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Reciprocal functions of the Drosophila Yellow and Ebony proteins in the development and evolution of pigment patterns

Patricia J. Wittkopp^*, John R. True^*, and Sean B. Carroll

Howard Hughes Medical Institute, Laboratory of Molecular Biology, University of Wisconsin, 1525 Linden Drive, Madison, WI 53706, USA
^* These authors contributed equally to this work
Present address: Department of Ecology and Evolution, State University of New York at Stony Brook, Stony Brook, NY 11794-5245, USA

View larger version (103K): [in a new window]   Fig. 1. yellow and ebony are required for proper pigment patterning in D. melanogaster. (A) Wild type D. melanogaster females produce a stripe of dark pigment (arrowhead) near the posterior edge of abdominal segments A2-A6. (B) In yellow null mutants, black melanin is lost within the stripe, leaving a brown pigment, and cuticle anterior to the stripe has a tan appearance relative to wild type. (C) ebony1 mutants retain a distinct pigment stripe and the cuticle anterior to the stripe is much darker than wild type. (D) yellow; ebony1 double mutants have brown pigment throughout the abdomen and the stripe is no longer distinct. Similar changes in pigmentation are observed in other structures. Wild-type flies are a uniform color in the thorax (E) and wings (I). In yellow mutants, these structures become tan (F,J). In ebony1 mutants, the thorax (G) and wings (K) are more darkly pigmented, and new pigment patterns are visible (arrowheads). yellow; ebony double mutants also show these patterns, but the black pigment is absent, and they consist of two shades of brown pigment (H,L, arrowheads). In all panels, anterior is upwards and dorsal cuticle is shown. Abdominal segments A3-A5 are shown in A-D.

View larger version (38K): [in a new window]   Fig. 2. Antibodies specific for the Drosophila Yellow and Ebony proteins. Western blots of 60-72 hour pupal protein extracts. Antibodies to the Yellow protein produced in rabbits (A) and rats (B) recognize a 60 kDa protein in protein extracts from animals with a wild-type y gene (CantonS, eAFA), but not from y mutants (y170, yw, ywac). Both antibodies also recognize a similar size protein in D. biarmipes. (C) Antibodies raised in rabbits against the Ebony protein recognize a 94 kDa protein (arrowheads) in wild-type animals (CantonS), but not e mutants (e1, eAFA). Several smaller proteins in both wild-type and e mutant flies are also detected (data not shown). A protein of approximately 94 kDa is recognized in extracts from D. biarmipes. A strong band just below the presumptive Ebony band also appears in both wild-type and e mutant extracts. (A-C) The panel below each blot indicates relative protein loading.

View larger version (134K): [in a new window]   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.

View larger version (53K): [in a new window]   Fig. 4. Ebony protein is expressed widely and does not correlate with a single pigment. (A) Immunohistochemical staining of eAFA with the Ebony antibody does not detect any staining at approximately 72 hours APF. In pharate adults, a weak signal is produced in epidermal cells (data not shown). (B) From approximately 72 to 90 hours APF, Ebony protein (green) is expressed in cells associated with mechanosensory bristles (arrow), but not in epidermal cells (arrowhead). This expression may not function in pigmentation because e mutants have wild-type bristle color. (C) Beginning at approximately 90 hours APF, low levels of Ebony protein (green) are present in all epidermal cells of each abdominal segment. Bracket indicates the future location of the pigment stripe and staining of cells near the top of the panel is in a different focal plane. (D) In the thorax, highest levels of Ebony expression are in epidermal cells that produce the ‘trident’ in e mutants (arrow). The strong staining seen near the top of the panel is background signal from underlying tissues.

View larger version (89K): [in a new window]   Fig. 5. Changing Yellow and Ebony expression is sufficient to alter pigment patterns. (A) Ectopic expression of Yellow results in a subtle increase of black pigment in the thorax relative to wild type. (B) In the abdomen, this expression causes a slight widening of the pigment stripe (arrowhead indicates the border between wild-type and ectopic pigmentation), as well as the dorsal midline pigment (arrow). (C) Ectopic expression of Ebony in the same cells results in a tanning of the thorax and a removal of melanin in the abdominal stripe (D). (E) In ebony1 mutants, ectopic expression of Yellow induces black pigment in the thorax (arrow) and the abdomen (F). (G,H) Co-expression of Yellow and Ebony results in a phenotype more similar to wild type than does ectopic expression of either protein alone. (B,D,F,H) Brackets indicate the approximate boundaries of pannier-Gal4 expression.

View larger version (64K): [in a new window]   Fig. 6. Complementary patterns of Yellow and Ebony expression correlate with the formation of a novel, male-specific, black melanin pattern in D. biarmipes wings. (A) A spot of black melanin (arrow) is present in the wings of D. biarmipes males. (B,D) Yellow protein (purple) is expressed at higher levels in the cells that produce this spot (arrow) than in the surrounding wing. (C,D) Ebony expression (green) is lower in these cells (arrow) than in the rest of the wing. (E-H) The boundaries between expression levels of Yellow and Ebony coincide (F-H, arrows) and correlate with the boundary of pigment in adult wings (E, arrow). (I) D. biarmipes females, typically do not produce a pigment spot in the wing, and both Yellow (J,L) and Ebony (K,L) proteins are uniform throughout the wing in most females. In some adult females, a small, faint pigment spot is observed (data not shown). Consistent with this phenotypic variation, a few cells expressing higher levels of Yellow are present in a small proportion of the female pupal wings (data not shown).

View larger version (28K): [in a new window]   Fig. 7. Proposed biochemical and molecular mechanisms for pigment synthesis and patterning. (A) Dopa-melanin, dopamine-melanin and NBAD scelerotin are products of discrete branches of a common biochemical pathway. Yellow, Tan and Ebony proteins are rate-limiting enzymatic steps in the formation of black, brown and tan pigments, respectively. The ‘?’ indicates the activity of an unknown gene that regulates pigment patterns in the absence of tan (P. J. W., unpublished). (B) Development of the abdominal pigment stripe in D. melanogaster requires spatial regulation of Yellow, Ebony and Tan. Boxes represent a section of abdominal tergite as shown in the far right panel. Yellow expression is gray, Ebony expression is yellow and our prediction of Tan expression is brown. The combined action of these patterns is to induce the formation of black melanin in the stripe where Yellow is expressed, tan pigment anterior to the stripe where Ebony is expressed alone, and brown melanin in the stripe where both Ebony and Tan are present. The combination of black and brown melanins produces the final appearance of the stripe.