I.3. Cellular basis of Hydra regeneration
Sources of cells that are used for regeneration vary greatly in different regenerative animals. At the moment it is accepted that there are three main mechanisms that provide new cells during regeneration: (1) Stem cell activation, where resident stem cells start to divide and produce more cells like itself, following by differentiation into the required cellular types (Figure 2A, top) (Weissman et al., 2001). Clonogenic Neoblasts (cNeoblasts) in planarian regeneration (Wagner et al., 2011) and i-‐cell progenitors in Hydra mid-‐gastric apical regeneration (Chera et al., 2009b) are a good example of stem cell activation. (2) De-‐differentiation is a process where differentiated cells temporarily lose their differentiated characters, re-‐enter the cell cycle and produce cells that can now act as progenitor cells that continue to proliferate for a while to form a blastema and subsequently differentiate to form the missing structure (Figure 2A, middle) (Jopling et al., 2011). Cellular de-‐
differentiation is a main source for regeneration in zebrafish heart (Jopling et al., 2010), but also in bone regeneration in zebrafish fin (Knopf et al., 2011). (3) New cells can be a result of a process called trans-‐differentiation, during which a cell changes a state from one cell type to another, and this can occur without cell division (Figure 2A, bottom) (Jopling et al., 2011). Trans-‐differentiation is much less common than the previously mentioned mechanisms. Some invertebrates such as jellyfish have high trans-‐differentiation potential, but this is heavily reduced in vertebrate regeneration (Shen et al., 2004). Although not naturally occurring, but rather induced, common examples of this mechanism are the formation of the lens of the eye in the chick (Eguchi and Okada, 1973; Araki and Okada, 1977), or newt where pigmented
epithelial cells can de-‐differentiate and then re-‐differentiate into missing lens cells (Jopling et al., 2011).
Additionally, stem cells can be multipotent or be restricted for their contribution to the novel regenerated structure. Planarian cNeoblast are an example of classical pluripotent stem cells, while Hydra’s i-‐cells are undifferentiated multi-‐potent stem cell that when needed can provide many different cellular types, such as nematocytes, nerve or gland cells (Nishimiya-‐Fujisawa and Kobayashi, 2012) (Figure 2B), while for example in salamander and axolotl, limb regeneration is occurring in a much more restricted fashion (Figure 2C). Axolotl regenerates its limb using different stem cells that show lineage restriction, and there is no contribution of, for example, muscle cells to epidermis regeneration (Kragl et al., 2009).
Figure 2. Sources of new cells in regeneration
(A) Stem cells can have three distinct action patterns during regeneration: activation (top), de-‐differentiation (middle) and trans-‐differentiation (bottom). (B) cNeoblasts (S.
mediterranea) and i-‐cells (Hydra) show multi-‐potency, while in axolotl, muscle, skeleton or Schwan cells are lineage-‐restricted during regeneration (C). Scheme after (Tanaka and Reddien, 2011)
It is important to state that apical and basal regeneration in Hydra are very different.
While apical regeneration results in the formation of a complex head structure, basal regeneration results in a simpler structure, the foot. Also apical regeneration is simpler to follow, since it is visually easier to monitor the morphological changes such as the appearance of tentacle rudiments (Bode, 2003), especially with kinetics-‐
type experiments, and thus it was studied much more. On the level of cellular
A B
C
remodeling, Hydra apical regeneration can be divided into four different phases:
early, early-‐late, late and very late (Figure 3) (Galliot, 2013).
During the immediate phase (up to 2 hours post amputation; hpa) (Figure 3, top-‐
left), when the wound-‐healing process is launched, several important events take place. I-‐cells, located in epidermis undergo apoptosis while gastrodermal ESCs lose their typical morphology. In the early phase, between 2 and 12 hpa (Figure 3, bottom-‐left), apoptotic i-‐cells are engulfed by the gastrodermal ESCs, that now transiently lost their epithelial organization, which they re-‐gain in the early-‐late phase (Figure 3, top-‐right). Something similar to these cellular changes can be seen during the Hydra re-‐aggregation process (Murate et al., 1997). After the wound is successfully healed during the earlier phases, the late phase is characterized by a visible re-‐construction event, with the appearance of tentacle rudiments that become visible from 40 hpa (Figure 3, bottom-‐right) (Galliot, 2013).
Figure 3. Phases of cellular remodeling during Hydra apical regeneration
Hydra successfully performs the wound healing process during immediate to early phases in regeneration. ESCs in gastrodermis are shown in gray with red nuclei, and i-‐
cells as green spots in white epidermal ESCs. I-‐cells that undergo apoptosis are shown as stars, which are later being engulfed by gESCs that transiently lost their epithelial organization (bottom-‐left). First regeneration visual markers can be seen during the late phase (bottom-‐right), where tentacle rudiments appear, followed by formation of hypostome (Explained in details in the text). Scheme after (Galliot, 2013)
For some time, it was considered that Hydra undergoes only mophallaxis – a regenerative program that does not rely on cell proliferation (Bosch, 2007). However,
Immediate (0-2 hpa) Early (2-12 hpa) Early-late (>16 hpa) Late (>40 hpa)
most of the research on Hydra regeneration was based on decapitation, which was shown indeed to be mostly morphallactic. In 2009, the laboratory of B. Galliot has shown that upon mid-‐gastric bisection (an amputation made at 50% of animal body length) but not decapitation, proliferating cells accumulate in the head-‐regenerating tips immediately underneath the amputation site. Following mid-‐gastric bisection a wave of apoptosis occurs in the apical-‐regenerating (AR) tip of the animal, not present in the basal-‐regenerating (BR) one. Independently of this apoptotic event, progenitors of interstitial cells migrate towards the wound, thus accumulating under the apoptotic layer. The signals released by the dying cells push the progenitors to rapidly synchronously divide in the AR tips (Chera et al., 2009b). This report demonstrated the rapid asymmetrical cellular response on each side of the cut and introduced in the regeneration field the developmental role of the dying cells on regeneration.