II.1. Basics of the programmed cell-death machinery
Apoptosis or how it is also called ‘programmed cellular suicide’ is a tightly regulated process in the cell. If the machinery does not work optimally it can compromise the cell or not execute biological processes correctly. The common part for all apoptosis-‐
related processes is a special class of aspartate-‐specific cysteine proteases, called caspases (Degterev et al., 2003). They are divided in two groups, initiator (long) and effector (short) caspases based on the length of their pro-‐domain. When becoming active, the initiator caspases process the inactive effector caspase via proteolytic cleavage, and from there effector caspases, now active, are cleaving different cellular substrates causing apoptotic program to be launched in the cell (Kumar, 2007).
Together with how apoptosis is tightly regulated biological process goes the fact that there are seven different caspase genes in Drosophila genome, (Salvesen and Abrams, 2004). In homeostasis, caspases are constantly under the inhibition of specialized proteins, most important being Drosophila inhibitor-‐of-‐apoptosis (Diap1), which blocks the initiator caspase Dronc. When apoptosis is triggered in the cell with the proper stimuli, 3 different proteins named Reaper, Hid and Grim are degrading Diap1, thus making Dronc available to initiate apoptosis (Hay et al., 1995; Laundrie et al., 2003; Salvesen and Abrams, 2004; Cashio et al., 2005; Xu et al., 2005; Hay and Guo, 2006; Muro et al., 2006; Xu et al., 2006; Kumar, 2007).
II.2. Discovery of cell death-induced compensatory proliferation
Pioneer work connecting apoptosis with wound healing and regeneration started in Drosophila melanogaster. Apoptotic cells were shown to provide different kind of signals that are directly in charge of wound healing and regeneration. Already in the
’70 it was known that larval imaginal disks in flies can regenerate even after more than 50% of cells that were destroyed via radiation (Haynie and Bryant, 1977).
Additional information came few years after in the early ’80 when a group of radiologists found out by using electron microscopy that not only apoptosis, but radiation-‐induced necrosis contributes to the massive lesions in imaginal disk, but still does not impact the proper regeneration of wings in the flies (Abbott, 1983).
Induced cell death was shown to launch the process of cellular proliferation of nearby living tissue (Graves and Schubiger, 1982; Ijiri and Potten, 1986; Ruifrok et al., 1997).
These observations led to the hypothesis that dying cells have a specific way of signaling with nearby living tissue, pointing to the phenomena initially named as
“Altruistic cell death”, a process widely used across metazoan and non-‐metazoan organisms (Kondo, 1988) that is today known as compensatory proliferation.
However as the dying cells are promptly removed via phagocytosis, the study of the mechanisms driving how dying cells exactly execute compensatory proliferation was not so easy. In the early 2000’ Drosophila geneticists exploited a main strength of Drosophila model to study cellular pathways, a fact that it is very easy to precisely inhibit or over-‐express certain genes. In this case, over-‐expression of the caspase-‐
inhibitor p35 was used to tackle this issue. P35 protein is the inhibitor of effector caspases, named DrICE and Dsp-‐1 in Drosophila (Bump et al., 1995; Xue and Horvitz, 1995; Yoo et al., 2002; Yu et al., 2002). Even when apoptosis is initiated in the cell, p35 blocks the cell from dying by inhibition of its actual execution. And because of that, cells are practically kept alive in the ‘undead’ state, in which they constantly produce signals that trigger the compensatory proliferation of nearby tissue, leading to an overgrowth phenotype. This very elegant experimental setup allowed researchers to further characterize components of the apoptotic pathway and their exact function regarding compensatory proliferation (Huh et al., 2004; Perez-‐Garijo et al., 2004; Ryoo et al., 2004; Kondo et al., 2006; Wells et al., 2006). In the past 15 years or so, the non-‐autonomous role of programmed cell death – apoptosis was investigated in developmental and regenerative contexts and its critical role in the initiation of regeneration was established in several model organisms (Bergmann and Steller, 2010; Vriz et al., 2014; Perez-‐Garijo and Steller, 2015).
II.3. Molecular signaling involved in cell death-induced compensatory proliferation
Several following studies were performed using compensatory proliferation as a model, mostly to help characterize which components of the apoptotic pathway are involved in this process (Figure 5). In Drosophila, several independent studies converged to show the importance of the initiator caspase Dronc (Huh et al., 2004;
Kondo et al., 2006; Wells et al., 2006) and the inhibitor-‐of-‐apoptosis protein Diap1
of the regenerative program in planarians (Hwang et al., 2004; Pellettieri et al., 2010), newts (Vlaskalin et al., 2004), Xenopus (Tseng et al., 2007), zebrafish (Gauron et al., 2013). Similarly, in mouse, the caspases 3 and 7 (both effector caspases) play crucial roles in epidermal wound healing and liver regeneration, as mice mutated for these two genes show reduced cell proliferation and are deficient for skin wound healing and liver regeneration (Li et al., 2010).
Cell death machinery in Hydra
The Boettger lab convincingly demonstrated the global conservation of the cell death machinery in Hydra over the past 20 years (Cikala et al., 1999; David et al., 2005; Lasi et al., 2010a; Lasi et al., 2010b). From components mentioned above, Hydra does express hyCARD1/2 and hyCaspA predicted as orthologous to initiator caspases Dronc in Drosophila or Caspase-‐9 in mammals. HyDEDCasp and hyDDCasp are predicted to perform the similar function of Caspase-‐10 in mammals. Concerning the effector caspases, Hydra does express hyCaspB/C as a predicted ortholog to Drosophila’s DrICE and Dsp-‐1 and mammalian Caspase-‐3. As explained before, DIAP is an important anti-‐apoptotic protein in the fruit fly, and hyIAP is presumed to perform the same function in Hydra.