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doi: 10.1242/10.1242/dev.00605

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Echinoid is essential for regulation of Egfr signaling and R8 formation during Drosophila eye development

Department of Molecular Biology and Pharmacology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8103, St Louis, MO 63110, USA

View larger version (114K): [in a new window]   Fig. 1. Patterned expression of the bHLH transcription factor Atonal is essential for R8 formation in wild-type discs. (A) Atonal expression is patterned in the morphogenetic furrow of wild-type eye imaginal discs (red); terminally differentiated R8 cells express the protein Boss (green). (B,C) A close-up of Atonal immunostaining shows initial expression in proneural groups; the nuclei of two to three cells at the posterior edge of these groups rise apically, forming the R8 equivalence group. One cell from this group retains Atonal expression and differentiates as the R8 photoreceptor. (D) Scanning electron micrograph of a wild-type adult eye shows normal pattern formation. (E) EgfrEllipse is an activated allele of Egfr; EgfrEllipse/+ flies have slightly smaller, mispatterned eyes. (F) Loss of one copy of echinoid in trans to EgfrEllipse enhances the rough eye phenotype. Anterior is towards the left.

View larger version (119K): [in a new window]   Fig. 2. Mutations in echinoid lead to formation of multiple R8 cells per ommatidium. (A) Atonal expression (green) narrows to a single cell posterior to the morphogenetic furrow in wild-type discs. (B) In homozygous edl(2)k01102 eye discs, multiple cells often retain Atonal expression (arrows). (C,D) Similarly, when edl(2)k01102 patches of tissue are created using the FLPFRT technique (loss of green GFP marker), Atonal expression (red) within the patch fails to narrow to a single cell. (E) In wild-type tissue, one mature R8 photoreceptor is present in each ommatidium, as visualized with an antibody against Boss (red). (F) Multiple Boss-expressing cells are present in many ommatidia of edl(2)k01102discs. (G,H) Using the FLP-FRT technique to create homozygous edl(2)k01102 patches of tissue (loss of green) shows a similar multiple R8-phenotype by Boss staining (red). (I,J) In wild-type tissue (I), an antibody against Senseless detects one R8 photoreceptor per ommatidium, but in edl(2)k01102/edslA12 discs (J) many ommatidia contain multiple R8 cells (arrow). (K,L) In adult eyes, both R7 and R8 cells are distinguished by a small inner rhabdomere; the rhabdomere of the R7 cell is present in the apical portion of the eye, while the R8 rhabdomere is visible in more basal sections. Sections of adult eyes containing FLP-FRT-mediated clones of echinoid tissue show multiple inner rhabdomeres present in basal sections (arrow, K), but not necessarily in apical sections of the same ommatidia (arrow, L). Because the mutation used for these experiments is marked with the white gene, clonal boundaries cannot be discerned in these sections.

View larger version (75K): [in a new window]   Fig. 3. Loss of echinoid leads to sustained ERKA phosphorylation. (A,B) High levels of phosphorylated ERKA, a readout of Egfr signaling, are present in a single row of cell clusters within the morphogenetic furrow of wild-type discs (anti-dpERK, red, A,B); these clusters correspond to the Atonal-expressing proneural clusters (anti-Atonal, green, B). (C,D) In homozygous edl(2)k01102 eye discs, these clusters fail to lose dpERK signaling (red) as cells emerge from the morphogenetic furrow and retain high levels of signaling for several rows. Groups containing high levels of dpERK retain Atonal expression in two or more cells (anti-Atonal, green). (E,F) Patches of edl(2)k01102 tissue created using the FLP-FRT technique (absence of green in F) retain high levels of phosphorylated ERKA relative to surrounding wild-type tissue (anti-dpERK in red).

View larger version (105K): [in a new window]   Fig. 4. echinoid and argos act in concert to inhibit Egfr signaling, but through different mechanisms. (A,B) Mutations in argos enhance the echinoid phenotype. (A) Discs homozygous for edl(2)k01102 show the multiple R8 phenotype when stained with anti-Atonal antibodies. An example of a pair of R8s is indicated by the arrow. (B) Mutations in argos (argosW11/argosW11)further enhance the edl(2)k01102 phenotype. (C,D) Same genotypes as in A,B. Enhancement of the multiple R8 phenotype is also apparent in more mature R8 cells as visualized with antibodies against Boss. (E-H) Echinoid-FLAG driven by the actin promoter was overexpressed in clonal patches of tissue using the FLP-out technique (green in F and H; see Materials and Methods). Unlike Argos, overexpression of Echinoid had no effect on the pattern of dpERK phosphorylation (E,F) or Boss expression (G,H).

View larger version (38K): [in a new window]   Fig. 5. Immunoprecipitation of Echinoid reveals homophilic interactions and interactions with Egfr. (A) Cultured S2 cells were transfected with EdFLAG and Edmyc to evaluate homophilic interactions. Immunoprecipitation with anti-FLAG Sepharose co-precipitates Edmyc; co-precipitation is not significantly affected by transfection with Egfr. (B) Analysis of immunoprecipitated Echinoid-FLAG (EdFLAG) revealed that a clipped form of Echinoid is also present, corresponding approximately to the 500 C-terminal amino acids (arrow, left blot). The amount of the clipped form is not altered by Egfr transfection. An antibody against the N-terminal region of Echinoid (Bai et al., 2001) detects a smaller form of Echinoid in media from these cells (right blot); this may represent shedding of the immunoglobulin and fibronectin III domains of Echinoid into the extracellular space. (C) Echinoid associates with Egfr. (Left panel) Cultured S2 cells were transfected with 1.8 µg Egfr DNA and variable amounts of EdFLAG DNA. Immunoprecipitation of EdFLAG co-precipitated increasing amounts of Egfr. (Middle and right panels) Cultured S2 cells were transfected with Egfr and EdFLAG or Edmyc DNA. Immunoprecipitation with FLAG-Sepharose or myc-Sepharose co-precipitated Egfr. Transfection of Egfr and other FLAG and myc-tagged proteins (morgueFLAG, erkmyc) did not lead to co-precipitation of Egfr. (D) Immunoprecipitation of Egfr from cultured S2 cells transfected as in (C) co-precipitated EdFLAG. (E) Echinoid constructs used in these experiments: full-length Echinoid contains seven Ig repeats and a FN3 repeat on its extracellular face. EdFLAGN lacks the Ig repeats and the N-terminal part of the FN3 domain (amino acids 69-787) but not the signal sequence. EdFLAGC lacks the tyrosine-rich intracellular domain (amino acids 1078-1332).

View larger version (26K): [in a new window]   Fig. 6. Echinoid is phosphorylated in response to Egfr. (A) Immunoprecipitated EdFLAG from S2 cells transfected with EdFLAG and/or Egfr were analyzed by western blot using an antibody specific for phosphotyrosine. Echinoid was phosphorylated in response to Egfr transfection. This phosphorylation was further increased by addition of an active form of the Egfr ligand Spitz (sSpi) to the tissue culture medium. The clipped C-terminal form of Echinoid also becomes phosphorylated (arrow). (B) Echinoid lacking the intracellular domain (EdFLAGC) was not phosphorylated in response to Egfr signaling, but Echinoid lacking the extracellular motifs (EdFLAGN) was phosphorylated. (C) Echinoid immunoprecipitated from imaginal discs was also tyrosine-phosphorylated in response to Egfr signaling: Echinoid from w1118 imaginal discs shows a low-level of tyrosine phosphorylation. Activation of Egfr signaling by transient overexpression of Rhomboid increases phosphotyrosine associated with Echinoid; inhibition of Egfr by transient overexpression of Argos decreases phosphotyrosine.

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