The blastoporal groove of the early gastrula of the treefrog, Hyla regilla, was examined with the electron microscope. The innermost extension of the groove is lined with invaginating flask-and wedge-shaped cells of entoderm and mesoderm. The distal surfaces of these cells bear microvilli which are underlain with an electron-opaque layer composed of fine granular material and fibrils. The dense layer and masses of vesicles proximal to it fill the necks of the cells. In flask cells bordering the forming archenteron the vesicles are replaced by large vacuoles surrounded by layers of membranes. The cells lining the groove are tightly joined at their distal ends in the region of the dense layer. Proximally, the cell bodies are separated by wide intercellular spaces. The cell body, which is migrating toward the interior of the gastrula, contains the nucleus plus other organelles and inclusions common to amphibian gastrular cells. A dense layer of granular material, vesicles, and membranes lies beneath the surface of the cell body and extends into pseudopodium-like processes and surface undulations which cross the intercellular spaces. A special mesodermal cell observed in the dorsal lining of the groove is smaller and denser than the surrounding presumptive chordamesodermal cells. A long finger of cytoplasm, filled with a dense layer, vesicles and membranes, extends from its distal surface along the edge of the groove, ending in a tight interlocking with another mesodermal cell. Some correlations between fine structure and the mechanics of gastrulation are discussed, and a theory of invagination is proposed, based on contraction and expansion of the dense layer and the tight junctions at distal cell surfaces.
Recently, an antihapten antibody bridge method was proposed for specific labeling of cell surface antigens for electron microscopy (1). Hapten groups are coupled to specific antibodies and to certain markers, which are linked at the site of the designated cellular antigen with antihapten antibody. The idea of a hapten-sandwich labeling technique had also occurred to us, and we have developed a similar approach which can be applied simply to the amplification of any immunoglobulin fraction used for detecting surface antigens, including alloantigens. We report here hapten-sandwich labeling of Ig, thymus associated, or H-2 antigens on mouse lymphocytes, visualized by fluorescence and transmission electron microscopy. The use of the same technique for specific labeling of cells observed with scanning electron microscopy is reported in another paper2Our procedure succeeds in discriminating with high specificity between different cellular antigens in double-labeling experiments, an application suggested, but not realized, when the antihapten bridging method was projected (1). 2 Abbreviations used in this paper: anti-ars, p-azophenyl arsonate; anti-lac, p-azophenyl ~-D-lactoside; anti-MBr, rabbit antimouse brain; anti-MIg, polyvalent antimouse IgG; BSA, bovine serum albumin; fer, ferritin; Fl, fluorescein; KLH, keyhole limpet hemocyanin; LPS, lipopolysaccharide; PHA-P, phytohemagglutinin P; Rh, rhodamine; SAS, saturated ammonium sulfate; TMV, tobacco mosaic virus; VBS, Veronal-buffered saline.
Materials and Methods
Antisera and Purified Antibodies.--Antibodies
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