Surface contour, contact relations, and cortical structure of marginal cells of the enveloping layer (EVL) and of the yolk syncytial layer (YSL) of Fundulus heteroclitus were studied before and during epiboly with transmission and scanning electron microscopy. The contacts of the marginal cells of the EVL with the underlying YSL involve only the most marginal part of each cell and consist of a mixture of tight and close junctions apically and wider appositions more proximally. This junctional complex is very extensive (2.1–2.4 μm) prior to the onset of epiboly, very restricted (0.5–0.8 μm) during early epiboly up to a mid-gastrula, more extensive again (0.8–1.0 μm) at late midgastrula, and still more extensive (2.0–2.3 μm) at late gastrula toward the end of epiboly. At this time, the margin of each marginal cell is embedded in the YSL. Several lines of evidence suggest that these marginal contacts are stable and therefore that epiboly of the EVL occurs passively, in response to pull exerted by the independently expanding YSL.
As the external YSL (E-YSL) narrows during the earliest phase of epiboly, its surface becomes more and more convoluted. Since the network of 4–6 nm microfilaments in the cortical cytoplasm of the E-YSL thickens with increasing convolution of its surface, it seems possible that a contractile force resides in the E-YSL cortex which simultaneously throws the surface of the E-YSL into folds, narrows the E-YSL, and exerts tension on the attached margin of the EVL. Networks of thin microfilaments are also found in the cortex of EVL cells, especially in the leading edge of the marginal cells (as in fibroblasts and epithelial cells in vitro) and in the cortex of the yolk cytoplasmic layer (YCL), where they are presumably responsible for the contractile tension of this layer; 10-nm microfilaments are also present but have a different distribution. They are arranged in bundles in both the marginal cytoplasm of each marginal cell and in the YSL beneath the marginal contact, running parallel to the contact and circumferentially relative to the whole egg. This arrangement in this location coincides with constriction of the egg in this marginal region and suggests that these thick filaments might provide the contractile force for the constriction, along with the thin filaments with which they are associated. The morphological relationship between the E-YSL and the YCL during epiboly is also described.
SEM reveals that the surface of the internal YSL (I-YSL) is covered with long microvilli at the beginning of gastrulation and that they disappear and are replaced by shorter microvilli as epiboly progresses. Estimation of the amount of surface in the long microvilli at the beginning of epiboly indicates that there is enough membrane on the surface of the I-YSL at this time to account for epibolic expansion up to a late gastrula. Although it is not known how the surface membrane in these microvilli might be redistributed, the presence within them of abundant microfilaments that appear to insert in the plasma membrane suggests that this process might be accomplished by these presumed contractile elements.
Finally, measurements of cell surface expansion and calculations of cell number show that the number of cells in the EVL remains approximately constant during epiboly. Clearly, cell division is not a factor in the epibolic expansion of the enveloping layer. Instead, there is a marked thinning of individual cells.