Detection of Cell Death in Planarians

Background

Cell renewal in adult organisms is a complex mechanism based on three processes: (a) the elimination of selected cells by cell death; (b) the replacement of eliminated cells through cell division, typically involving adult stem cells and their descendants; and (c) the differentiation of newly generated cells and their integration with preexisting tissue (Pellettieri and Sanchez Alvarado, 2007; González-Estévez and Saló, 2010). In planarians, cell renewal must be continuously coordinated, since they grow and degrow depending on food availability and temperature (Baguñá and Romero, 1981). It is known that the changes in size result mainly from changes in cell number, rather than in cell size, so the ratio of dying/proliferating cells is controlled by environmental conditions (González-Estévez and Saló, 2010). Planarians are able to tolerate long starvation periods, and during this time, they degrow up to minimum sizes. Under these stressful conditions, food reserves from gastrodermis and mesenchyma are the firsts to be used, and at more extreme points, the sexual strains digest the sexual organs, and become asexual (González-Estévez and Saló, 2010; Miller and Newmark, 2012). When food is available, planarians are able to grow back, and in the sexual strains, the reproductive organs reappear. These cycles of grow and degrow occur throughout planarian lives without damage to the animal.

During planarian starvation, cell death increases to re-organize the organs and structures, and planarian adult stem cells (neoblasts) self-renewal is maintained at basal levels, resulting in a decrease of planarian body size (Figures 1A and 1B) (González-Estévez et al., 2012). The tissue remodeling is critical during planarian starvation because it maintains a proportioned planarian body. It was shown that JNK signaling, and Gtdap-1 are controlling the planarian body re-scaling during degrowing through the modulation of apoptotic cell death (González-Estévez et al., 2007; Almuedo-Castillo et al., 2014).

Because cell death is a relevant process in planarian, in the last few years molecular techniques to detect and measure cell death have been developed and optimized. Here, we will explain step-by-step the two main protocols used to detect cell death in planarians: measurement of Caspase-3 activity and TUNEL assay.


Figure 1. Planarian homeostasis. A. Planarians are able to grow and degrow during their lives, maintaining their body proportions and functionality. Image provided by Gustavo Rodriguez-Esteban. B. After a stimulus, proliferation and/or cell death can change in planarians. After feeding, neoblast proliferation increases throughout planarian body, and cell death is reduced to minimum levels, resulting in the increase of animal’s size. Conversely, when planarians are in starvation, neoblast proliferation is maintained at basal levels and cell death increases, which not only results in a decrease in body size but allows the reorganization of the tissues. Image from Nídia de Sousa Ph.D. thesis (de Sousa, 2017).