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.     

The in vitro synthesis and characteristics of ribosomal RNA in imaginal discs of Drosophila melanogaster

Summary Late third instar imaginal discs of Drosophila melanogaster cultured in vitro in Robb's tissue culture medium synthesize 38S, 28S and 18S ribosomal RNAs which are qualitatively indistinguishable from their in vivo synthesized counterparts (Fig. 1). As found in other insect systems, the 38S molecule appears to be the precursor for both the 28S and 18S rRNAs (Figs. 2, 3 and 4). The 28S rRNA and a portion of the 38S pre-rRNA shift in sedimentation value upon exposure to heat or dimethylsulfoxide (Figs. 5 and 8). Studies of the thermal denaturations of these molecules (Figs. 6, 7 and 9) indicate the existence of a single class of 28S rRNA, but three classes of 38S pre-rRNAs. The addition of -ecdysone to the in vitro culture medium stimulates the net amount of rRNA synthesized, increases the rate of processing of the 38S precursor and increases the relative amount of 18S material produced (Figs. 10 and 12).This work was supported in part by grants from the National Science Foundation (GB-8176) and from the Atomic Energy Commission (AT-04-3-34).Predoctoral Trainees, PHS Training Grant No. 2-Tl-GM367 from Research Training Grants Branch, National Institute of General Medical Sciences.1 For purposes of simplification we shall refer to the rRNA molecules of D. melanogaster as being 38S, 30S, 28S and 18S; however, it should be noted that these values are approximate (see Hastings and Kirby, 1966; Greenberg, 1969; Tartof and Perry, 1970).     

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.