Ectoderm and mesoderm interactions in the limb bud of the chick embryo studied by transfilter cultures: cartilage differentiation and ultrastructural observations

Development ◽  
1980 ◽  
Vol 59 (1) ◽  
pp. 157-173
Author(s):  
Madeleine Gumpel-Pinot
Keyword(s):  
Chick Embryo ◽  
Pore Size ◽  
Limb Bud ◽  
Mesoderm Cell ◽  
Cartilage Differentiation ◽  
Cell Processes ◽  
Upper Face ◽  
Wing Bud

The wing mesoderm of the chick embryo cultured in vitro without ectoderm is able to differentiate into cartilage from stage 17 (Hamburger & Hamilton, 1951). But before this stage the presence of ectoderm is necessary. In transfilter cultures of wing-bud ectoderm and mesoderm, the mesodermal response as measured by chondrogenesis was directly related to the pore size (0·2–1 μm) of the filter. Filters of 0·2 μm pore size and 10 μm thickness gave no increase in chondrogenesis over that of mesoderm cultures alone. The lower face of filters on the upper face of which mesoderm or ectoderm had been cultured was observed by scanning electron microscopy. With ectoderm, no cell processes crossed the filter. In contrast, with mesoderm, cell processes crossed the filter and this was also related to pore size. A good correlation was observed between the mass and density of processes crossing the filter and the mesodermal response. It is concluded that induction of cartilage in limb mesoderm cannot be classified as a ‘long-range transmission’ system. It requires ectoderm and mesoderm to be separated by a very narrow gap and this condition can be brought about in vitro by extension of mesodermal processes through the filter close to the ectoderm. The results are discussed in relation to a possible role of the basement membrane and associated extracellular matrix in limb cartilage induction.

The relationship between intracellular calcium levels and limb bud chondrogenesis in vitro

Development ◽  
1987 ◽  
Vol 100 (1) ◽  
pp. 73-81
Author(s):  
J.A. Bee ◽  
R. Jeffries
Keyword(s):  
Phenotypic Expression ◽  
Culture Conditions ◽  
Limb Bud ◽  
Concentration Effects ◽  
Cartilage Differentiation ◽  
Standard Culture ◽  
The Relationship ◽  
Plateau Level

Under standard culture conditions, chondrogenic expression by stage-21 embryonic chick limb bud mesenchyme is dependent upon high cell plating densities. Alternatively, when cultured in suspension aggregating limb bud cells differentiate exclusively as cartilage. We have previously demonstrated that the aggregation of prechondrogenic limb bud cells is specifically mediated by a Ca2+ -dependent mechanism. In the present paper, we examine the involvement of calcium cations in chondrogenic expression in vitro. During cartilage differentiation, we demonstrate that limb bud cells elevate their intracellular Ca2+ levels to achieve a conserved plateau level. This increase in intracellular Ca2+ levels does not occur in sparse cell cultures, which also fail to demonstrate cartilage differentiation. Although elevation of extracellular Ca2+ concentration effects precocious chondrogenesis, ultimately this is substantially lower than in control cultures. In contrast, elevation of intracellular Ca2+ levels by the addition of 0á1 μm-A23187 readily stimulates precocious and extensive cartilage differentiation. 0á1μm-A23187 initially elevates intracellular Ca2+ levels to that required for cartilage differentiation but this then continues to increase concomitant with a reduction in cartilage nodule size. 10μm-retinoic acid completely inhibits chondrogenesis in vitro and elevates intracellular Ca2+ to particularly high levels. Our data indicate the central role of controlled intracellular Ca2+ levels to normal chondrogenic expression. Deviation from this level by cells that either fail to achieve or that exceed it inhibits subsequent cartilage development, and can cause a loss of phenotypic expression by differentiated cartilage.

Studies on the possible role of cyclic AMP in limb morphogenesis and differentiation

Development ◽  
1980 ◽  
Vol 56 (1) ◽  
pp. 91-105
Author(s):  
Robert A. Kosher ◽  
Mary P. Savage
Keyword(s):  
Cyclic Amp ◽  
Molecular Level ◽  
Mesenchymal Cells ◽  
Limb Bud ◽  
Limb Morphogenesis ◽  
Cartilage Differentiation ◽  
Negative Effect ◽  
Cyclic Amp Derivatives

Recent studies indicate that one of the major functions of the apical ectodermal ridge (AER) of the embryonic chick limb bud is to maintain mesenchymal cells directly subjacent to it (i.e. cells extending 0·4–0·5 mm from the AER), in a labile, undifferentiated condition, and that when mesenchymal cells are freed from the AER's influence either artificially or as a result of normal polarized proximal to distallimb outgrowth, they are freed to commence cyto-differentiation. In a preliminary attempt to investigate at a molecular level the mechanism by which the AER exerts its ‘negative’ effect on the cytodifferentiation of subjacent mesenchymal cells, we haveexamined the effect of a variety of agents that elevate cyclic AMP levels on the morphogenesis and differentiation of the unspecialized subridge mesoderm in an organ culture system. In vitro in the presence of the AER, undifferentiated subridge mesoderm explants undergo remarkably normal morphogenesis characterized primarily by progressive polarized proximal to distal outgrowth and changes in the contour of the developing explant. In the presence of cyclic AMP derivatives, explants fail to undergo the polarized outgrowth and contour changes characteristic of control explants. In fact, in the presence of dibutyryl-cyclic AMP and theophylline, AER-directed morphogenesis essentially ceases during the first day of culture. The cessation of AER-directed morphogenesis inthe presence of cyclic AMP derivatives is accompanied by the histochemically and biochemically detectable precocious chondrogenic differentiation of the subridge mesenchymal cells. In control explants, cartilage differentiation only occurs in those proximal cellsof the explant which gradually become located greater than 0·4–0·5 mm from the AER. In contrast, in the presence of cyclic AMP derivatives, cartilage differentiation by cells within 0·4–0·5 mm of the AER is detectable from the first day of culture, and by the third day cartilage formation has occurred throughout the entire explant. Overall, these results indicate that elevating the cyclic AMP content of the subridge mesenchymal cells enables the cells to overcome negative influences on cytodifferentiation and the positive influences on morphogenesis being imposed upon them by the AER. On the basis of this observation and previous studies, a testable model on the role of cyclic AMP in limb morphogenesis and differentiation is proposed.

Ectoderm-mesoderm interactions in relation to limb-bud chondrogenesis in the chick embryo: transfilter cultures and ultrastructural studies

Development ◽  
1981 ◽  
Vol 65 (1) ◽  
pp. 73-87
Author(s):  
Madeleine Gumpel-Pinot
Keyword(s):  
Chick Embryo ◽  
Basement Membrane ◽  
Culture Conditions ◽  
Limb Bud ◽  
Mesodermal Cell ◽  
Ultrastructural Studies ◽  
Cartilage Differentiation ◽  
Mesodermal Origin ◽  
Cell Processes

Limb ectoderm induces cartilage differentiation in mesoderm from chick embryo limb buds. Transfilter cultures have shown that this interaction requires ‘contact’ conditions and cannot take place at a distance. In vivo, a basement membrane is always present between ectoderm and mesoderm. The present paper demonstrates that the relationship between ectoderm and mesoderm is similar in vivo and in transfilter cultures. In culture conditions, the filter appears to be infiltrated by mesodermal cell outgrowths which form a continuous mesodermal cover on the filter. A basement membrane is always present between the mat of mesodermal cell processes and the ectoderm. Mesodermal cell processes are able to cross the Nuclepore filters (pore size 0·6–0·8 µm) within 15 min. After 2 h in culture, the surface of the filter opposite to the mesodermal explant is completely covered with mesodermal outgrowths. The extracellular material accumulating at the ectoderm-mesoderm interface appears to be mainly of mesodermal origin.

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

Development ◽  
1993 ◽  
Vol 118 (1) ◽  
pp. 95-104 ◽  
Author(s):  
B.B. Riley ◽  
M.P. Savage ◽  
B.K. Simandl ◽  
B.B. Olwin ◽  
J.F. Fallon
Keyword(s):  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Limb Development ◽  
Limb Bud ◽  
Fibroblast Growth Factor 2 ◽  
Fibroblast Growth ◽  
Donor Tissue ◽  
Wing Bud

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

scholarly journals Stimulation of corneal differentiation by interaction between cell surface and extracellular matrix. I. Morphometric analysis of transfilter "induction".

1975 ◽  
Vol 66 (2) ◽  
pp. 275-291 ◽  
Author(s):  
L Meier ◽  
E D Hay
Keyword(s):  
Extracellular Matrix ◽  
Electron Microscope ◽  
Cell Surface ◽  
Pore Size ◽  
Collagen Synthesis ◽  
Lens Capsule ◽  
Physical Contact ◽  
Corneal Epithelial ◽  
Cell Processes

The present study was undertaken to determine whether or not physical contact with the substratum is essential for the stimulatory effect of extracellular matrix (ECM) on corneal epithelial collagen synthesis. Previous studies showed that collagenous substrata stimulate isolated epithelia to produce three times as much collagen as they produce on noncollagenous substrate; killed collagenous substrata (e.g., lens capsule) are just as effective as living substrata (e.g., living lens) in promoting the production of new corneal stroma in vitro. In the experiments to be reported here, corneal epithelia were placed on one side of Nucleopore filters of different pore sizes and killed lens capsule on the other, with the expectation that contact of the reacting cells with the lens ECM should be limited by the number and size of the cell processes that can tranverse the pores. Transfilter cultures were grown for 24 h in [3H]proline-containing median and incorporation of isotope into hot trichloroacetic acid-soluble protein was used to measure corneal epithelial collagen production. Epithelial collagen synthesis increases directly as the size of the pores in the interposed filter increases and decreases as the thickness of the filter layer increases. Cell processes within Nucleopore filters were identified with the transmission electron microscope with difficulty; with the scanning electron microscope, however, the processes could easily be seen emerging from the undersurface of even 0.1-mum pore size filters. Morphometric techniques were used to show that cell surface area thus exposed to the underlying ECM is linearly correlated with enhancement of collagen synthesis. Epithelial cell processes did not pass through ultrathin (25-mum thick) 0.45-mum pore size Millipore filters nor did "induction" occur across them. The results are discussed in relation to current theories of embryonic tissue interaction.

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)

Retinoic acid signaling is required during early chick limb development

Development ◽  
1996 ◽  
Vol 122 (5) ◽  
pp. 1385-1394 ◽  
Author(s):  
J.A. Helms ◽  
C.H. Kim ◽  
G. Eichele ◽  
C. Thaller
Keyword(s):  
Retinoic Acid ◽  
Limb Development ◽  
Acid Synthesis ◽  
Apical Ectodermal Ridge ◽  
Limb Bud ◽  
Retinoid Receptor ◽  
Limb Morphogenesis ◽  
Wing Bud ◽  
Zone Of Polarizing Activity

In the chick limb bud, the zone of polarizing activity controls limb patterning along the anteroposterior and proximodistal axes. Since retinoic acid can induce ectopic polarizing activity, we examined whether this molecule plays a role in the establishment of the endogenous zone of polarizing activity. Grafts of wing bud mesenchyme treated with physiologic doses of retinoic acid had weak polarizing activity but inclusion of a retinoic acid-exposed apical ectodermal ridge or of prospective wing bud ectoderm evoked strong polarizing activity. Likewise, polarizing activity of prospective wing mesenchyme was markedly enhanced by co-grafting either a retinoic acid-exposed apical ectodermal ridge or ectoderm from the wing region. This equivalence of ectoderm-mesenchyme interactions required for the establishment of polarizing activity in retinoic acid-treated wing buds and in prospective wing tissue, suggests a role of retinoic acid in the establishment of the zone of polarizing activity. We found that prospective wing bud tissue is a high-point of retinoic acid synthesis. Furthermore, retinoid receptor-specific antagonists blocked limb morphogenesis and down-regulated a polarizing signal, sonic hedgehog. Limb agenesis was reversed when antagonist-exposed wing buds were treated with retinoic acid. Our results demonstrate a role of retinoic acid in the establishment of the endogenous zone of polarizing activity.

scholarly journals MICROFILAMENTS AND CELL LOCOMOTION

1971 ◽  
Vol 49 (3) ◽  
pp. 595-613 ◽  
Author(s):  
Brian S. Spooner ◽  
Kenneth M. Yamada ◽  
Norman K. Wessells
Keyword(s):  
Protein Synthesis ◽  
Plasma Membrane ◽  
Cytochalasin B ◽  
Cell Movement ◽  
Leading Edge ◽  
Complete Recovery ◽  
Cell Locomotion ◽  
Cell Processes

The role of microfilaments in generating cell locomotion has been investigated in glial cells migrating in vitro. Such cells are found to contain two types of microfilament systems: First, a sheath of 50–70-A in diameter filaments is present in the cytoplasm at the base of the cells, just inside the plasma membrane, and in cell processes. Second, a network of 50-A in diameter filaments is found just beneath the plasma membrane at the leading edge (undulating membrane locomotory organelle) and along the sides of the cell. The drug, cytochalasin B, causes a rapid cessation of migration and a disruption of the microfilament network. Other organelles, including the microfilament sheath and microtubules, are unaltered by the drug, and protein synthesis is not inhibited. Removal of cytochalasin results in complete recovery of migratory capabilities, even in the absence of virtually all protein synthesis. Colchicine, at levels sufficient to disrupt all microtubules, has no effect on undulating membrane activity, on net cell movement, or on microfilament integrity. The microfilament network is, therefore, indispensable for locomotion.

scholarly journals Molecular analysis of external genitalia formation: the role of fibroblast growth factor (Fgf) genes during genital tubercle formation

Development ◽  
2000 ◽  
Vol 127 (11) ◽  
pp. 2471-2479 ◽  
Author(s):  
R. Haraguchi ◽  
K. Suzuki ◽  
R. Murakami ◽  
M. Sakai ◽  
M. Kamikawa ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Gene Knockout ◽  
External Genitalia ◽  
Neutralizing Antibody ◽  
Protein S ◽  
Limb Bud ◽  
Urethral Plate ◽  
Genital Tubercle

The molecular mechanisms underlying the development of the external genitalia in mammals have been very little examined. Recent gene knockout studies have suggested that the developmental processes of its anlage, the genital tubercle (GT), have much in common with those of limb buds. The Fgf genes have been postulated as regulating several downstream genes during organogenesis. Fgf8 was expressed in the distal urethral plate epithelium of the genital tubercle (GT) together with other markers such as the Msx1, Fgf10, Hoxd13 and Bmp4 expressed in the mesenchyme. To analyze the role of the FGF system during GT formation, an in vitro organ culture system was utilized. It is suggested that the distal urethral plate epithelium of GT, the Fgf8-expressing region, regulates the outgrowth of GT. Ectopic application of FGF8 beads to the murine GT induced mesenchymal gene expression, and also promoted the outgrowth of the GT. Experiments utilizing anti-FGF neutralizing antibody suggested a growth-promoting role for FGF protein(s) in GT outgrowth. In contrast, despite its vital role during limb-bud formation, Fgf10 appears not to be primarily essential for initial outgrowth of GT, as extrapolated from Fgf10(−/−) GTs. However, the abnormal external genitalia development of Fgf10(−/−) perinatal mice suggested the importance of Fgf10 in the development of the glans penis and the glans clitoridis. These results suggest that the FGF system is a key element in orchestrating GT development.

In vitro studies on the morphogenesis and differentiation of the mesoderm subjacent to the apical ectodermal ridge of the embryonic chick limb-bud

Development ◽  
1979 ◽  
Vol 50 (1) ◽  
pp. 75-97
Author(s):  
Robert A. Kosher ◽  
Mary P. Savage ◽  
Sai-Chung Chan
Keyword(s):  
Organ Culture ◽  
Chondrogenic Differentiation ◽  
Mesenchymal Cells ◽  
Apical Ectodermal Ridge ◽  
Limb Bud ◽  
Embryonic Chick ◽  
Marked Contrast ◽  
Culture Cells ◽  
Wing Bud

It has been suggested that one of the major functions of the apical ectodermal ridge (AER) of the embryonic chick limb-bud is to maintain mesenchymal cells directly subjacent to it (i.e. cells extending 00·4–00·5 mm from the AER) in a labile, undifferentiated condition. We have attempted to directly test this hypothesis by subjecting the undifferentiated subridgemesoderm of stage-25 embryonic chick wing-buds to organ culture in the presence and absence of the AER and the ectoderm that normally surrounds the mesoderm dorsally and ventrally. During the period of culture, control explants comprised of the subridge mesoderm capped by the AER and surrounded by the dorsal/ventral ectoderm undergo progressivemorphogenesis characterized by polarized proximal to distal outgrowth and changes in the contour of the developing explant, and ultimately form a structure grossly resembling a normal distal wing-bud tip. In contrast, explants from which the AER and dorsal/ventral ectoderm have been removed (minus ectoderm explants) or from which just the AER has been removed (minus AER explants) form compact, rounded masses exhibiting no signs of morphogenesis. During the polarized proximal to distal outgrowth control explants undergo during the first 3 days of culture, as cells of the explant become located greater than 0·4– 0·5 mm from the AER, they concomitantly undergo a sequence of changes indicative of their differentiation into cartilage. However, those cells which remain 0·4–0·5 mm from the AER during this period retain the characteristics of non-specialized mesenchymal cells. In marked contrast to control explants, virtually all of the cells of minus ectoderm explants initiate chondrogenic differentiation during the first day of culture. Cells comprising the central core of minus AER explants also initiate chondrogenic differentiation during the first day of culture, but in contrast to minus ectoderm explants, non-chondrogenic tissue types form along the periphery of the explants subjacent to the dorsal/ventral ectoderm. These results indicate that the AER maintains cells directly subjacent to it in a labile, undifferentiated condition, and that when mesenchymal cells are freed from the AER's influence either artificially or as a result of normal polarized outgrowth, they are freed to commence cytodifferentiation. The results further suggest that the dorsal/ventral ectoderm may have an influence on the differentiation of the mesenchymal cells directly subjacent to it, once the cells have been removed from the influence of the AER.

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