Figure 4. Epithelial affinity.

(A) Explants of deep IMZ plus NIMZ tissue were made prior to the onset of gastrulation and stabilized by adhering them to fibronectin-coated cover glass below the explant; strips of AC epithelium large enough to span the entire animal-vegetal extent of the deep tissue were subsequently grafted onto its surface and kept in place with cover glass above the explant. (B) In the examples shown (lower IMZ below magenta dashed line, upper IMZ between magenta and green, NIMZ & AC above green dashed line; boundaries between the three regions identified by cell, motility, size and gray cast), the movie (Video 11*, where these events can be most easily seen) begins 10 min prior to the onset of gastrulation (G-10m). The epithelium adhered and spread over all regions (G + 20 m), after which the upper cover glass was removed. Immediately afterward, epithelium over both NIMZ and IMZ regions contracted slightly, then that over the NIMZ re-spread, while that over the IMZ contracted more strongly and continuously, often to the point of becoming detached from the deep tissue, and in many cases retracted from the lower (vegetal) edge of the deep tissue (G + 2 h). Tissue over the IMZ remained contracted, while that over the NIMZ tended to spread (G + 4.5 h). (C) The relative area of spreading or contraction of the epithelial tissue over different deep tissue regions over time was quantitated, beginning after cover glass removal (each time point based on measures from 2 to 8 explants at sequential time points; error bars = SEM). (D) In a variation on this assay, explants of deep ventral IMZ plus NIMZ tissue were cultured on BSA coated cover glass (below) and patches of epithelium were lapped across the NIMZ/IMZ boundary and held apposed to the deep tissue with an agarose coated cover glass (above) throughout the experiment. In the example (E), the epithelium initially over the deep IMZ (below green line) first contracts animally (G + 2 h), eventually retracting across the boundary (G + 4 h), then respreads on the deep NIMZ (G + 6 h) (Video 12*, where these events can be most easily seen). During this time, upper deep IMZ cells (mesoderm) spread over deep NIMZ cells (ectoderm), such that the NIMZ/IMZ boundary on the surface of the deep tissue is shifted from its original position (dashed orange line). The deep NIMZ/IMZ boundary is based on differential cell size and motility, which are evident in the movie.