Estimation of Cell Sizes in Normal ! / l ~ ld-type Embryos

In Volvox carteri development, visibly asymmetric cleavage divisions set apart large embryonic cells that will become asexual reproductive cells (gonidia) from smaller cells that will produce terminally differentiated somatic cells. Three mechanisms have been proposed to explain how asymmetric division leads to cell specification in Volvox: (a) by a direct effect of cell size (or a property derived from it) on cell specification, (b) by segregation of a cytoplasmic factor resembling germ plasm into large cells, and (c) by a combined effect of differences in cytoplasmic quality and cytoplasmic quantity. In this study a variety of V. carteri embryos with genetically and experimentally altered patterns of development were examined in an attempt to distinguish among these hypotheses. No evidence was found for regionally specialized cytoplasm that is essential for gonidial specification. In all cases studied, cells with a diameter > ,x,8 #m at the end of cleavage-no matter where or how these cells had been produced in the embryo-developed as gonidia. Instructive observations in this regard were obtained by three different experimental interventions. (a) When heat shock was used to interrupt cleavage prematurely, so that presumptive somatic cells were left much larger than they normally would be at the end of cleavage, most cells differentiated as gonidia. This result was obtained both with wild-type embryos that had already divided asymmetrically (and should have segregated any cytoplasmic determinants involved in cell specification) and with embryos of a mutant that normally produces only somatic cells. (b) When individual wild-type blastomeres were isolated at the 16-cell stage, both the anterior blastomeres that normally produce two gonidia each and the posterior blastomeres that normally produce no gonidia underwent modified cleavage patterns and each produced an average of one large cell that developed as a gonidium. (c) When large cells were created microsurgically in a region of the embryo that normally makes only somatic cells, these large cells became gonidia. These data argue strongly for a central role of cell size in germ/soma specification in Volvox carted, but leave open the question of how differences in cell size are actually transduced into differences in gene expression. V ISIBLY asymmetric cell division is associated with cellular differentiation in organisms as diverse as bacteria (Stragier, 1991), budding yeasts (Nasmyth and Shore, 1987), higher plants (Raven et al., 1986), and animals (Davidson, 1986). In animal embryos, visibly asymmetric cleavage divisions are frequently accompanied by segregation of demonstrably different cytoplasmic components to sister blastomeres (reviewed in Davidson, 1986). In at least some of these cases it can be demonstrated experimentally that it is the difference in cytoplasmic quality of the large and small cells produced by asymmetric division-and not their difference in size-that is the cause of their differential specification (for example see Henry, 1986). Therefore, even though there are many studies indicating that cells of many kinds do monitor their size and regulate their behavior in a size-dependent way (Fankhauser, 1945; Prescott, 1956; Johnston et al., 1977; Newport and Kirschner, 1982; Edgar et al., 1986; Hayles and Nurse, 1986; Cossins, 1991; Henery, et al., 1992), it is frequently assumed that the visible difference in size between the products of an unequal division is probably accompanied by some invisible qualitative difference that is the actual cause of their divergent development (Horvitz and Herskowitz, 1992). Recent evidence indicates, however, that size differences may act rather directly to trigger gene expression differences. In the bacterium Bacillus subtilis, for example, the first visible step in spore formation is an asymmetric division that produces a large cell destined to become a spore mother cell and a small cell destined to become a spore. After asymmetric division, a spore-specific sigma factor that was uniformly distributed before division is selectively activated in the small cell; it then initiates a cascade of differential gene expression that causes sister cells to develop entirely different phenotypes. It has now been postulated that this key © The Rockefeller University Press, 0021-9525/93/10/191/18 $2.00 The Journal of Ceil Biology, Volume 123, Number 1, October 1993 191-208 191 on Jne 5, 2017 D ow nladed fom Published October 1, 1993