Fig. 1. Morphological changes during segmental boundary formation. (A) Schematic drawing of gene expression patterns in a horizontal section through one segmental unit. The position of the segmental boundary is marked with a vertical bar. (B) Schematic drawing of the fusion protein used to label cell outlines under the EM (under UAS control). It comprises the signal peptide from Wingless, human CD2 (without signal peptide) and HRP. (C-K) TEM images showing the changes in cell morphology as segmental grooves form and regress. Embryos were stained with DAB and sectioned horizontally through the ventral aspect of the embryo. Although some staining appears at the surface of non-expressing cells (maybe as a result of membrane shedding from expressing cells), we were able to confidently identify expressing cells after a bit of practice. An annotated version of this figure highlighting expressing cells is provided at http://dev.biologists.org/supplemental/. (C) Shortly after germ-band retraction is initiated, a small dip (arrow) appears between engrailed-expressing and non-expressing cells. (D) Apical contact appears to loosen (arrow). (E) The most posterior engrailed-positive cell constricts apically and moves inwards in relation to surrounding cells. (F) This cell finds itself at the bottom of the forming groove and neighbouring cells follow this inward motion. (G) More cells have moved in and the groove is now two to three cell diameters deep. (H) The groove at its deepest reaches at least three cell diameters in depth. (I) At this stage the bottom engrailed-expressing cell is bottle shaped and severely constricted apically (arrow). (J) The disappearance of grooves is a very rapid event, which allows the cells to return to their original position. (K) The embryo eventually becomes almost flat ventrally. (L-N) Grooves are much deeper laterally (L) than ventrally (M), and posterior grooves (between abdominal segments 2 and 3; N) are not as deep as anterior ones (between abdominal segments 7 and 8; M). Scale bars: 500 nm.