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First published online 15 March 2006
doi: 10.1242/dev.02334

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smoothened and thickveins regulate Moleskin/Importin 7-mediated MAP kinase signaling in the developing Drosophila eye

Alysia D. Vrailas¹, Daniel R. Marenda¹, Summer E. Cook¹, Maureen A. Powers¹, James A. Lorenzen^2,3, Lizabeth A. Perkins² and Kevin Moses^1,*,

¹ Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA.
² Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Harvard Medical School Boston, MA 02114, USA.
³ Department of Pediatric Gastroenterology, Medical College of Wisconsin, Milwaukee, WI 53226, USA.

View larger version (105K): [in a new window]   Fig. 1. Three phases in eye development. Third instar eye-imaginal disc, stained for Atonal (green) and Elav (red) to show the positions of phases 0, 1 and 2, as indicated (see text). Anterior is to the right.

View larger version (136K): [in a new window]   Fig. 2. Cell proliferation and survival requires msk in the developing eye. (A-L) Third-instar (A-I) and 60-hour pupal (J-L) eye-imaginal discs containing clones of msk null cells, revealed by negative marking using GFP (D,G,J); outlined in (D-I). Anterior is to the right; A-C and D-L are the same scale as indicated in A and D, respectively. In E,H,K, antigens are indicated on the left; F,I and L are merged images showing GFP (green) and antigens (red); A-C shows a time course following the hsp70-driven induction of clones. Brightly labeled cells are homozygous wild-type `twin-spots' (white filled arrows), gray cells are heterozygous and black cells are homozygous msk nulls (black filled arrows). Note, late clones (48 hours before dissection in A) are numerous, small, equally distributed on both sides of the furrow, and are accompanied by twin spots of roughly equal size; earlier clones (72 hours before dissection in B) are rare, smaller than their twin spots, and are present only posterior to the furrow. Note also that very early clones (96 hours before dissection in C) are absent, with only rare, large twin spots remaining. Cell-cycle markers (BrdU for S-phase (D-F) and Cyclin E for G1 (G-I) are not eliminated by msk loss of function (arrows). Note, msk null cells posterior to the furrow eventually die during pupal life, as revealed by activated Caspase 3 antigen (arrows in J-L).

View larger version (91K): [in a new window]   Fig. 3. msk is not required for cell-type specification in the developing eye but does contribute to ommatidial rotation. (A,D,G,J,M,P) Third-instar eye-imaginal discs containing clones of msk null cells, revealed by negative marking using GFP. (B,E,H,K,N,Q) Antigens visualized by the immunostain indicated at the left. (C,F,I,L,O,R) Merged images, with GFP in green and antigens in red (Elav, blue in R). Anterior is to the right; all images are at the same scale, see bar in P. (A-C) msk loss-of-function does not affect the neural marker Elav (arrows). (D-F) Senseless expression in R8 cells (arrow). (G-I) Spalt expression in R3 and R4 (arrows). (J-L) Cut expression in the cone cells (arrow). (M-R) msk loss-of-function affects ommatidial rotation (M-O), as revealed by BarH1 antigen labelling of R1 and R6 (normal orientation is indicated by an arrowhead and reversed by an arrow), but not chirality (P-R), as revealed by E(Spl)M-delta-0.5:lacZ expression in R4 cells (reversed ommatidia have reversed a R3/4 position, arrow). Note, occasionally two cells are specified as R4 in msk clones (arrowhead in P-R).

View larger version (128K): [in a new window]   Fig. 4. Ectopic Msk affects late eye development. All panels show anterior right, dorsal up; genotypes and temperatures are indicated above panels. (A,D,G,J) GMR:Gal4, raised at 18°C; (B,E,H,K) GMR:Gal4 UAS:Msk, raised at 18°C; (C,F,I,L) GMR:Gal4 UAS:Msk, raised at 25°C. (A-C) Adult compound eyes viewed by light microscopy. Note the reduced red pigment upon the addition of ectopic Msk (compare B and C with A). (D-F) Adult compound eyes viewed by scanning electron microscopy. Note the disrupted facet pattern upon the addition of ectopic Msk (compare E and F with D). (G-I) Adult compound eye sections. Note the reduced numbers of photoreceptor and pigment cells upon the addition of ectopic Msk (compare the area indicated by the arrow in H with G). (J-L) 60-hour pupal eyes, stained for F-actin. Note the disrupted pattern upon the addition of ectopic Msk (compare K and L with J). Note, all phenotypes are accentuated by elevated Msk expression (through increased Gal4 activity) at 25°C (C,F,I,L).

View larger version (147K): [in a new window]   Fig. 5. Msk protein is apically sequestered in the morphogenetic furrow and ectopically expressed Msk at the furrow reduces both pMAPK and Atonal. Third instar eye-imaginal discs, anterior right. A,B and C-J are at the same scale, as indicated. (A,B) Apical (A) and basal (B) view of Msk staining (Dim7 antigen, black) at the morphogenetic furrow (arrowheads) Note the apical Msk expression (arrows, A), and that this is lost from cells surrounding the preclusters (black asterisk, B). Note also the higher level of Msk antigen anterior to the furrow (white asterisks). (C-J) pMAPK (black, C-F) and Atonal (white, G-J) expression at the morphogenetic furrow (arrowheads). pMAPK antigen stained with Ni/Co intensified DAB; Atonal by immunofluorescence. Genotypes and heat-induction times are indicated above the panels. pMAPK antigen is lost from the furrow after one hour of hsp70 promoter-driven Msk expression (compare F with arrows in C-E. Atonal expression is greatly reduced by the same treatment, on the anterior side of the furrow (arrows, J).

View larger version (100K): [in a new window]   Fig. 6. msk loss-of-function does not affect Atonal or pMAPK in the furrow. (A-F) Third instar eye-imaginal discs with msk null clones (outlined), stained for (A,D) GFP, which negatively marks the clones, (B) pMAPK, (C) pMAPK (red) and GFP (green), (E) Atonal, and (F) Atonal (red) and GFP (green). Anterior is to the right, scale as indicated in A. Atonal and pMAPK expression is normal in clones (arrows).

View larger version (107K): [in a new window]   Fig. 7. smo and tkv signaling affect pMAPK and Atonal expression. Third instar eye-imaginal discs with mutant null clones (outlined); anterior to the right; all to the same scale as indicated in L. Genes homozygous in the clones are indicated on the left, antigens/stains above; arrowheads indicate the furrow. (A-D) smo null clones showing reduced Atonal and pMAPK expression (asterisk). Note, residual Atonal is strongest in the lone R8 cells (arrow in B,D). (E-H) tkv null clones showing of Atonal and pMAPK (arrows). (I-L) smo tkv double-mutant clone in which Atonal and pMAPK expression is lost (asterisk).

View larger version (95K): [in a new window]   Fig. 8. msk is epistatic to smo and tkv in the furrow. (A-H) Third instar eye-imaginal discs with triply mutant smo, tkv and msk null clones (outlined), stained for (A,E) GFP, which negatively marks the clones, (B,F) Atonal and (C,G) pMAPK. D and H show merged images, colors as indicated. Anterior right, scale as indicated in A. Atonal and pMAPK expression is normal in the clones in the furrow (black arrow, compare with Fig. 7I-L). Note the ectopic pMAPK expression anterior to the furrow (white arrows in G,H).

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