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doi: 10.1242/10.1242/dev.00556

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Nuclear translocation of activated MAP kinase is developmentally regulated in the developing Drosophila eye

Justin P. Kumar^*, Frank Hsiung, Maureen A. Powers and Kevin Moses

Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA

View larger version (103K): [in a new window]   Fig. 1. MAP kinase phosphorylation in the Atonal-positive intermediate groups in phase 1. Wild-type third instar eye-imaginal discs, anterior rightwards and (B,C) apical upwards. Arrowheads indicate furrow in all panels. (A) Antigen dpErk (which indicates MAPK activation); arrows indicate cell clusters with elevated dpErk. (B,C) dpErk in the large clusters is predominantly cytoplasmic (white in B, red in C). Arrows indicate unstained nuclei. (D-F) Co-localization of dpErk (red) and Atonal (green). Note that dpErk clusters correspond to the Atonal-expressing intermediate groups (arrows).

View larger version (52K): [in a new window]   Fig. 2. MAP kinase-Gal4/Vp16 fusion constructs and their activity in the developing eye. (A) MAP kinase-Gal4/Vp16 constructs. Protein linear maps drawn to scale. NLS, SV40 nuclear localization sequence; MAPK, entire 286 residue natural sequence of the Drosophila rolled MAP kinase gene (Biggs and Zipursky, 1992); GAL4, residues 2-147 of the S. cerevisiae Gal4 (includes DNA-binding domain, but no known NLS sequence); VP16, residues 413-490 of HSV protein 16 (Sadowski et al., 1988); T, 11 residue epitope tag from HSV glycoprotein D (Novagen). (B-E) Eye discs are shown; anterior rightwards, dorsal upwards. In B-E, the fusion construct carried is indicated in the bottom left-hand corner and in all cases activity is detected antigenically using UAS:lacZ. Arrowheads indicate the furrow.

View larger version (142K): [in a new window]   Fig. 3. Neither dpErk antigen or the MG fusion protein are detected in cell nuclei in the morphogenetic furrow. Eye discs are shown; anterior rightwards. A,C and B,D are pairs at the same magnification. Genotypes are indicated in the bottom left-hand corner; antigens are indicated in the top right-hand corner. (A,B) The activity of the MG fusion protein in activating reporter genes (UAS:GFP and UAS:lacZ as indicated), relative to the endogenous dpErk antigen. Note that not only is reporter gene activity later than the high level dpErk antigen cell clusters in the furrow, but that the pattern of clusters is not reiterated by the reporter gene. (C,D) The same specimen showing the MG fusion protein expressed specifically behind the furrow (GMR:MG). Note that although the MG protein can be detected directly in most or all cells from the furrow (the leading edge of the TAG, arrow), MG activity is detected later and in a more restricted subset of cells (GFP, arrowhead).

View larger version (105K): [in a new window]   Fig. 4. Calibration of the delay between driver and reporter expression. Eye discs are shown; anterior rightwards, dorsal upwards. (A,B) GMR directly driving Gal4 (GMR:G); (C,D) GMR driving the MAP kinase-Gal4/Vp16 fusion (GMR:MG); (E,F) GMR driving the nuclear localized MAP kinase-Gal4/Vp16 fusion (GMR:NMG). Reporters (A,C,E) UAS:lacZ, (B,D,F) UAS:GFP. In all cases, Glass antigen is colocalized with the reporter antigen (ß-galactosidase or GFP) as indicated. Leading edges of Glass expression (arrowheads) and of reporter expression (arrows).

View larger version (81K): [in a new window]   Fig. 5. MAP kinase-Gal4/Vp16 fusion protein expression suppresses the phenotype of a MAP kinase loss-of-function mutant. (A,B) Adult compound eyes. (C,D) Adult retinal tangential sections. (E,F) Forty-two-hour-old pupal retina stained for F-actin. (G,H) Third larval instar retina stained for F-actin. In all panels, anterior rightwards and dorsal is upwards. Genotypes as indicated. (Below) Histograms showing analysis of numbers of rhabdomeres per ommatidium in wild type, rl1 homozygotes and HS:MG rl1 double homozygotes. All animals were raised at 29°C.

View larger version (105K): [in a new window]   Fig. 6. MAP kinase-Gal4/Vp16 fusion protein drives reporter gene expression in all cell types in the developing larval eye. Eye discs are shown; anterior rightwards, dorsal upwards. (A,B,D) HS:MG driver; (C,E-O) GMR:MG driver. Reporter expression shown green (white in H,K) and other antigen in red (or white in G,J) as indicated. Note that reporter expression in A,B follows downregulation of Atonal; in C, it can be detected in the first seven types of photoreceptor cells (arrowheads); in D, it colocalizes with Elav in a subset of cells; in E, it colocalizes with Elav in many more cells, but from a later time; in F, it colocalizes with Glass (similar to Elav); in G-I, it is detected in late R8 cells together with Sens (two examples indicated by arrows); in J-L, it is detected in some R7 cells together with Pro (two examples indicated by arrows); in M it is detected in some R1 and R6 cells together with BarH1 (two examples indicated by arrowheads); in N, it is detected in strongly in R3 and R4 cells, but later than SalM; and in O, it is detected in some cone cells together with Cut (four examples indicated by arrowheads).

View larger version (109K): [in a new window]   Fig. 7. MAP kinase-Gal4/Vp16 nuclear translocation activity is not upregulated by loss of Egfr, Notch, hedgehog or atonal function. Eye discs are shown; anterior rightwards, dorsal upwards. HS:MG activity detected by the UAS:lacZ reporter in the genetic background of temperature-sensitive mutations for Egfr (A,B), Notch (C) and hh (D) at restrictive temperature, and in ato3 (loss-of-function, antigen positive) clones (I-L). (E-H) In ato3 clones, MAP kinase phosphorylation is lost (dpErk antigen, as previously reported) (Chen and Chien, 1999). Note that reporter gene expression is not de-repressed in any of these genetic conditions.

View larger version (89K): [in a new window]   Fig. 9. Nuclear localization of MAPK detected directly with an epitope tag. Eye discs are shown; anterior rightwards, dorsal upwards. All stained immediately after 1 hour of heat induction. (A-D) Homozygous HS:MG larvae; (E-H) HS:NM larvae. (A,E) Stained to show DNA; (B,F) stained for the HSV epitope tag; (C,G) stained for Atonal; (D,H) Merges. Note that in HS:M, the epitope is expressed at a general and low level and does not appear to be nuclear, except in the final two columns of Atonal-positive cells, where is does appear to be nuclear (three examples indicted by arrows in A-D). Also note that in HS:NM the epitope is expressed at a uniform, low level, and does appear to be nuclear in both Atonal-positive (arrowheads) and -negative (arrow) cells.

View larger version (79K): [in a new window]   Fig. 8. Expression of MAPK directed to the nucleus affects development in the morphogenetic furrow. Eye discs are shown; anterior rightwards, dorsal upwards. A-D are at the same magnification; E,F are at the same magnification; G,H are at the same magnification. (AD) Atonal (green), Elav (red). (E,F) F-actin expression. (G,H) Elav expression. Note that at the end of the induction period (t=0) in HS:NM (C), Atonal expression is markedly reduced in the furrow (arrow in C), compared with wild type (A) or HS:M (B). Note that in HS:NM (D), 1 hour later Atonal expression has recovered. In addition, in HS:NM at t=0 the tight apical localization of F-actin markedly reduced in the furrow (arrow in F), compared with wild type (E, arrow). Elav expression appears precociously in the proto-R8 cells (arrowheads in H) compared with its normal first appearance in the proto-R2 and R5 cells (arrowheads in G).

View larger version (41K): [in a new window]   Fig. 10. MAPK cytoplasmic hold in the furrow. A diagram showing a section through the furrow, apical upwards and anterior rightwards. The furrow is moving from left to right. Phase 1 and phase 2 are indicated. Green nuclei are Atonal positive, yellow nuclei are Elav positive. Green cells are fated to become R8 photoreceptors, blue cells will become other photoreceptors. Red cells are the intermediate group, and have both high levels of Ras pathway activity and also MAPK cytoplasmic hold.