Epithelial morphogenesis in developing Artemia: The role of cell replication, cell shape change, and the cytoskeleton

Abstract

The roles of cell replication and shape change as morphogenetic forces in epithelial invagination were examined in instar II Artemia. The epidermal cells underwent a fixed pattern of cell division during the first 5 hr of instar II. Greater cell replication in the thoracopod bud (ThB) than in the arthrodial membrane (AM) region resulted in a higher density of epidermal cells in the ThB region (differential cell density). The ratio of cell density ( AM ThB ) declined from 1.0 to less than 0.80 by Hour 2 of instar II. Invagination of the AM occurred during Hour 4 when the AM ThB reached 0.75. A 2-hr pulse with 5′-fluorodeoxyuridine (FudR) during instar I delayed completion of the cell replication pattern and development of transverse cell files in the ThB region for a period equal to the length of the exposure. The delay in the cell division program resulted in a cell density ratio of 0.93 at Hour 4, a value normally observed in Hour 2 larvae, and evagination of the epidermis did not occur at apolysis (Hour 4). The FudR treatment did not perturb the cytoskeleton or the initial steps in cell shape change and the larvae formed small segments during instar III. Cell shape change within the AM began during Hour 4 as this region became significantly thinner than the neighboring ThB region (thickness ratio, AM ThB = 0.77 ). Before apolysis the AM cells became wedge shaped, a change which occurred when the basal region of the cell enlarged. The microtubules and microfilaments were reorganized from the apical cytoplasm to the lateral border of apposing AM cells. Following apolysis (Late Hour 4) shape change was completed as the cells attained a thin spindle form, with microtubule- and microfilament-rich filopodial extensions which overlapped adjacent AM cells. As contact with ThB cells shifted from lateral to apicolateral, the AM cells formed the innermost edge of the invagination. Microtubules in the differentiating AM cells contained tyrosinated, detyrosinated, and acetylated α-tubulin isoforms. Treatment with nocodazole, colchicine, taxol, or cytochalasin B blocked AM cell shape change and inhibited segmentation, but did not affect the mitotic pattern or differential cell density. We conclude that the specific pattern of cell division led to differential cell density which, along with AM cell shape change, established the conditions necessary to achieve epidermal evagination.