Mechanism of closure of experimental excision-wounds in the bare upper layer of the chick blastoderm.

The closure of experimental excision-wounds in the upper layer of the gastrulating chick blastoderm was studied by time-lapse videography and videomicrography and by light microscopy, transmission electron microscopy and scanning electron microscopy. One experimental excision-wound was made in the upper layer of stages 4V to 6V blastoderms (Vakaet, Arch. Biol. (Liège) 81:387-426, 1970), in the proamnion where no middle layer cells are present. The deep layer was previously discarded, so that the wounds were made in the bare upper layer. They closed within 2 to 6 hours and further development was normal by in vitro standards. With videography, global movements of the upper layer towards the wound were observed. With videomicrography, the wound submarginal region cells were seen to move like sheep in a flock: individual cells in different directions, the whole flock towards the wound. During closure the shape of the wound edge was irregular. The structure of the epithelium of the wound submarginal region was unchanged throughout closure: a pseudostratified columnar epithelium in which cell divisions occur at its dorsal side and are parallel to its surface. The basal lamina was absent below the edge of the wounds. We propose that the cells of the upper layer are mobile against one another and are not confined to a specific part of the basal lamina. During wound closure the movements of the cells on the basal lamina would be driven by mitotic pressure. This is the horizontal pressure exerted by the addition of daughter cells and their parting during anaphase and telophase.(ABSTRACT TRUNCATED AT 250 WORDS)

Fate mapping the neural plate and the intraembryonic mesoblast in the upper layer of the chicken blastoderm with xenografting and time-lapse videography.

The disposition of the Anlage fields of the neural plate and the intraembryonic mesoblast in the upper layer of the chicken blastoderm was studied at the primitive streak stage prior to the regression of Hensen's node (stages 5V to 6V, L. Vakaet (1970) Arch. Biol. 81, 387-426). Chicken blastoderms were cultured by New's technique on a mixture of thin egg white and agar. The anterior half of the deep layer was reflected with a tungsten needle. A circular fragment of the upper layer was punched out with a pulled out Pasteur pipette and discarded. It was replaced with an isotopic and isopolar piece of quail upper layer that was punched out with the same pipette. The deep layer was replaced and the chimeras were reincubated for 24 hours. The xenografts were followed with time-lapse videography. After fixation, the quail cells were located using Le Douarin's quail nucleolar marker technique. Integrating the observations with time-lapse videography and the results of Feulgen stained sections, we have drawn a new fate map of the disposition of the Anlage fields in the upper layer of the chicken blastoderm at stages prior to the regression of Hensen's node (stages 5V to 6V). The disposition of the neural plate and of the notochord, somites, nephrotome and lateral plates was therefore determined before the Anlage fields are morphologically discernible. The pathway of the fields in the upper layer towards their disposition was documented with time-lapse videography in chimeric chicken blastoderms that developed normally.

The effects of partial hypoblast removal on the cell morphology of the epiblast in the chick blastoderm.

The hypoblast of early-primitive-streak-stage chick blastoderms was partially removed. This experiment provokes a reaction in the epiblast which curls up and becomes even at its ventral surface. The basal lamina underlying the epiblast is also dependent upon the presence of hypoblast. During culture after partial hypoblast removal, active hypoblast wound healing is observed. Where the hypoblast underlies the epiblast again, the effects of the removal disappear and normal development proceeds. The results suggest that the normal epiblast morphology is dependent upon the presence of hypoblast. This influence of hypoblast on epiblast is thought to be concerned with the morphology of the epiblast and not directly with its morphogenesis.

Wound healing in the primitive deep layer of the young chick blastoderm.

Wound healing in the primitive deep layer of stage 4 chick blastoderms was studied in vitro by cinemicrophotography of living cultures and by photomicroscopy, scanning- and transmission electron microscopy after fixation. Experimental wounds with an average diameter of 0.3 mm healed completely within 2 to 4 h through migration of the cells at their rims. Healing occurred in mesenchyme-free areas, providing us with a purely epithelial reaction. The rim cells of the primitive deep layer formed extensions at their free flank, described as fila, filopodia, lamellae and lamellipodia. They were already present in blastoderms fixed at the earliest after the intervention. This reaction was ascribed to the elimination of a normal fellow cell at the side of the rim cell facing the defect. Movement of the rim cell ceased upon meeting another cell with the same polarity. At this moment lamellipodia disappeared as suddenly as they had formed, and the number of fila and filopodia decreased. We believe that the chick blastoderm's primitive deep layer might be appropriate for analysis of the factors governing primary epithelial wound healing.

Cell size and mutual cell adhesion. I. Increase in mutual adhesivenes of HeLa cells from density-inhibited suspension cultures by hypotonic treatment.

HeLa cells harvested from density-inhibited or fast growing suspension cultures, were incubated in NaCl solutions of different tonicity. Cell size enlargement produced by hypotonicity is accompanied by an increased sedimentation rate of the density-inhibited cells, whereas no appreciable change is observed in the sedimentation rate of fast growing cells. Hypotonicity also has no effect on the sedimentation rate of density-inhibited cells which previously had been treated with neuraminidase or trypsin. It is shown that the effect of hypotonicity on density-inhibited cells cannot be ascribed to release of cell surface sialic acids during hypotonic incubation. Several arguments are presented which indicate that the changes in sedimentation rate, as measured in the rotating suspension system, are not the direct consequence of the alterations in cell size, but rather must be attributed to differences in intercellular adhesiveness resulting from the size alterations. Analogous changes in intercellular adhesiveness and cell size are shown to occur during growth in isotonic suspension culture. The results can be explained by assuming that changes in cell size affect the intercellular adhesiveness by modifying the extent to which cell surface sialic acids counteract adhesion.

Cell size and mutual cell adhesion. II. Evidence for a relation between cell size, long-range electrostatic repulsion and intercellular adhesiveness during density-regulated growth in suspension.

The strength of the long-range electrostatic repulsion forces on HeLa cells is measured by agglutinative titration using low molecular weight polylysine (M.W. 11,000). Repulsion forces, found to be present on the smaller HeLa cells from density-inhibited suspension cultures, are weakened by incubation of the cells in hypotonic NaCl solutions. Repulsion forces, found to be absent on the larger cells from fast growing cultures, can be induced on these cells by incubation in hypertonic NaCl solutions. Both effects of anisotonicity are reversible, and disappear on restoration of the medium to normal tonicity. Induction of repulsion forces on fast growing cells is prevented by previous treatment of the cells with neuraminidase. Neuraminidase also abolishes repulsion on density-inhibited cells. It is proposed that alterations of the cell size, produced by anisotonicity or occurring during growth in isotonic suspension medium, affect mutual cell adhesiveness by modifying the strength of the repulsion forces generated by cell surface sialic acids.