Additional file 1:

echinus mutants have a decrease in IOC apoptosis. TUNEL staining of (A) OreR, (B) ec^PlacZ and (C) ec^Δ9 pupal retinas (29–30 hr APF). Anti-active caspase-3 immunostaining in (D) OreR and (E) ec⁵⁶ and (F) ec^EPΔ4 pupal retinas (30 hr APF). Apoptosis is reduced, though not completely absent, in ec mutant pupal retinas.

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Additional file 2:

Phenotype of the ec^3c3 allele in trans to a deficiency for the region containing echinus. Scanning electron micrographs and pupal retinas of several different genotypes are shown. ec^3c3 placed in trans to a deficiency that removes echinus, Df(1)HC244, shows a more severe rough eye phenotype than homozygous ec^3c3 flies. Pupal retinas show a significant increase in the number and improper sorting of IOCs. This genetic observation suggests ec^3c3 represents a partial loss-of-function allele.

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Additional file 3:

Three echinus splice forms, ec-SF1, ec-SF2 and ec-SF3, are expressed in the pupal retina during the stage of IOC death. Gel image shows the results of RT-PCR analysis using primers for a positive control (rp49), primers specific to each echinus splice form (SF1, SF2, SF3), and primers that recognize all three splice forms (all). M= Size Marker; – and + designate the absence or presence of pupal retinal RNA template; expected product sizes are as follows: 91 bp (rp49), 104 bp (SF1), 111 bp (SF2), 147 bp (SF3), 135 bp (all).

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Additional file 4:

Genetic interactions between echinus and components of several signaling pathways. The indicated genotypes were introduced into the ec^Δ4 background, or into a wildtype background in the presence of GMR-ec-SF1. For each genotype tested, similar phenotypes were observed in the presence of GMR-ec-SF2 (data not shown). Genotypes not discussed in the text are indicated below. The EGFR^ELP mutation is a hypermorphic allele of the EGF receptor [81,82]. However, genetically it behaves as a partial loss-of-function allele in the eye because it induces the expression of high levels of the EGFR inhibitor Argos [83]. Downstream Ras pathway components tested include Ras^N17 (a dominant negative version of Ras driven by the sevenless promoter) Sina and Yan^act (a version of Yan that is not inhibited by MAPKinase phosphorylation). GMR-GAL4-UAS-Delta expresses the Notch ligand Delta in every cell behind the morphogenetic furrow [84]. N^fa-g removes Notch activity specifically in pigment cells [85], leading to a failure of 1° pigment cells to differentiate, and a decrease in IOC death [86]. Notch and echinus are both on the X chromosome. To search for interactions between N^fa-g and echinus loss-of-function mutations we took advantage of flies carrying an autosomal insertion of GMR-CG2904-RNAi that targets transcript sequences common to all echinus splice forms, and that phenocopies echinus (Fig. 2C,I). GMR-ΔN-DCP1 expresses under GMR control a version of the caspse DCP-1 that lacks the N-terminal prodomain. GMR-GAL4-UAS-dTAK flies express under GMR control the kinase TAK1, an activator of JNK signaling. GMR-Strica flies express the long prodomain caspase under GMR control.

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Additional file 5:

echinus does not interact with roughest. Eye-specific Gain-(GMR-ecSF1) and loss-of-function (GMR-ec-RNAi) of echinus mutants were introduced into gain (GMR-GAL4-UAS-rst) and loss-of-function (rst^CT) roughest mutant backgrounds. No significant interactions were observed between these genes.

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