A gradation of hyaluronate accumulation along the proximodistal axis of the embryonic chick limb bud

Development ◽  
1981 ◽  
Vol 63 (1) ◽  
pp. 85-98
Author(s):  
Robert A. Kosher ◽  
Mary P. Savage ◽  
Kordasey H. Walker
Keyword(s):  
Chondroitin Sulphate ◽  
Distal Segment ◽  
Limb Bud ◽  
Central Core ◽  
Embryonic Chick ◽  
Limb Buds ◽  
Progressive Decline ◽  
Negative Effect ◽  
Core Tissue ◽  
Region Segment

We are currently investigating the mechanism by which the apical ectodermal ridge (AER) of the embryonic chick limb bud exerts its negative effect on the cytodifferentiation of limb mesenchymal cells directly subjacent to it, and the mechanism by which cytodifferentiation is triggered when the cells leave the influence of the AER. Since there is a gradation of differentiation along the proximodistal axis of the limb bud, we have dissected limb buds into discrete segments along the proximodistal axis, and biochemically examined the accumulation of hyaluronate (HA) and other glycosaminoglycans (GAG) in each segment. The unspeciajized subridge region of stage-25 limb buds was separated into distal (segment 1) and proximal (segment 2) regions, and the remaining proximal portion of the limb was separated into four segments (segments 3, 4, 5 and 6) along the proximodistal axis. Stage-24 limb buds were separated into corresponding regions. Since in the proximal regions of the limb (segment 3 through 6), only the cells comprising the central cores of the limb are involved in chondrogenic differentiation, the central core tissue was surgically separated from the peripheral tissue. We have found that HA is by far the predominant GAG accumulated by cells comprising the distal subridge region (representing greater than 50% of the total GAG accumulated during a 90-min labelling period with [3H]glucosamine), and that there is a progressive decline in HA accumulation along the proximodistal axis. The relative and total amount of HA accumulated is highest in the distal subridge region (segment 1), intermediate i(i the proximal subridge region (segment 2) and lowest in the proximal central core regions Of the limb (segments 3 through 6). The striking decrease in HA accumulation in the central core of segment 3 is accompanied by a striking increase in the accumulation of chondroitin sulphate, one of the major constituents of cartilage matrix. In contrast to the central core regions of segments 3 through 6, the relative and total amount of HA accumulated by the peripheral non-chondrogenic regions of these segments remains relatively high, being similar to the accumulation observed in the proximal subridge region. These results indicate that there is a gradation of HA accumulation along the proximodistal axis of both stages-24 and -25 limb buds which correlates with distance of cells from the AER and the state of differentiation of the cells.

scholarly journals Heterogeneity of proteoglycans in developing chick limb cartilage

1977 ◽  
Vol 164 (1) ◽  
pp. 179-183 ◽  
Author(s):  
N S Vasan ◽  
J W Lash
Keyword(s):  
Limb Bud ◽  
Embryonic Chick ◽  
Regulatory Factor ◽  
Limb Buds ◽  
Link Protein

Proteoglycan heterogeneity was studied during the maturation of embryonic-chick limb cartilage in vivo. The results suggest that during the differentiation of limb-bud cartilage the aggregated forms of proteoglycans increase between stages 24 and 35, whereas the non-aggregated or monomeric forms decrease. Only one link protein is found in stage-24 limb buds, whereas two are present at stage 35. Evidence suggests that the synthesis of link proteins may be a regulatory factor in limb chondrogenesis.

Conservation of Pitx1 expression during amphibian limb morphogenesis

2006 ◽  
Vol 84 (2) ◽  
pp. 257-262 ◽  
Author(s):  
W Y Chang ◽  
F KhosrowShahian ◽  
M Wolanski ◽  
R Marshall ◽  
W McCormick ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Xenopus Laevis ◽  
Expression Patterns ◽  
Limb Bud ◽  
Eleutherodactylus Coqui ◽  
Limb Buds ◽  
Homeodomain Transcription Factor ◽  
Limb Morphogenesis ◽  
Pelvic Limb

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.

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

Development ◽  
1992 ◽  
Vol 115 (2) ◽  
pp. 629-637 ◽  
Author(s):  
C.N. Coelho ◽  
W.B. Upholt ◽  
R.A. Kosher
Keyword(s):  
Limb Development ◽  
Ectopic Expression ◽  
Limb Bud ◽  
Chick Embryos ◽  
Limb Buds ◽  
Hox Gene ◽  
Wing Bud ◽  
Coated Bead ◽  
Experimentally Induced

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

The spatial and temporal distribution of polarizing activity in the flank of the pre-limb-bud stages in the chick embryo

Development ◽  
1991 ◽  
Vol 111 (3) ◽  
pp. 725-731 ◽  
Author(s):  
A. Hornbruch ◽  
L. Wolpert
Keyword(s):  
Posterior Part ◽  
Temporal Distribution ◽  
Early Embryo ◽  
Limb Bud ◽  
Posterior Edge ◽  
Limb Buds ◽  
Avian Embryos ◽  
Low Levels ◽  
Clear Change ◽  
Somitic Mesoderm

The presence of polarizing activity in the limb buds of developing avian embryos determines the pattern of the anteroposterior axis of the limbs in the adult. Maps of the spatial distribution and the strength of the signal within limb buds of different stages are well documented. Polarizing activity can also be found in Hensen's node in the early embryo. We have mapped the distribution of polarizing activity as it emerges from Hensen's node and spreads into the flank tissue of the embryo. There is a clear change in the local pattern of expression of polarizing activity between stage 8 and 18. Almost no activity is measured for stages 8 and 9. More or less uniform levels of around 10% are spread along the flank lateral to the unsegmented somitic mesoderm from somite position 12 to 22 in stage 10 embryos. Some 6 to 8 h later at stage 12, there is a distinct peak of activity at somite position 18, the middle of the wing field. This peak increases at stages 13 to 15 and its position traverses to the posterior edge of the wing field. Full strength of activity is reached shortly before the onset of limb bud formation at stage 16 to 17. Stages 16 to 18 were investigated for polarizing activity in the wing and the leg field. Low levels of polarizing activity are present in the anterior leg field at stages 16 and 17 but have disappeared by stage 18 and all activity is confined to the posterior part of the leg bud.

The appearance of muscle protein and myofibrils within the embryonic chick limb-bud

Development ◽  
1973 ◽  
Vol 30 (3) ◽  
pp. 673-679
Author(s):  
P. V. Thorogood
Keyword(s):  
Banding Pattern ◽  
Hind Limb ◽  
Muscle Protein ◽  
Ventral Surface ◽  
Ultrastructural Level ◽  
Limb Bud ◽  
Embryonic Chick ◽  
Immunofluorescence Technique ◽  
Flexor Muscles

Myotubes are present in the developing hind limb of the embryonic chick at 5 days. An immunofluorescence technique was used to detect actomyosin within the myotubes. The earliest detectable appearance of this muscle protein was at six days of development, at sites located peripherally beneath the flattened dorsal and ventral surface of the limb. These dorsal and ventral loci are interpreted as representing the primordial extensor and flexor muscles. At the ultrastructural level the cytoplasm of the myotubes contains fibrillar components which are apparently aggregating to form myofibrils. A rudimentary banding pattern can be distinguished.

Extracellular matrix synthesis in the chick embryo lateral plate prior to and during limb outgrowth

Development ◽  
1980 ◽  
Vol 59 (1) ◽  
pp. 71-87
Author(s):  
Trent D. Stephens ◽  
N. S. Vasan ◽  
James W. Lash
Keyword(s):  
Extracellular Matrix ◽  
Chick Embryo ◽  
Molecular Basis ◽  
Limb Bud ◽  
Matrix Synthesis ◽  
Lateral Plate ◽  
Limb Buds ◽  
Cartilage Development ◽  
Limb Outgrowth ◽  
Lateral Plates

Little is known at the present time about the molecular basis and mechanisms of morphogenesis. The present study is an attempt to determine what influence the extracellular matrix has on the initial outgrowth of the limb bud. Stage -12 to -18 chick embryo lateral plates were examined in relation to proline and sulfate incorporation into collagen and proteoglycan. The flank and limbs incorporated the same amount of labeled proline and sulfate before stage 16. At stage 16 the flank began to incorporate more of both isotopes until at stage 18 there was twice as much incorporation into the flank as into the limbs. The flank and limbs contained the same type of collagen during the period examined. The limbs contained both large and small proteoglycans but the flank contained only small proteoglycans. These data suggest that the extracellular matrix in the flank and limb regions may play a role in limb outgrowth and that the limb buds at these stages may be more inclined toward cartilage development.

Analysis of the intermediate size proteoglycans from the developing chick limb buds11Presented in part at 72nd Annual Meeting of the American Society of Biological Chemists, St Louis, Missouri, U.S.A. 31 May to 4 June, 1981.

Development ◽  
1982 ◽  
Vol 70 (1) ◽  
pp. 61-74
Author(s):  
Nagaswamisri Vasan
Keyword(s):  
Chondroitin Sulphate ◽  
Binding Capacity ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Limb Bud ◽  
Side Chain ◽  
Intermediate Size ◽  
Proteoglycan Aggregates ◽  
American Society ◽  
Hyaluronic Acid Binding

Limb-bud proteoglycans are heterogeneous molecules which vary in their chemical and physical properties with development. This report describes proteoglycan intermediates (PG-I) that predominate in stage-34 limbs, and compares them with proteoglycan aggregates (PG-A) in stage-38 limbs. We analysed proteoglycans and their components extracted with guanidinium chloride by subjecting them to density gradient centrifugation, molecular sieve chromatography, electrophoretic separation, and selective enzymatic degradation. PG-I and PG-A have similar chondroitin sulphate composition, amino sugars, chondroitin sulphate side-chain length, glycoprotein link factors, and hyaluronic acid binding capacity, and both cross react with antisera prepared against cartilage-specific chick sternal proteoglycans. However, PG-I has lower molecular weight, lower buoyant density, and fewer chondroitin sulphate side chains on the protein core. The PG-I in the developing limb can be considered a mixture of smaller aggregates and cartilage-specific large monomers in which the former predominate.

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.

The genesis of membrane bone in the embryonic chick maxilla: epithelial-mesenchymal tissue recombination studies

Development ◽  
1980 ◽  
Vol 56 (1) ◽  
pp. 269-281
Author(s):  
Mary S. Tyler ◽  
David P. McCobb
Keyword(s):  
Bone Formation ◽  
Hind Limb ◽  
Bone Grafts ◽  
Chick Embryos ◽  
Embryonic Chick ◽  
Limb Buds ◽  
Mesenchymal Tissue ◽  
Membrane Bone ◽  
Tissue Recombination

In the present study, the question of whether a relatively non-specific epithelial requirement exists for membrane bone formation within the maxillary mesenchyme was investigated. Organ rudiments from embryonic chicks of three to five days of incubation (HH 18–25) were enzymatically separated into the epithelial and mesenchymal components. Maxillarymesenchyme (from embryos HH 18–19) which in the absence of epithelium will not form bone was recombined with epithelium from maxillae of similarly aged embryos (homotypichomochronic recombination) and of older embryos (HH 25) (homotypic-heterochronicrecombination). Heterotypic recombinations were made between maxillary mesenchyme (HH 18–19) and the epithelium from wing and hind-limb buds (HH 19–22). Recombinants were grown as grafts on thechorioallantoic membranes of host chick embryos. Grafts of intact maxillae, isolated maxillary mesenchyme, and isolated epithelia from the maxilla, wing-, and hind-limb buds weregrown as controls. The histodifferentiation of grafted intact maxillae was similar to that in vivo; both cartilage and membrane bone differentiated within the mesenchyme. Grafts of maxillary mesenchyme (from embryos HH 18–19) grown in the absence of epithelium formed cartilage but did not form membrane bone. Grafts of maxillary mesenchyme (from embryos HH 18–19) recombined with epithelium in homotypichomochronic, homotypic-heterochronic, and heterotypic tissue combinations formed membrane bone in addition to cartilage. These results indicate that maxillary mesenchyme requires the presence of epithelium to promote osteogenesis and that this epithelial requirement is relatively non-specific in terms of type and age of epithelium.

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