Experiments concerning the cleavage stimulus in sand dollar eggs.

It is now clear that the mitotic figure of dividing echinoderm eggs plays a causal role in the determination of the furrow. Since Hiramoto ('56) showed that removal of the mitotic apparatus before appearance of the furrow did not prevent division, it is convenient to consider the apparatus the source of a stimulus which is the immediate trigger for cytokinesis. Neither the area of the cell which receives the stimulus nor the mechanism of division is known. Information concerning one aids in the understanding of the other. Detailed information concerning the particular areas of the mitotic apparatus necessaiy for the establishment of the furrow is highly desirable. Such information could be derived from study of healthy cells in which some part of the apparatus had been deleted. Since direct manipulation or destruction of a portion of the mitotic appartus may result in general cell damage, it was decided to attempt the creation of artificial, incomplete mitotic figures by bringing together regions of the two mitotic figures in a dividing, binucleate sand dollar egg. The spherical egg was distorted to a torus or doughnut shape by pressing a glass sphere through it. In the first division, the cleavage plane severed the cell in the region between the asters on one side of the torus. The cell thus became horseshoeshaped and binucleate. In subsequent divisions, as cells divided from the free ends of the horseshoe, the asters in the binucleate cell were brought together. There ensued a cleavage furrow which cut between the polar regions of the two separate mitotic figures in an area which had never been in close proximity to a spindle or chromosomes. Furrows of this kind could be elicited and closely observed between asters of the second, third and fourth divisions. The furrows were morphologically normal and appeared in concert with the division of other cells of the embryo. The modification of cell geometery which was employed to produce binucleate cells also yielded additional information bearing upon the source and nature of the stimulus. In two recent hypotheses (Swann and Mitchison, '58; Wolpert, '60) concerning the mechanism of cytokinesis it has been proposed that the telophase nuclei or asters are the sources of the immediate stimulus for cytokinetic activity. These structures are considered the origin of a substance which modifies the poles of the cell but does not reach the part of the cortex that will become the walls and head of the furrow. The proper operation of this mechanism requires sharp diffusion gradients. The form of the furrow would also be altered concomitantly with the distance between the sources. In some of the experiments mentioned above, it was noted that an apparently normal furrow could be established between asters separated by a greater than normal distance. The need €or further investigation of the capacity of echinoderm eggs to divide under conditions that would make difficult the establishment of sharp diffusion gradients was indicated. To this end, cells were kneaded by repeated compression or extrusion during the period when the cleavage furrow is determined. This treatment did not prevent temporally and morphologically normal division.