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MASK, a large ankyrin repeat and KH domain-containing protein involved in Drosophila receptor tyrosine kinase signaling

Rachel K. Smith, Pamela M. Carroll^*, John D. Allard and Michael A. Simon

Department of Biological Sciences, Stanford University, 385 Serra Mall, Stanford, CA 94305-5020, USA
^* Present address: Department of Applied Genomics, Bristol-Myers Squibb, P.O. Box 5400, Princeton, NJ 08543-5400, USA
Present address: Inflammatory Diseases Unit, Roche Bioscience, 3401 Hillview Ave, Palo Alto, CA 94304-1397, USA

View larger version (90K): [in a new window]   Fig. 1. mask was identified as a modifier of the cswCS phenotype. (A-C,I-L) Scanning electron micrographs of adult eyes. (D-F) Tangential sections of adult eyes. (A,D) w. (B,E) w; sev-cswCS/+. (C,F) w; sev-cswCS/+; mask10.22/+. (G,H) The number of inner and outer photoreceptors per ommatidium was tabulated in w; sev-cswCS/+ (white bars) and w; sev-cswCS/+; mask10.22/+ (black bars) eyes. (I-L) mask genetically interacts with ras. (I) rasV12/+ (J) rasV12/ mask10.22 (K) w; rasN17/+ (L) w; rasN17/mask10.22. All constructs are expressed under the control of the sev promoter. In all figures, anterior is to the left, posterior is to the right.

View larger version (93K): [in a new window]   Fig. 2. mask tissue is reduced in size. (A) Wild-type (w) third instar eye imaginal disc stained with -BP104, a membrane marker for differentiating photoreceptors (Hortsch et al., 1990). (B) w; mask6.3/mask7.29 third instar eye imaginal disc stained with -BP104. (C) Loss-of-function mask clones lacking GFP were much smaller than the wild-type twin spots containing two copies of the GFP transgene. (D) For 21 clones examined, the size of the mutant clone was on average 20.8% the size of the twin spot.

View larger version (80K): [in a new window]   Fig. 3. Loss of MASK increases programmed cell death and reduces proliferation. (A-C) mask clones in third instar larval eye imaginal discs. Non-clonal cells are marked with GFP (green) and apoptotic nuclei are marked with TUNEL-TMR (red). (D,E) Third instar eye imaginal discs are stained with antibodies against Elav, which is expressed in photoreceptor nuclei (red), and TUNEL-fluorescein, which marks the nuclei of apoptotic cells (green). White arrows mark the morphogenetic furrow. (D) Wild type (w); (E) w; mask6.3/mask7.29. (F,G) Alimentary canal tissue from third instar larvae stained for apoptotic nuclei with TUNEL-fluorescein (green). (F) Wild type (w); (G) w; mask6.3/mask7.29. (H-J) Third instar eye imaginal discs. As above, -Elav marks photoreceptor nuclei (red) and TUNEL-fluorescein marks apoptotic nuclei (green). White arrows mark the morphogenetic furrow. H) w, csw114 In these mutants, the morphogenetic furrow does not progress farther than is shown. (I) w; sev-cswCS/+; (J) w; sev-cswCS/mask10.22. (K,L) Mutant clones (marked as cells not expressing GFP; green) in eye imaginal discs were stained with -phosphohistone H3 (red) to visualize nuclei in M phase. (K) Clones generated using hs-flp were small and rarely contained any mitotic cells. (L) Clones generated using ey-flp were larger and contained more cells undergoing mitosis. White arrowheads mark zones of mitosis before and after the morphogenetic furrow.

View larger version (73K): [in a new window]   Fig. 4. Loss of MASK inhibits photoreceptor differentiation. (A,B) Third instar larval eye imaginal discs stained with -Elav to mark photoreceptor nuclei. (A) Optical section of a wild-type disc; each ommatidial cluster contains eight photoreceptor nuclei. (B) Optical section of a mask6.3/mask7.29 disc. Yellow arrows indicate ommatidial clusters containing fewer than eight photoreceptor nuclei. (C-E) A third instar larval eye imaginal disc containing a mask10.22 clone. Arrows mark incomplete ommatidia. (C) Photoreceptor nuclei are stained with -Elav. (D) The clone is identified as those cells not expressing GFP. (E) Color merge: Elav is in red and GFP is in green. (F,G) Tangential sections of adult eyes. (F) Small mask10.22 clone induced by the expression of hs-flp. White arrows indicate ommatidia missing photoreceptors. (G) A portion of a large mask10.22 clone induced by the expression of ey-flp. Almost all ommatidia are missing at least one photoreceptor.

View larger version (120K): [in a new window]   Fig. 5. Loss of MASK affects RTK-dependent processes other than eye development. (A) Dorsal appendages produced by wild-type follicle cells. (B,C) Dorsal appendages produced by mask10.22 clones in the follicle cells.

View larger version (126K): [in a new window]   Fig. 6. MASK is not required for MAPK activation or translocation. (A) A stacked confocal image of a wild-type third instar eye imaginal disc stained with -diphospho-MAPK. In all panels an arrow indicates the location of the morphogenetic furrow; arrowheads mark nuclei containing activated MAPK. (B) A single confocal slice showing cytoplasmic activated MAPK in a mask10.22 clone. (C) A single confocal slice showing nuclear activated MAPK in a mask10.22 clone. (B',C') High magnification of -diphospho-MAPK staining. (B'',C'') GFP (B''',C''') Color merge. (D) mask10.22 suppresses the ectopic R7 phenotype of rlSEM.

View larger version (77K): [in a new window]   Fig. 7. MASK is a large protein containing ankyrin repeats and a KH domain. (A) Genomic structure of the mask locus (GenBank accession number AF425651). The light gray boxes represent the stretches of ankyrin repeats, and the dark gray box represents the KH domain. Arrowheads indicate the positions of amino acids that have been changed to stop codons in mutant alleles. (B) Stretches of homology between MASK and predicted proteins from cDNA sequences from other species. Dark gray shading indicates identical residues, and light gray shading indicates similar residues.

View larger version (147K): [in a new window]   Fig. 8. MASK is localized in the cellular cytoplasm. (A,B) mask10.22 clones. The MASK antibody (A) only stains the GFP-marked cells containing wild-type MASK (B). (C) Third instar wild-type eye disc stained with -MASK. The morphogenetic furrow is marked with white arrows. (D) A section at high magnification showing increased -MASK staining around the nuclei of R8 photoreceptors. (E,F) S2 cells expressing signaling proteins, stained with -MASK. (E) hs-sevS11 (F) hs-sevS11 kinase dead.