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doi: 10.1242/10.1242/dev.00568

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The Drosophila dual-specificity ERK phosphatase DMKP3 cooperates with the ERK tyrosine phosphatase PTP-ER

Zoologisches Institut der Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland

View larger version (51K): [in a new window]   Fig. 7. Ommatidial development in the presence and absence of Dmkp3. (A) Schematic representation of the emergence of preclusters in successive rows posterior to the morphogenetic furrow (at row 0). Starting from a `rosette' and `arch' stage (rows 0 and 1), the preclusters in row 3 contain initially equivalent R3/R4 precursors and 1-2 intervening mystery cells (M). The R3/R4 equivalence group is able to sense a graded Frizzled signal from the equator such that the cell closer to the equator becomes R3, the other R4. Subsequently, the preclusters undergo a rotation and thereby achieve chirality. Ultimately, R3 and R4 adopt asymmetrical positions in the ommatidium conveying the typical trapezoidal shape (right, symbolizing the arrangement of rhabdomeres). (B) Between row 3 and row 4 the mystery cell is expelled from preclusters like a melon-seed. We propose that in the absence of DMKP3 function cells from the surrounding pool and also R3 and R4 display altered adhesive properties, thereby affecting the exit of the mystery cell(s) from the precluster.

View larger version (96K): [in a new window]   Fig. 3. Dmkp3 genomic organization, alleles, protein structure and expression. (A) The Dmkp3 gene consists of five exons and is characterized by a large intron and extensive 5' and 3' UTRs (open rectangles). A putative alternative (incomplete) transcript is denoted and the site of the Dmkp35 splice-acceptor mutation is indicated (arrowhead). EP insertion sites and orientations are symbolized by wands. (B) The DMKP3 protein has three human functional homologs: MKP-3, MKP-4 and MKP-X, which are 52-58% similar. MKP-X sequence has been delineated from partial cDNAs. Underlined are the N-terminal Cdc25-homology (CH2) domain implicated in MAPK binding (gray) and the C-terminal catalytic domain (black). The broken line marks the ERK docking motif and the dotted line the core catalytic site. These central sites are presumably disrupted in the DMKP37 and DMKP310 gene products. With the possible exception of Dmkp36, the other alleles are null mutations as well (see amino-acid replacements and allele numbers above the alignment). The arrow marks the site of the Dmkp35 mutation. (C) Dmkp3 is weakly expressed just posterior to the morphogenetic furrow. Negative and positive controls are shown in D and E, respectively.

View larger version (118K): [in a new window]   Fig. 1. DMKP3 is an ERK phosphatase. Eye sections (left), wings (middle) and cuticle preparations (right) of strains overexpressing the indicated phosphatases by sev-GAL4, en-GAL4 and 69B, respectively. Wild-type controls are shown in A-C. Eyes and wings are not affected by overexpression of mammalian M3/6 and Drosophila puckered (D,E,G,H), but by MKP-3 and Dmkp3 (J,K,P,Q). The latter two do not impair embryogenesis (L,R), but the JNK phosphatases cause a dorsal closure defect (F,I). CL100 interferes with all processes (M-O) and even leads to wing duplications (N, lower). Driver lines were sev-GAL4K25 (D,G,J), sev-GAL4KK12/3 (M,P), en-GAL433 (K,N — upper wing) and en-GAL454 (E,H,Q,N — lower wings). en-GAL4 is expressed in the posterior wing region. The wing shown in K harbors two copies of the MKP-3 transgene and the wing in Q is derived from a male.

View larger version (141K): [in a new window]   Fig. 2. Epistasis analysis of Dmkp3 overexpression. A direct sev-Dmkp3 transgene causes rough eyes (section in D, compare to wild-type in Fig. 1A), which are enhanced by rl10A (G). Note that the direct sev-Dmkp3 construct causes the loss of outer and central photoreceptors, whereas the sev-GAL4 driven Dmkp3 mainly affects the R3/R4 pair, indicating that the time-window for ERK-dependent differentiation is shorter in R7 cells than in R3/R4 cells (Fig. 1P). The construct almost completely rescues sevS11 (E,H) and rlSEM (F,I) eyes. However, the rlSEM wing phenotype (B) is unaffected by en-GAL433-driven UAS-Dmkp3 (A,C). (The line marks the anterior-posterior wing boundary.) Specimens shown in A-C,F,I are from males.

View larger version (86K): [in a new window]   Fig. 4. The Dmkp3 wing phenotype is sensitive to RAS pathway activity. (A) Dmkp3 mutant wings exhibit additional vein material at four different sites marked by arrowheads. This phenotype is strongly enhanced by Ellipse (compare C to D) and it is very reminiscent of UAS-rlSem (B). Relative numbers exhibiting extra veins at each of the four sites are given in E. rl10A dominantly suppresses and PTP-ER dominantly enhances the phenotype. Between 100 and 235 wings were counted.

View larger version (130K): [in a new window]   Fig. 5. DMKP3 affects different cellular decisions from PTP-ER. (A) Section through Dmkp3 mutant eyes reveals misdifferentiations affecting the outer R3 and R4 cells, as well as a central cell that is normally not incorporated into ommatidia. Yellow dots mark mutant ommatidia and the different types and corresponding percentages are shown on the right. (609 ommatidia were analyzed, 4% of which could not be unambiguously assigned.) 1, Wild-type; 2-6,8,9, symmetrical ommatidia; 2,7-9, with an extra R7; 6, containing an extra outer photoreceptor; 2,4,8, R3/R3-type; 5,9, R4/R4 type; 3,7, devoid of an R3/R4-type photoreceptor; 4 and 5 are sometimes hard to distinguish and therefore percentages are combined. Cartoons to the right symbolize the different mutant classes with R3 rhabdomeres in red, R4 rhabdomeres in green and rhabdomeres of unclear identity in yellow. (B) The eye phenotype is almost completely suppressed by rl10A. The only mutant ommatidium out of 700 is marked. (D) In Dmkp3- eye imaginal discs two (arrows) or no (arrowhead) cells per cluster (green) may differentiate into R4 cells (red, single channel in C). (E) PTP-ER- eyes are rough because of the recruitment of extra R7 cells (red dots), and PTP-ER- Dmkp3- pharate adults display ommatidia characteristic of either single mutant (yellow and red dots in F). (G) The double mutant is viable when rl/ERK dose is reduced and shows a wing phenotype characteristic of enhanced ERK activity.

View larger version (101K): [in a new window]   Fig. 6. DMKP3 functions both in the R3/R4 pair and outside of the photoreceptors. (A) A section through a mosaic eye and a magnification and further examples of mosaic ommatidia in insets. Pigmented cells are Dmkp3+ (arrowheads), tissue devoid of pigment granules is Dmkp3-. Cartoons represent the genotype with Dmkp3+ photoreceptors in red and Dmkp3- cells in pink. (B) Quantitative analysis of 73 phenotypically Dmkp3- mosaic ommatidia (cartoons to the left, compare to Fig. 5A) showing both Dmkp3- (pink in the bar diagram) and Dmkp3+ photoreceptors (red) at any position. Data are from both males and females and absolute numbers are given above and below the chart, respectively. Genetically wild-type ommatidia exhibiting a Dmkp3- shape can also be observed close to a Dmkp3- clone (section through a male eye in C and insets). (D) Concordantly, an additional photoreceptor (blue) can differentiate into an R4 cell (red) even outside of a Dmkp3- clone (marked by the absence of GFP, green) (higher magnification in E-H). The misdifferentiated cell is marked by an arrow. R4 cells (red) are also observed outside of ommatidial clusters (green) in a sevS11 background (arrow in I-K) providing an explanation for the occurrence of extra outer photoreceptors in sevS11 eye-sections (arrow in L).