Fig. 3. The spatial pattern and subcellular distribution of the Yellow protein is temporally dynamic. The final distribution of Yellow in late pupal stages correlates with the location and intensity of black melanin in the adult. (A) Immunohistochemical staining with the anti-Yellow antibody does not recognize any proteins in a yellow mutant. Abdominal segments A3 and A4 from a pupa 72 hours after puparium formation (APF) are shown. (B) Wild-type (CantonS) pupa approximately the same age as in A. In the A3 segment, Yellow protein is present almost exclusively in the cells that secrete the pigment in the stripe (bracket). Yellow is expressed in this pattern in all segments at later developmental stages. In the A4 segment, Yellow protein is present in cells that underlie the future pigment stripe (bracket), as well as in more anterior cells that produce significantly less black melanin. During earlier pupal stages, the distribution of Yellow in all segments resembles the A4 segment shown. The change in the spatial distribution of Yellow protein occurs first in A2 and progresses posteriorly to A6. The pupa shown in B has undergone this refinement in A3, but not yet in A4. (C) In the thorax, at approximately 80 hours APF, Yellow protein is present in cells that produce the thoracic pigment pattern in ebony mutants (arrowhead; see Fig.1C). Additionally, Yellow is expressed in a cell associated with each mechanosensory bristle (arrows). (D) Expression of UAS-GFP (green) shows that the pannier-Gal4 driver is expressed in dorsal cells along the length of the fly. (E-I) Co-expression of UAS-Yellow and UAS-Ebony activated by pannier-Gal4. (E-H) Ebony (green) is present in all cells within the pannier-Gal4 expression domain, whereas, Yellow protein (red) is only present in a subset of these cells. (E,F) Abdominal segments A3 and A4 are shown with the dorsal midline at the left edge, and the lateral midline at the right edge. Arrowhead indicates the edge of the pannier-Gal4 expression. Endogenous Yellow protein underlying the pigment stripes (brackets) and endogenous Ebony expression (arrow) are also detected. (G,H) Initially, ectopic Yellow is present in the cytoplasm of a subset of cells in which it is transcribed. The presence of ectopic Ebony protein indicates transcriptional activation of UAS by pannier-Gal4. TOPRO staining (blue) shows the location of both epidermal nuclei and the larger bristle cell nuclei. Later in development, cytoplasmic expression of Yellow forms foci within the cell (data not shown) which are subsequently exported and evenly distributed among neighboring cells (I). The transition of the Yellow protein from cytoplasmic to extracellular occurs in an anterior-to-posterior wave, similar to the change in spatial expression pattern. In the A4 segment shown in F, Yellow expression is still predominantly cytoplasmic near the posterior of the segment, but becomes diffuse foci in the more anterior cells and in the A3 segment. (J) An optical cross section shows that after it is exported, Yellow protein (red) becomes evenly distributed above the apical side of epidermal cells that directly underlie the developing cuticle (arrowhead). TOPRO staining (blue) and Ebony expression (green) show the nuclear and cytoplasmic boundaries, respectively. Apical is towards the left. Scale bars: in A-F, 100 µm.
