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.

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.

Interactions between mesoderm cells and the extracellular matrix following gastrulation in the chick embryo

1991 ◽  
Vol 99 (2) ◽  
pp. 431-441
Author(s):  
A.J. Brown ◽  
E.J. Sanders
Keyword(s):  
Extracellular Matrix ◽  
Chick Embryo ◽  
Basement Membrane ◽  
Primitive Streak ◽  
Cell Binding ◽  
Fab Fragment ◽  
Fibronectin Receptor ◽  
In Vitro Methods

In the gastrulating chick embryo, the mesoderm cells arise from the epiblast layer by ingression through the linear accumulation of cells called the primitive streak. The mesoderm cells emerge from the streak with a fibroblastic morphology and proceed to move away from the mid-line of the embryo using, as a substratum, the basement membrane of the overlying epiblast and the extracellular matrix. We have investigated the roles of fibronectin and laminin as putative substrata for mesoderm cells using complementary in vivo and in vitro methods. We have microinjected agents into the tissue space adjacent to the primitive streak of living embryos and, after further incubation, we have examined the embryos for perturbation of the mesoderm tissue. These agents were: cell-binding regions from fibronectin (RGDS) and laminin (YIGSR), antibodies to these glycoproteins, and a Fab' fragment of the antibody to fibronectin. We find that RGDS, antibody to fibronectin, and the Fab' fragment cause a decrease in the number of mesoderm cells spread on the basement membrane, and a perturbation of cell shape suggesting locomotory impairment. No such influence was seen with YIGSR or antibodies to laminin. These results were extended using in vitro methods in which mesoderm cells were cultured in fibronectin-free medium on fibronectin or laminin in the presence of various agents. These agents were: RGDS; YIGSR; antibodies to fibronectin, fibronectin receptor, laminin and vitronectin; and a Fab' fragment of the fibronectin antiserum. We find that cell attachment and spreading on fibronectin is impaired by RGDS, antiserum to fibronectin, the Fab' fragment of fibronectin antiserum, and antiserum to fibronectin receptor. The results suggest that although the RGDS site in fibronectin is important, it is probably not the only fibronectin cell-binding site involved in mediating the behaviour of the mesoderm cells. Cells growing on laminin were perturbed by YIGSR, RGDS and antibodies to laminin, suggesting that mesoderm cells are able to recognise at least two sites in the laminin molecule. We conclude that the in vivo dependence of mesoderm cells on fibronectin is confirmed, but that although these cells have the ability to recognise sites in laminin as mediators of attachment and spreading, the in vivo role of this molecule in mesoderm morphogenesis is not yet certain.

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.

MHox: a mesodermally restricted homeodomain protein that binds an essential site in the muscle creatine kinase enhancer

Development ◽  
1992 ◽  
Vol 115 (4) ◽  
pp. 1087-1101 ◽  
Author(s):  
P. Cserjesi ◽  
B. Lilly ◽  
L. Bryson ◽  
Y. Wang ◽  
D.A. Sassoon ◽  
...  
Keyword(s):  
Creatine Kinase ◽  
Homeobox Genes ◽  
Cell Types ◽  
Limb Bud ◽  
Homeodomain Protein ◽  
Muscle Creatine Kinase ◽  
Mesodermal Cell ◽  
Adult Mice ◽  
Mesodermal Origin ◽  
Muscle Creatine

Myogenic helix-loop-helix (HLH) proteins, such as myogenin and MyoD, can activate muscle-specific transcription when introduced into a variety of nonmuscle cell types. Whereas cells of mesodermal origin are especially permissive to the actions of these myogenic regulators, many other cell types are refractory to myogenic conversion by them. Here we describe a novel homeodomain protein, MHox, that binds an A+T-rich element in the muscle creatine kinase (MCK) enhancer that is essential for muscle-specific transcription and trans-activation by myogenic HLH proteins. MHox is completely restricted to mesodermally derived cell types during embryogenesis and to established cell lines of mesodermal origin. In contrast to most other homeobox genes, MHox expression is excluded from the nervous system, with the highest levels observed in limb bud and visceral arches. In adult mice, MHox is expressed at high levels in skeletal muscle, heart and uterus. The DNA-binding properties and pattern of MHox expression are unique among homeobox genes and suggest a role for MHox as a transcriptional regulator that participates in the establishment of diverse mesodermal cell types.

Chondrogenesis in chick embryo somites grafted with adjacent and heterologous tissues

Development ◽  
1972 ◽  
Vol 27 (1) ◽  
pp. 229-234
Author(s):  
M. J. O'Hare
Keyword(s):  
Chick Embryo ◽  
Basement Membrane ◽  
Membrane Material ◽  
Grafting Technique ◽  
Cartilage Differentiation ◽  
Sulphated Glycosaminoglycans ◽  
Embryonic Ectoderm

A variety of heterologous tissues have been tested for the ability to promote cartilage differentiation in isolated chick-embryo somites, using a modified chorioallantoic grafting technique. Of the 12 tissues tested only 3- and 4-day embryonic ectoderm promoted somite chondrogenesis in somites that fail to chondrify when grafted in isolation. This activity of ectoderm was evident in grafts of somites isolated with adjacent ectoderm, and in grafts of somites recombined with ectoderm derived from several sources. Four-day embryonic limbbud ectoderm, including the apical ridge, was capable of promoting somite chondrogenesis, but to no greater extent than dorsal trunk ectoderm of the same age. It is suggested that the ability of embryonic ectoderm to promote cartilage differentiation in isolated somites is associated with its ability to synthesize basement membrane material (sulphated glycosaminoglycans and collagen), in association with adjacent somite mesoderm.

Cytokeratin expression in rat mesothelial cells in vitro is controlled by the extracellular matrix

1990 ◽  
Vol 95 (1) ◽  
pp. 97-107
Author(s):  
A.M. Mackay ◽  
R.P. Tracy ◽  
J.E. Craighead
Keyword(s):  
Extracellular Matrix ◽  
Basement Membrane ◽  
Intermediate Filament ◽  
Culture Conditions ◽  
Mesothelial Cells ◽  
Intermediate Filament Protein ◽  
Cellular Contact ◽  
Filament Protein

Rat mesothelial cells co-express vimentin and the simple epithelial cytokeratins. While cytokeratins predominate in situ, under most culture conditions vimentin is the major intermediate filament protein of the cells. This loss of cytokeratin production upon culture can be partly prevented by growing mesothelial cells on a basement membrane matrix. However, the basement membrane-promoted persistence of cytokeratin synthesis is not accompanied by expression of cytokeratin G (no. 19), the major acidic cytokeratin of mesothelium in vivo. While cells grown on plastic establish a prominent juxtanuclear assemblage of tonofilaments, those cultured on basement membrane exhibit cytokeratin filaments which are distributed throughout the cytoplasm and attach to neighboring cells at the plasma membrane. This latter pattern resembles that seen in the intact mesothelium. Intermediate filaments are markers of cellular differentiation, but their roles are obscure. The response of cultured mesothelial cells to different growth substrata supports the hypothesis that intermediate filament synthesis is influenced by cellular contact with the extracellular matrix.

Probing the ultrastructure of the mitotic and meiotic spindle in eggs: An expeditious approach based on semi-thin (0.25 μm) serial sections

1983 ◽  
Vol 41 ◽  
pp. 552-555
Author(s):  
Conly L. Rieder ◽  
S. Bowser ◽  
R. Nowogrodzki ◽  
K. Ross ◽  
G. Sluder
Keyword(s):  
Small Sample Size ◽  
Small Sample ◽  
Meiotic Spindle ◽  
Serial Sections ◽  
Mitotic Apparatus ◽  
Thin Sections ◽  
Cell Cycles ◽  
Ultrastructural Studies

Eggs have long been a favorite material for studying the mechanism of karyokinesis in-vivo and in-vitro. They can be obtained in great numbers and, when fertilized, divide synchronously over many cell cycles. However, they are not considered to be a practical system for ultrastructural studies on the mitotic apparatus (MA) for several reasons, the most obvious of which is that sectioning them is a formidable task: over 1000 ultra-thin sections need to be cut from a single 80-100 μm diameter egg and of these sections only a small percentage will contain the area or structure of interest. Thus it is difficult and time consuming to obtain reliable ultrastructural data concerning the MA of eggs; and when it is obtained it is necessarily based on a small sample size.We have recently developed a procedure which will facilitate many studies concerned with the ultrastructure of the MA in eggs. It is based on the availability of biological HVEM's and on the observation that 0.25 μm thick serial sections can be screened at high resolution for content (after mounting on slot grids and staining with uranyl and lead) by phase contrast light microscopy (LM; Figs 1-2).

Adenomatoid Tumor of the Testis, A Mesonephric Hamartoma

1971 ◽  
Vol 29 ◽  
pp. 318-319
Author(s):  
Raoul Fresco ◽  
Mary Chang-Lo
Keyword(s):  
Electron Microscopy ◽  
Epithelial Cells ◽  
Fibrous Tissue ◽  
Salient Feature ◽  
Luminal Surface ◽  
Adenomatoid Tumor ◽  
Ultrastructural Studies ◽  
Epithelial Origin ◽  
Brush Borders ◽  
Cell Processes

Confusion surrounds the nature of the “adenomatoid tumor” of the testis, as evidenced by the large number of synonyms which have been ascribed to it. Various authors have considered the tumor to be of endothelial, mesothelial or epithelial origin. There appears to be no controversy as to the stromal elements of the tumor, which consists mainly of smooth muscle and fibrous tissue. It is the irregular gland-like spaces which have given rise to the numerous theories as to its histogenesis, and even recent ultrastructural studies fail to agree on the origin of these structures.Electron microscopy of a typical intrascrotal adenomatoid tumor showed the gland-like spaces to be lined by epithelial cells (Fig. 1), rich in cytoplasmic tonofibrils and united to each other by numerous desmosomes (Fig. 2). The most salient feature of these epithelial cells was the presence on their luminal surface of numerous long and repeatedly branching microvillous structures of the type known as stereocilia (Fig. 3). These are extremely long slender cell processes which are as much as three to four times the length of those in brush borders.

Osseointegration interface of calcified bone and endosseous implants

1992 ◽  
Vol 50 (2) ◽  
pp. 1096-1097
Author(s):  
K.E. Krizan ◽  
J.E. Laffoon ◽  
M.J. Buckley
Keyword(s):  
Structure And Function ◽  
Titanium Implants ◽  
En Bloc ◽  
Endosseous Implants ◽  
Ultrastructural Studies ◽  
The Past ◽  
Cacodylate Buffer ◽  
One Year ◽  
And Function

With increase use of tissue-integrated prostheses in recent years it is a goal to understand what is happening at the interface between haversion bone and bulk metal. This study uses electron microscopy (EM) techniques to establish parameters for osseointegration (structure and function between bone and nonload-carrying implants) in an animal model. In the past the interface has been evaluated extensively with light microscopy methods. Today researchers are using the EM for ultrastructural studies of the bone tissue and implant responses to an in vivo environment. Under general anesthesia nine adult mongrel dogs received three Brånemark (Nobelpharma) 3.75 × 7 mm titanium implants surgical placed in their left zygomatic arch. After a one year healing period the animals were injected with a routine bone marker (oxytetracycline), euthanized and perfused via aortic cannulation with 3% glutaraldehyde in 0.1M cacodylate buffer pH 7.2. Implants were retrieved en bloc, harvest radiographs made (Fig. 1), and routinely embedded in plastic. Tissue and implants were cut into 300 micron thick wafers, longitudinally to the implant with an Isomet saw and diamond wafering blade [Beuhler] until the center of the implant was reached.

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