Fig. 3. dpp expression, neuronal determination and precluster formation in hnt mutant eye discs. (A-D) Expression of a dpp-lacZ reporter in wild-type (A) and hnt mutant (B-D) eye tissue. (A) In a late third instar disc from a dpp-lacZ/+ larva, reporter expression occurs along the furrow and is abruptly downregulated posterior to the furrow. The arrow marks the posterior border of the furrow. The reporter was visualized by X-Gal staining. (B) A disc with a hnt^XE81 clone visualized by the lack of -HNT antibody staining (brown) behind the furrow. The dpp-lacZ reporter expression, visualized by X-Gal staining, looks normal. The same result was obtained with hnt^EH704a clones (not shown). (C) A disc from a hnt^peb/Y larva raised at the restrictive temperature, showing that the dpp-reporter expression is no longer tightly downregulated at the posterior edge of the furrow but persists in some of the emerging clusters in the eye field (reporter was visualized by X-Gal staining). (D) A disc with a hnt^XO01 clone, showing that the dpp-reporter expression (blue-black staining), again persists posterior to the furrow, when it would normally be shut off. In this case, reporter expression was visualized with -ß-gal antibody. (E) Eye disc containing a hnt^XE81 clone visualized by lack of immunostaining with -HNT antibody (HNT-expressing cells are green). -HRP staining (red) labels clusters of neuronally determined R cell precursors. Within the hnt tissue, the neuronal determination in the clusters is delayed relative to the adjacent wild-type clusters. The white arrowheads mark the first neuronal cells in the mutant patch versus in the surrounding wild-type tissue; note that the arrowhead is two rows closer to the furrow in the wild-type tissue. (F) Eye disc containing a hnt^XE81 clone visualized by lack of immunostaining with -HNT antibody (HNT-expressing cells are red). The outlines of emerging R cell clusters are highlighted using -PY antibody (green). Inside the mutant patch, the arc stage of development looks normal (arrow), but defects in cell arrangement and number can be observed in the preclusters (arrowheads). (G) Eye disc from a wild-type larva stained with the -Glass antibody, which labels all of the presumptive R cells. Cells of the definitive precluster are labeled with red arrowheads. (H) Eye disc from a hnt^peb larva, raised at the restrictive temperature and stained with the -Glass antibody. Many of the preclusters (yellow arrowheads) have defects, including missing or extra Glass-positive cells and displaced nuclei within a cluster. Red arrowheads point to preclusters with a normal complement of cells.