As reported previously 3, full-length Eiger expressed in the Drosophila eye under the control of GMR-Gal4 induced a severe apoptotic eye phenotype (Figure 1A-i and 1C-i). Expression of the entire extracellular domain (ecto-Eiger-60) also resulted in complete eye loss (Figure 1A-ii and 1C-iv). As anticipated, the THD that contains the receptor-binding domain in all vertebrate TNF ligands 8 was required for the activity (Figure 1A-vii and 1A-viii). However, shorter forms of soluble Eiger containing the THD alone (Figure 1A-v and 1A-vi) or corresponding to predicted processed forms (Figure 1A-iii and 1A-iv) were inactive despite protein expression, and also failed to interfere with death induced by full-length Eiger or ecto-Eiger-60 (data not shown). Moreover, Eiger lacking the stalk also failed to induce apoptosis (Figure 1A-ix). All these data establish both the THD and the membrane-proximal portion of the stalk (sequence 60-145) as essential determinants of Eiger's activity.

The JNK pathway mediates the apoptotic activity of Eiger 2, 3, 7 (Figure 1B). Accordingly, the apoptotic activity of ecto-Eiger-60 was totally inhibited when expressed in the eye of dTAK1 hemizygous mutant flies (Figure 1C-v), similar to full-length Eiger expression (Figure 1C-ii). Interestingly, a novel “hanging eye” phenotype was observed when ecto-Eiger-60 was co-expressed in the eye with Puckered, a cell-autonomous inhibitor of Basket 9 (Figure 1C-vi and 1C-vi′). This phenotype, which was not observed when full-length Eiger was co-expressed with Puckered 3 (Figure 1C-iii and 1C-iii′), suggests that soluble Eiger (ecto-Eiger-60) can kill the surrounding tissue that is not protected by Puckered. This was confirmed by clonal analysis in the Drosophila wing imaginal disc. In this experiment, GFP-positive clones co-expressing ecto-Eiger-60 and Puckered induced caspase-3 activation in neighbouring cells where Puckered was not overexpressed (Figure 1D-iv, 1D-v and 1D-vi). In contrast, full-length Eiger failed to induce obvious activation of caspase-3 within or outside the clone when co-expressed with Puckered (Figure 1D-i, 1D-ii and 1D-iii). These results demonstrate that both full-length Eiger and ecto-Eiger-60 induce a JNK-mediated cell death, but that soluble Eiger is able to act over longer distances.

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