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First published online 5 October 2005
doi: 10.1242/dev.02058

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Genetic and biochemical analysis of the role of Egfr in the morphogenetic furrow of the developing Drosophila eye

Aloma B. Rodrigues, Erica Werner and Kevin Moses^*,

Department of Cell Biology, Emory University School of Medicine, 615 Michael Street NE, Atlanta, GA 30322-3030, USA

View larger version (60K): [in a new window]   Fig. 1. Time course of Spitz induction of Egfr signaling in S2 cells. (A) Protocol used in B,C, see text. (B) Blot probed for Egfr. Second, non-specific band (asterisk) serves as a loading control. The DNA used and the times after Spitz induction are indicated above. (C) Blot for pMAPK shows pathway activation (same gel as is shown in B); asterisk indicates the level of pMAPK activation after 5 minutes. Note, the pMAPK intensity at 0 minutes (no time with Spitz induction) is indistinguishable from that of the untransfected controls. Also note, there is no detectable pMAPK (above controls) in the Egfrtsla transfections, whereas there is detectable Egfr antigen. (D) Protocol used in E-H, see text. Lysates were prepared by immunoprecipitation using anti-Egfr. (E,F) Blot probed for phospho-Tyrosine (`pTyr'). Note that, at 18°C (E), the anti-Egfr precipitable pTyr signal is detectable over control (black asterisk) for the Egfr+ and Egfrtsla transfections (white asterisks), whereas at 30°C (F), Egfr+ yields detectable anti-Egfr precipitable pTyr signal (white asterisk), but Egfrtsla does not (black asterisk). (G,H) The same gels as in E,F, re-probed for Egfr antigen.

View larger version (59K): [in a new window]   Fig. 2. Egfrtsla is rapidly made inactive by a shift to 30°C. (A) Diagram of the protocol, see text. (B) Blot probed for Egfr. The DNA used and the time incubated at 30°C are indicated. (C) Blot probed for pMAPK shows pathway activation (same gel as is shown in B). Note, pMAPK levels appear constant over 60 minutes at 30°C for wild-type Egfr. However, for Egfrtsla, although pMAPK is detected at 18°C, after the shift to 30°C, the level falls rapidly down to background levels. Asterisks in B and C indicate examples of the 60-minute time-point bands used in the quantification shown in Fig. 3. (D) Same blot as in C, re-probed for total MAPK (loading control).

View larger version (22K): [in a new window]   Fig. 3. Quantification of the temperature-shift Egfr-activity data from S2 cells. Histograms for two experiments shown in Fig. 2, showing 0-minute at 18°C and 60-minute at 30°C time points (see text). Data are shown as a percentage of wild type at 30°C. Error bars indicate s.e.m.

View larger version (95K): [in a new window]   Fig. 4. Nuclear pMAPK following Spitz activation in S2 cells. S2 cells were stained for Egfr antigen, pMAPK antigen and DNA as indicated. Conditions are as in Fig. 2A and Fig. 3, see text. Transfected DNA and temperature are indicated on the left. Transfected cells express elevated Egfr antigen (arrowheads), untransfected control cells do not. Note that cells transfected with Egfrtsla, at 30°C, do not express pMAPK over background levels. Arrows indicate increased levels of pMAPK antigen. Scale bar: 5 µm.

View larger version (45K): [in a new window]   Fig. 5. Egfr antigen is removed from the cell surface in Egfrtsla after temperature shift. (A) Diagram of the protocol used in B,C, see text. (B) Biotinylated material, probed for Egfr. (C) Total lysate, probed for Egfr. Note that after the temperature shift, biotinylated Egfrtsla antigen is lost more quickly than is total Egfr antigen.

View larger version (81K): [in a new window]   Fig. 6. Egfr antigen localization is rapidly altered in Egfrtsla after temperature shift. Eye discs with mosaic clones homozygous for Egfrtsla were stained for Egfr antigen. In all figures, anterior is shown to the right. (A,C,E) Posterior region; (B,D,F-H) the furrow. Times and temperatures are indicated left, see text. A granular, non-nuclear stain in all parts of the disc (white arrows) and a strong additional stain in the furrow between specific cells (white arrowheads in B) is observed. Note that rapidly after the shift to 30°C the furrow cell border stain is lost and is replaced by a heavy antigen stain in larger granules. (G,H) Eye discs stained for (G) Armadillo (Arm), and for (H) Hrs (green) and Egfr (red). Note, Armadillo junctional stain persists after temperature shift (arrow in G), but that Egfr and Hrs do not co-localize (white arrowhead in H indicates an Hrs-positive granule and black arrowhead in H indicates an Egfr-positive granule). Scale bar: 5 µm.

View larger version (74K): [in a new window]   Fig. 7. Egfrtsla clones affect cell growth, late R8 spacing and neural differentiation. (A-H) Eye discs with ey:Flp induced mosaic clones are shown; clones are negatively marked with GFP and are outlined in E-H. (A-D) Homozygous clone genotypes and the times incubated at each temperature are indicated (see text). (E-H) Egfrtsla clones, raised at 18°C, and shifted to 30°C 24 hours before dissection. (E,F) Senseless; note the presence of Senseless-positive R8 cells, sometimes immediately adjacent to each other (arrow). (G,H) Elav; note that although most Elav-positive cells are lost (asterisk), some persist for some time (arrows).

View larger version (101K): [in a new window]   Fig. 8. Egfrtsla clones do not affect the speed of furrow progression or Atonal patterning. Eye discs with Egfrtsla clones, stained for Atonal antigen. (A,C,D,F) GFP; (B,C,E,F) Atonal. Mosaic clones are negatively marked by GFP and are outlined. (A-C) ey:FLP-induced clone homozygous for Egfrtsla, raised at 18°C, then at 30°C for 24 hours. Note that the position of the furrow is the same in the clone (black arrowheads) and in the flanking territory (white arrowheads). (D-F) Same genotype, raised continuously at 30°C. Note the normal appearance of Atonal expression in the mutant territory, both anterior (white arrow) and posterior (black arrow) to the furrow. Scale bar: 20 µm.

View larger version (90K): [in a new window]   Fig. 9. Two different Minute mutations affect Atonal expression, whereas Egfr alleles do not. Eye discs stained for Atonal are shown inA-C,E,F,H,I,K,L,N,O,Q,R. Clones are negatively marked by ß-gal (white in D,G,J,M,P, blue in F,I,L,O,R) and are outlined. (A-C) Egfr alleles in trans to wild type; note normal Atonal expression. (D-R) hs:Flp-induced clones induced in trans to Minute chromosomes. (D-O) ß-gal-positive territories are heterozygous for the Egfr allele and for the Minute mutation. Black (ß-gal negative) territories are homozygous for the Egfr allele and are wild type for the Minute mutation. (P-R) ß-gal-positive territories are heterozygous for the Minute mutation. Black (ß-gal negative) territories are genetically (but not phenotypically) wild type. No Minute– homozygous cells survive. Mutations are indicated on the left. Note, the Atonal pattern is not wild type in either the Egfr homozygous (black in D-O) or the Egfr heterozygous territories (compare the ß-gal-positive regions to the heterozygous examples shown in A-C). Also, the Atonal pattern is not wild type in either the wild-type territories (white arrows in P-R) or the adjacent Minute heterozygous cells (black arrows in P-R). Note that Minute(2)53 and Minute(2)56i have different, but self-consistent dominant effects on Atonal expression. Atonal expression in the Egfrtsla Minute+ clones is indistinguishable from that in the two Egfr null alleles. Scale bar: 10 µm.