Nikolas Zagris

Spatial and Temporal Expression of Perlecan in the Early Chick Embryo

Perlecan is a major heparan sulfate proteoglycan that binds growth factors and interacts with various extracellular matrix proteins and cell surface molecules. The expression and spatiotemporal distribution of perlecan was studied by RT-PCR, immunoprecipitation and immunofluorescence in the chick embryo from stages X (morula) to HH17 (29 somites). Combined RT-PCR and immunohistochemistry demonstrated the expression of perlecan as early as stage X and its presence may be fundamental to the first basement membrane assembly on the epiblast ventral surface at stage XIII (blastula). Perlecan fluorescence was intense in the cells ingressing through the primitive streak and was strong lining the epiblast ventral surface lateral to the streak at stage HH3–4 (gastrula). At stage HH5–6 (neurula), perlecan fluorescence was low in the neuroepithelium and stronger in the apical surface of the neural plate. At stage HH10–11 (12 somites), perlecan fluorescence was intense in the neuroepithelium and was then essentially nondetectable in the neuroepithelium, and the intensity had shifted to the basement membranes of encephalic vesicles by stage HH17. Perlecan immunofluorescence was intense in neural crest cells, strong in pharyngeal arches, intense in thymus and lung rudiments, intense in aortic arches and in dorsal aorta, strong in lens and retina and intense in intraretinal space and in optic stalk, strong in the dorsal mesocardium, myocardium and endocardium, strong in dermomyotome, low in sclerotome in somites, intense in mesonephric duct and tubule rudiments, intense in the lining of the gut luminal surface. Inhibition of the function of perlecan by blocking antibodies showed that perlecan is crucial for maintaining basement membrane integrity which mediates the epithelialization, adhesive separation and maintenance of neuroepithelium in brain, somite epithelialization, and tissue architecture during morphogenesis of the heart tube, dorsal aorta and gut. An intriguing possibility is that perlecan, as a signaling molecule that modulates the activity of growth factors and cytokines, participates in the signaling pathways that guide gastrulation movements and neural crest cell migration, proliferation and survival, cardiac cell proliferation and paraxial mesoderm (somitic) cell proliferation and segmentation.

Decorin developmental expression and function in the early avian embryo.

Decorin, a proteoglycan, interacts with extracellular matrix proteins, growth factors and receptors. Decorin expression and spatio-temporal distribution were studied by RT-PCR and immunofluorescence, while decorin function was examined by blocking antibodies in the early chick embryo. Decorin was first detectable at stage XIII (late blastula). During gastrulation (stage HH3-4), decorin fluorescence was intense in epiblast cells immediately adjacent to the streak, and in migrating cells. Decorin fluorescence was intense in endoderm and strong at mesoderm-neural plate surfaces at stage HH5-6 (neurula). At stage HH10-11 (12 somites), decorin fluorescence was intense in myelencephalon and then showed distinct expression patterns along the myelencephalon axes by stage HH17. Decorin fluorescence was intense in neural crest cells, dorsal aorta, heart, somite and neuroepithelial cells apposing the somite, nephrotome, gut and in pancreatic and liver primordia. Antibody-mediated inhibition of decorin function affected the head-to-tail embryonic axis extension, indicating that decorin is essential for convergent extension cell movements during avian gastrulation. Decorin was also essential for retinal progenitor cell polarization, neural crest migration, somite boundary formation and cell polarization, mesenchymal cell polarization and primary endoderm displacement to the embryo periphery. The embryonic blood vessels were deformed, the dorsal mesocardium was thinned and the cardiac jelly was abnormally thickened in the heart. Decorin is known to modulate collagen fibrillogenesis, a key mechanism of matrix assembly, and cell proliferation. Decorin also appears to be essential for the coordination of cell and tissue polarization, which is an important feature in organ patterning of the embryo.

Developmentally regulated expression and functional role of α7 integrin in the chick embryo

Integrin α7â1 is a specific cellular receptor for laminin. In the present work, we studied the distribution pattern of the α7 subunit by immunofluorescence and immunoprecipitation and the role of the integrin by blocking antibodies in early chick embryos. α7 immunoreactivity was first detectable in the neural plate during neural furrow formation (stage HH5, early neurula, Hamburger & Hamilton 1951 ) and its expression was upregulated in the neural folds during primary neurulation. The α7 expression domain spanned the entire neural tube by stage HH8 (4 somites), and was then downregulated and confined to the neuroepithelial cells in the germinal region near the lumen and the ventrolateral margins of the neural tube in embryos by the onset of stage HH17 (29 somites). Expression of α7 in the neural tube was transient suggesting that α7 functions during neural tube closure and axon guidance and may not be required for neuronal differentiation or for the maintenance of the differentiated cell types. α7 immunoreactivity was strong in the newly formed epithelial somites, although this expression was restricted only to the myotome in the mature somites. The most intense α7 immunoreactivity was detectable in the paired heart primordia and the endoderm apposing the heart primordia in embryos at stage HH8. In the developing heart, α7 immunoreactivity was: (i) intense in the myocardium; (ii) milder in the endocardial cushions of the ventricle; (iii) intense in the sinus venosus; (iv) distinct in the associated blood vessels; and (v) undetectable in the dorsal mesocardium of embryos at stage HH17. Inhibition of function of α7 by blocking antibodies showed that α7 integrin–laminin signaling may play a critical role in tissue organization of the neural plate and neural tube closure, in tissue morphogenesis of the heart tube but not in the directional migration of pre‐cardiac cells, and in somite epithelialization but not in segment formation in presomitic mesoderm. In embryos treated with α7 antibody, the formation of median somites in place of a notochord was intriguing and suggested that α7 integrin–laminin signaling may have played a role in segment re‐specification in the mesoderm.

Differential heat shock gene expression in chick blastula

SummaryThe component areas of chick blastula show differential expression of heat shock genes. The area opaca (ao), marginal zone (mz) and central area (ca) components of the epiblast display distinct quantitative and minor qualitative differences in the heat-induced and heat-repressible proteins, but are clearly different from the primary hypoblast (endoderm) in their expression of a given stress protein (hsp) as a response to heat shock. The major proteins synthesized in the component areas of epiblast in response to heat shock include hsp 18, 24, 70 and 89 Kd. Two-dimensional electrophoresis shows that each of these proteins consists of multiple charged species. The hypoblast expresses only hsp 70 Kd at non-significant levels and shows marked inhibition in the level of synthesis of heat-shock-repressible proteins. Heat shock during the blastula stage results in an increase in the size of the blastoderm and disrupts normal embryonic development. The heat shock genes provide an important molecular marker, which attests to regional specification in the chick blastula.

Induction of the primitive streak in the chick blastoderm embryo: patterns of protein synthesis

SummaryInduction of the primitive streak is correlated with specific qualitative and quantitative changes in protein synthesis in the component areas of chick blastoderm. Blastoderm embryos at the initial to intermediate primitive streak stage were labeled with L-[35S] methionine. Radioactively labeled proteins separated by two-dimensional sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis revealed differences in the number and density of spots among the component areas of the epiblast and hypoblast. Protein patterns of the area opaca, marginal zone and central area of the epiblast are very similar qualitatively but show distinct quantitative differences. A comparison between any of the component areas of the epiblast and the hypoblast in chick blastoderm embryos, however, reveals both qualitative and quantitative differences. A protein with a molecular weight of 30,000 unique to the component areas of the epiblast, and proteins with a molecular weight of 22,000 and 37,000 unique to the hypoblast are prominent and seem to be related to the initial appearance of the primitive streak.

THE EXPRESSION OF THE GENES FOR LAMININ IN THE EARLY EMBRYO

The extracellular matrix (ECM) is composed of glycoproteins, proteoglycans and glycosaminoglycans locally secreted by resident cells. It is assembled into an organized fibrillar network in the forming extracellular spaces before the organization of a basement membrane in the early embryo. The ECM interacts with cells (“dynamic reciprocity”) and promotes and regulates cellular functions such as migration, adhesion and proliferation which result in induction, differentiation and morphogenesis (Grobstein, 1954; Bissel et al., 1982; Martin and Timpl, 1987; Engel, 1989; Hay, 1991; Sage and Bornstein, 1991; Adams and Watt, 1993; Chung, 1993; DeSimone, 1994; Horwitz and Thiery, 1994). Laminin is the first ECM glycoprotein to appear in the matrix in the extracellular spaces from which it mostly disappears during development and is later organized in basement membranes (Leivo et al, 1980; Cooper and MacQueen, 1983; Dziadek and Timpl, 1985; Rogers et al., 1986; Kucherer-Ehret et al., 1990; Zagris and Chung, 1990). In the adult, the ECM is confined to basement membranes. This paper focuses on the biological activity of laminin and its expression in the early embryo. The appearance of laminin early in embryogenesis may implicate it in regulating cell migration, adhesion and differentiation.

Extracellular matrix organized in embryonic cavities during induction of the embryonic axis in chick embryo

Extracellular matrix (ECM) is detected as short, disorganized fibrils in the forming embryonic extracellular spaces shortly prior to the first morphogenetic cellular movements and interactions in the early chick embryo. As development progresses, the ECM is organized into an intricate network spanning the embryonic cavities. This dynamic entity undergoes relatively rapid changes in its organization pattern during the developmental period from morula to the induction of the neural plate. The ECM seems to preserve the exquisite architecture of the embryo and could guide migrating cells into defined pathways in the early embryo.

Gene expression in chick morula

SummaryThe polypeptides synthesized during the morula stage in the chick embryo are insensitive to transcriptional inhibition by α-amanitin. Protein synthesis seems to depend predominantely, if not exclusively, on the recruitment of maternal mRNA, rather than on embryonic gene expression in chick morula. The morula embryo expresses the heatshock polypeptides when stressed at 43°C. The heat-induced polypeptides are isoforms of polypeptides that are synthesized normally. These polypeptides are α-amanitin sensitive and appear to mark the first major expression of the embryonic genome in the chick embryo.

Communication between Primary Endoderm and Mesoderm for Erythroblast Differentiation in Early Chick Blastoderm

Blood islands are mesodermal cells which, in close association with the underlying endoderm, form blood cells and endothelium in the early embryo. The primary hypoblast (primary endoderm), separated from the epiblast of chick blastoderm at the full hypoblast stage, was labeled with 14C-protein hydrolysate. The labeled hypoblast was recombined with its denuded unlabeled epiblast. Radioautographs and uptake and incorporation measurements in the blood islands show that there is transport of label from the primary hypoblast to the erythroblasts or to some progenitor cell(s) of the erythroblasts in the epiblast. The observed cell communication between mesoderm and the endoderm is in concert with the description of the blood island cytoarchitecture in which mesodermal cells are making direct contact with the adjacent endodermal cells during erythroblast differentiation.

Monensin inhibits the first cellular movements in early chick embryo

SummaryIn early chick blastoderm at stage XIII, the interaction of the hypoblast with the epiblast triggers on the epiblast the first extensive cellular migrations, which result in formation of the primitive streak, the source of the axial mesoderm. During this period, extracellular material (ECM) is secreted and assembled into an organized network in the extracellular spaces and is implicated in regulating the behaviour of the cells that contact it. The first cellular migrations and inductions are inhibited when early chick blastoderm is treated with the glycosylation-perturbing ionophore monensin. The difference in amount and in organization of ECM between monensin-treated embryos and control embryos is striking. Even blastoderms at stage X, which are essentially free of ECM, show extensive ECM after monensin treatment. Monensin produces a substantial change in the polypeptide pattern with the induction or marked accentuation of multiple charged species (isoforms) of polypeptides different from those present in the control embryos. The interference of monensin with the migration and induction mechanisms is permanent in embryos before the primitive streak (PS) stage, and it seems that the respective signals or the sensitivity of the epiblast/hypoblast cells to them must be very stage specific. Monensin-treated embryos probably secrete abnormal ECM that does not provide the proper conditions for the hypoblast to interact with the epiblast cells.