Preservation of fine structure in vibratome-cut sections of the central nervous system stained for light microscopy

A technique without negative effects on tissue preservation that allows precise identification and subsequent removal of central nervous system nuclei for ultrastructural analysis is described. The procedure uses 200 microns thick Vibratome-cut sections of glutaraldehyde fixed brains. These sections are stained for 25 seconds with a methylene blue solution and stored for 4 hours in 0.2 M pH 7.4 phosphate buffer in 4% sucrose for optimal visualization at the light microscopic level. The stock solution of 1 g methylene blue and 1 g sodium borate in 100 ml of distilled water, is filtered through a Millipore filter and diluted 5:95 with distilled water immediately prior to use. Regions of specific interest are then processed for electron microscopy.     

Studies on the mechanism of polyethylene glycol-mediated cell fusion using fluorescent membrane and cytoplasmic probes

The mechanism by which polyethylene glycol (PEG) mediates cell fusion has been studied by examining the movements of membrane lipids and proteins, as well as cytoplasmic markers, from erythrocytes to monolayers of cultured cells to which they have been fused. Fluorescence and freeze-fracture electron microscopy and fluorescence recovery after photobleaching have yielded the following results: (a) In the presence of both fusogenic and nonfusogenic PEG membranes are brought together at closely apposed contact regions. (b) Fluorescent lipid probes quickly spread from the membranes of erythrocytes to cultured cells in the presence of both fusogenic and nonfusogenic PEG. (c) Proteins of the erythrocyte membranes were never observed to diffuse into the cultured cell membrane. (d) Water-soluble proteins did not diffuse from the erythrocyte interior into the target cell cytoplasm until the PEG was removed. These data suggest that the coordinate action of two distinct components is necessary for fusion as mediated by PEG. Presumably, the polymer itself promotes close apposition of the adjacent cell membranes but the fusion stimulus is provided by the additives contained in commercial PEG.     

Analysis of normal somite development

We describe how the first 6 somite pairs form, using the third somites as examples. This history is based upon time-lapse movies of carbon-marked embryos and histological studies by light and electron microscopy of embryos fixed in situ with glutaraldehyde and osmium tetroxide. At head-process stage a continuous sheet of mesoblast occupies the regions of the future third somites. Mesoblast cells attach either to hypoblast or to overlying neural plate which is already a simple pseudostratified columnar epithelium. Prospective somite cells are those attached to the neuroepithelium, and they extend laterally exactly as far as the neural plate does. By head-fold stage, regression of the node down the midline is shearing the sheet of mesoblast into right and left halves. Somite cells hang from the bottom of the neural plate. As the neural plate condenses toward the midline, attached somite cells are compacted. When the somite segments, somite cells are tightly apposed to one another, and, in addition to junctions binding their basal ends, new junctions appear between their apical ends. This leads to reorganization into the typical somite rosette configuration. Spaces filled with extracellular materials form around the whole somite.     

Development of neuronal locus specificity in Xenopus retinal ganglion cells after surgical eye transection after fusion of whole eyes

A developmental program is established in the stage 28–32 optic cup of Xenopus embryos, which specifies the permanent AP and DV reference axes for positional information in the retina, and thereby determines the pattern of spatial deployment of ganglion cell locus specificities subserving assembly of retinotopically organized connections in the tectum. This developmental program has previously proved unmodifiable in intact eye primordia submitted to a variety of rotation, transplantation, and tissue culture conditions. Here we report that the program can be modified by surgical transection of stage 32 eye primordia (with subsequent fusion of the disconnected halves to reconstitute a whole eye) and by fusion of whole stage 38 eyes, although most of the transected eyes did develop normal visuotectal projections. The remaining vertically transected eyes, and all eyes formed when a left and right stage 38 eye fused along apposed temporal edges, developed “double-nasal compound” projections to the tectum: the nasal and temporal halves of the adult retina each projected to the entire tectum, and each tectal locus was driven from two stimulus positions symmetrically disposed about the vertical meridian. The remaining horizontally transected eyes, and all eyes formed when a left and right stage 38 eye fused along apposed dorsal edges, developed “double-ventral compound” projections to the tectum: the dorsal and ventral halves of the adult retina each projected to the entire tectum, and each tectal locus was driven from two stimulus positions symmetrically disposed about the horizontal meridian. The results are considered in terms of (1) the kinds of cellular processes that could mediate the observed modifications in the original developmental program; (2) the nature and stability of the program; and (3) the general suitability of eye fragment-fusion experiments for analysis of the assembly of retinotectal connections.