also fail to react with the antibody in situ. These data suggest that the blastemal cells arising during normal regeneration have been altered by the nerve. This regulation may occur either at the time of amputation (when the antigen is expressed) or during development (when the limb is first innervated).
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Altabef, M., J. D. Clarke, and C. Tickle. "Dorso-ventral ectodermal compartments and origin of apical ectodermal ridge in developing chick limb." Development 124, no. 22 (November 15, 1997): 4547–56. http://dx.doi.org/10.1242/dev.124.22.4547.
Full textAbstract:
We wish to understand how limbs are positioned with respect to the dorso-ventral axis of the body in vertebrate embryos, and how different regions of limb bud ectoderm, i.e. dorsal ectoderm, apical ridge and ventral ectoderm, originate. Signals from dorsal and ventral ectoderm control dorso-ventral patterning while the apical ectodermal ridge (AER) controls bud outgrowth and patterning along the proximo-distal axis. We show, using cell-fate tracers, the existence of two distinct ectodermal compartments, dorsal versus ventral, in both presumptive limb and flank of early chick embryos. This organisation of limb ectoderm is the first direct evidence, in vertebrates, of compartments in non-neural ectoderm. Since the apical ridge appears to be confined to this compartment boundary, this positions the limb. The mesoderm, unlike the ectoderm, does not contain two separate dorsal and ventral cell lineages, suggesting that dorsal and ventral ectoderm compartments may be important to ensure appropriate control of mesodermal cell fate. Surprisingly, we also show that cells which form the apical ridge are initially scattered in a wide region of early ectoderm and that both dorsal and ventral ectoderm cells contribute to the apical ridge, intermingling to some extent within it.
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Schmiege, Donna L., Richard L. Ridgway, and Stacia B. Moffett. "Ultrastructure of autotomy-induced atrophy of muscles in the crab Carcinus maenas." Canadian Journal of Zoology 70, no. 5 (May 1, 1992): 841–51. http://dx.doi.org/10.1139/z92-120.
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In Carcinus maenas and other decapod crustaceans, limb autotomy results in pronounced atrophy of coxal and thoracic muscles located proximal to the fracture plane. These muscles were not denervated or otherwise injured by autotomy. The atrophic changes are reversed after the subsequent molt, when a regenerated limb becomes functional. In this study, we examined the ultrastructure of the coxal head of the anterior levator (ALc) muscle from the fifth pereiopod of intermolt crabs (control ALc) and from crabs bearing a limb bud (autotomized and contralateral ALc). Control muscles exhibit small regions of myofilament erosion that may be the result of normal turnover of structural elements. Characteristic features of autotomy-induced atrophy are (i) decrease in fiber area, (ii) increase in interfibrillar space, (iii) extensive myofilament erosion, (iv) loss of organelles, (v) disruption of sarcomere organization, and (vi) degeneration of neural and vascular tissues. Similar changes occur to a lesser degree in the contralateral ALc, where they may be characteristic of muscle in the premolt condition. We propose that the atrophic changes in muscles proximal to a limb bud are superimposed on generalized premolt catabolism of muscle protein, the rate of which is elevated in crabs regenerating limbs.
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Milenkovic, L., L. V. Goodrich, K. M. Higgins, and M. P. Scott. "Mouse patched1 controls body size determination and limb patterning." Development 126, no. 20 (October 15, 1999): 4431–40. http://dx.doi.org/10.1242/dev.126.20.4431.
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Hedgehog (Hh) proteins control many developmental events by inducing specific cell fates or regulating cell proliferation. The Patched1 (Ptc1) protein, a binding protein for Hh molecules, appears to oppose Hh signals by repressing transcription of genes that can be activated by Hh. Sonic hedgehog (Shh), one of the vertebrate homologs of Hh, controls patterning and growth of the limb but the early embryonic lethality of ptc1(−)(/)(−) mice obscures the roles of ptc1 in later stages of development. We partially rescued ptc1 homozygous mutant embryos using a metallothionein promoter driving ptc1. In a wild-type background, the transgene causes a marked decrease in animal size starting during embryogenesis, and loss of anterior digits. In ptc1 homozygotes, a potent transgenic insert allowed survival to E14 and largely normal morphology except for midbrain overgrowth. A less potent transgene gave rise to partially rescued embryos with massive exencephaly, and polydactyly and branched digits in the limbs. The polydactyly was preceded by unexpected anterior limb bud transcription of Shh, so one function of ptc1 is to repress Shh expression in the anterior limb bud.
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Wolpert, Lewis. "Pattern formation in epithelial development: the vertebrate limb and feather bud spacing." Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 353, no. 1370 (June 29, 1998): 871–75. http://dx.doi.org/10.1098/rstb.1998.0251.
Full textAbstract:
The ectoderm of the vertebrate limb and feather bud are epithelia that provide good models for epithelial patterning in vertebrate development. At the tip of chick and mouse limb buds is a thickening, the apical ectodermal ridge, which is essential for limb bud outgrowth. The signal from the ridge to the underlying mesoderm involves fibroblast growth factors. The non–ridge ectoderm specifies the dorsoventral pattern of the bud and Wnt7a is a dorsalizing signal. The development of the ridge involves an interaction between dorsal cells that express radical fringe and those that do not. There are striking similarities between the signals and genes involved in patterning the limb ectoderm and the epithelia of the Drosophila imaginal disc that gives rise to the wing. The spacing of feather buds involves signals from the epidermis to the underlying mesenchyme, which again include Wnt7a and fibroblast growth factors.