Fig. 2. A molecular map of midline cells during germband elongation. (A) Engrailed is expressed in two phases at the midline. Embryos expressing tau-GFP driven by engrailed-GAL4 were stained with anti-GFP (GFP, brown) and anti-Engrailed (En, blue). Midline cells that express Engrailed from gastrulation (early Engrailed) can be stained with anti-GFP and anti-Engrailed (dark brown cells). Midline cells that initiate Engrailed expression at stage 10 (late Engrailed) can be stained with anti-Engrailed but not yet with anti-GFP (blue cells, arrow). (B) Gooseberry-distal (Gsb-D, dark purple) expression coincides with midline cells expressing early Engrailed (dark brown, arrow), but not late Engrailed (light brown). (C) At stage 10, midline cells located directly posterior to the Engrailed domain (brown) express Lethal of scute (black, arrow) but not Engrailed. (D) About one hour later, all Lethal of scute-positive cells also express Engrailed (dark brown, arrow). The two midline cells comprising the early phase of Engrailed expression (arrowhead) continue to express Engrailed only (light brown). (E) Lethal of scute-positive midline cells express the Hedgehog receptor Patched. Expression of ß-galactosidase (red) from the patched promoter (ptc-lacZ) is very high in Lethal of scute (green)-positive midline cells (yellow cells, arrow). (F) Patched (Ptc) expression (purple) is upregulated in the anterior midline cells, labelled by the early expression of Odd (brown). Due to repression of Patched by Engrailed, the early Engrailed domain is visible as a white stripe. (G) Using Engrailed as a reference, the anteroposterior position of midline subsets (see Fig. 1) is superimposed on the expression map. Segmentation of the ectoderm is shown by Engrailed and Hedgehog expression. Engrailed and Odd have two distinct phases of midline expression. Parasegmental (arrowhead) and segmental borders (arrow) are indicated. Because MP1 interneurons and midline glia have a very similar anteroposterior position, it is also possible that MP1 interneurons are the most anterior cells in every segment. Ventral views, anterior to the left; brackets outline the midline.