Wound healing, cellular regeneration and plasticity: the elegans way

Regeneration and wound healing are complex processes that allow organs and tissues to regain their integrity and functionality after injury. Wound healing, a key property of epithelia, involves tissue closure that in some cases leads to scar formation. Regeneration, a process rather limited in mammals, is the capacity to regrow (parts of) an organ or a tissue, after damage or amputation. What are the properties of organs and the features of tissue permitting functional regrowth and repair? What are the cellular and molecular mechanisms underlying these processes? These questions are crucial both in fundamental and applied contexts, with important medical implications. The mechanisms and cells underlying tissue repair have thus been the focus of intense investigation. The last decades have seen rapid progress in the domain and new models emerging. Here, we review the fundamental advances and the perspectives that the use of C. elegans as a model have brought to the mechanisms of wound healing and cellular plasticity, axon regeneration and transdifferentiation in vivo.


Introduction
Regenerative and wound healing capacities allow organisms to repair and regrow body parts after injuries. In humans, epithelia, including skin, intestine and lung, can heal and regrow, and a few organs, such as the kidney, endometrium and liver exhibit some regenerative abilities (Barker, 2014;Chou et al., 2014;Gargett et al., 2012;Kotton and Morrisey, 2014; Creative Commons CC-BY. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creative-commons.org/licenses/), which permits you to Share (copy and redistribute the material in any medium or format) and Adapt (remix, transform, and build upon the material for any purpose, even commercially), providing you give appropriate credit, provide a link to the license, and indicate if changes were made. You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use. Printed in Spain (https://creativecommons.org/licenses/by/4.0/). Mann et al., 2011;Michalopoulos, 2007). Regenerative capacities exist within all the metazoan phyla (Bely and Nyberg 2010;Sanchez Alvarado and Tsonis, 2006) and intensive research has focused on the underlying mechanisms.

Diverse regenerative mechanisms have been revealed through cellular tracking
The origin of the regenerating cells and the cellular path(s) they undertake to replenish the missing tissues have been the subject of many studies, leading to conflicting reports on the cellular replacement strategies. Strategies involving proliferation of endogenous replacement cells, be it resident stem cells or duplications of mature cells, have been opposed to strategies involving cellular plasticity, either through the proliferation of a dedifferentiated intermediate or via cell type conversion (aka transdifferentiation or direct reprogramming). Initially, the absence of single cell markers and of accurate lineage-tracing methods led to controversies or uncertainties concerning the origin of cells involved in regeneration in organs. Recent technical advances have improved our ability to track cell fates. For example, cellular labelling techniques using fluorophores, such as GFP or mCherry, allowed endogenous induced cells or transgenic grafted cells to be followed to determine their cellular potential, meaning the number of specific cell fates they could produce (Tanaka and Reddien, 2011). The study of muscle regeneration revealed the diversity of mechanisms that can be observed for one tissue. Striated muscle of the Xenopus tadpole tail regenerate thanks to muscle stem cells, called satellite cells (Chen et al., 2006). While this seems also true during Axolotl limb regeneration, in another salamander, the newt, myofiber fragmentation resulting in proliferating mononucleated cells was also shown to contribute to skeletal muscle regeneration (Sandoval-Guzman et al., 2014). In addition, cells were found to exhibit varied degrees of cellular potential during regeneration. For instance, muscle cells have restricted developmental potential in the axolotl and can only give rise to other muscle cells, whereas in the newt they can potentially give rise to cartilage as well (Kragl et al., 2009;Morrison et al., 2010;Tanaka and Reddien, 2011). Regenerating fish fin cells were also found to be lineage-restricted using similar cell-tracing methods (Jopling et al., 2011).
Thus, different mechanisms can lead to regeneration of the same tissue in closely related species suggesting that regeneration can be achieved via multiple paths (Simon and Tanaka, 2013). Even in a given species, not one, but a mosaic of regenerative mechanisms has been observed, depending on the tissue examined. The initial controversies have therefore begun to be solved in recent years, and in-depth examination of these phenomena have further pointed to the influence of the injury type or its extent, on the regenerative cellular mechanism at play. For instance, natural regeneration of the liver is thought to occur mainly via induced cell division and hepatocytes duplication following acute injury (Sadri et al., possesses a collagen-based cuticle which can heal after physical injury. This healing process similarly involves activation of the innate immune system, notably via the production of antimicrobial peptides (AMPs), actin polymerization at the wound site and ROS production, making it a simple system in which to probe the cellular dynamics and key factors at play Xu and Chisholm, 2014b). This review will thus focus on how C. elegans has allowed to study the fundamental mechanisms underlying aspects of regeneration and wound healing. C. elegans is a transparent round worm roughly 1 mm long at adult stage. The advantages of the model are summarised in Table 1. Three chapters are developed below: in the first part, we review how a simple level of tissue repair is observed in C. elegans and has contributed to a better understanding of wound healing; in the second part, the principles of axon regeneration are reviewed; finally, lessons from natural cellular plasticity events, and natural transdifferentiation (Td) in particular, are reviewed in the third part.
C. elegans do not involve cell proliferation, as the worm adult somatic cells are post-mitotic, nor does it involve cell migration. The differences in epidermis structures between mammals and C. elegans are presented in Table 2. The Fig. 1 summarises the mechanisms involved in wound healing in C. elegans.

Wound closure by F-actin polymerization
First, wounding the worm epidermis with a needle or laser triggers the release of internally stored epidermal Ca 2+ (Xu and Chisholm, 2011). The Ca 2+ spreads within the syncytial epidermis from the injured site to over 1/3 of worm's length in a matter of seconds, and is required for survival after injury. The TRPM (transient receptor potential melastatin) channel GTL-2 is required for this Ca 2+ release, possibly by initiating an initial influx of Ca 2+ after wounding that would trigger the release of the internally stored Ca 2+ . The phospholipase Cβ EGL-8 and its regulatory Gαq protein EGL-30 likely act downstream of the GTL-2 channel, and are responsible for the release of Ca 2+ from internal stores by acting on the IP3 receptor ITR-1 ( Fig. 1) (Xu and Chisholm, 2011). Subsequently, the repair of the injury site involves a CDC-42 small GTPase and ARP2/3-dependent actin ring formation that progressively closes the wound within 2 hours (Xu and Chisholm, 2011). This Ca 2+triggered wound closure by local actin polymerization is reminiscent of the mechanism described for the sea urchin coelomocyte wound healing (Henson et al., 2002). In addition, actin polymerization is also seen in the formation of lamellipodia, protrusions that are necessary for the process of epithelial cell migration during epithelial wound closure in vertebrates such as zebrafish and adult mammals (Martin, 1997;Richardson et al., 2016).
Another mechanism of wound closure called the "purse string" mechanism has been described, that allows, for example, the closure of Xenopus oocyte epithelial wounds at the single cell level or at the multicellular level (Clark et al., 2009) and of embryonic epithelia (Begnaud et al., 2016). This mechanism is CDC42-and RHO-dependent and uses contractile actomyosin cables. It is interesting to note that non-muscle myosin and the RHO-1 GTPase are negative regulators of C. elegans epidermis wound closure (Xu and Chisholm, 2011). Therefore, actomyosin cable formation could be competing with actin polymerization in the context of the C. elegans epidermal wound healing, possibly underlying the mechanistic shift between a "purse string" and a lamellipodia-like powered model (Begnaud et al., 2016).

Reactive oxygen species production
ROS are produced by various cellular compartments and take part in different biological processes including wound healing (Dunnill et al., 2017). An increase in ROS was found to be produced by the mitochondria (mtROS) at the site of epidermal or laser wounding, ultimately promoting the F-actin mediated wound closure. Analysis showed that the woundinduced increase in intracellular Ca 2+ level led to Ca 2+ uptake by the mitochondria via the mitochondrial Ca 2+ uniporter. This event triggered mtROS release by opening the mitochondrial permeability transition pore (mPTP). The released mtROS, mostly superoxide, then directly inhibited the redox-sensitive motif of the RHO-1 GTPase, thereby reducing RHO-1 wound closure inhibition activity ( Fig. 1) (Xu and Chisholm, 2014a). This study implicated for the first time the mitochondria in the process of wound signalling. The relevance of these pathways for mammalian epithelial wound repair needs now to be assessed. As a start, it has recently been shown that a Ca 2+ -triggered increase in mtROS Vibert et al. Page 4 Int J Dev Biol. Author manuscript; available in PMC 2018 September 28.
Europe PMC Funders Author Manuscripts production facilitates actin-mediated wound closure of injured skeletal muscle cells in mice (Horn et al., 2017).

Production of antimicrobial peptides (AMPs)
C. elegans doesn't possess specialized immune cells per se, but injury and bacterial or fungal infections induce an innate immune response that includes the production of antimicrobial peptides (AMPs), especially, in the case of fungal infection of the epidermis, members of the NLP (neuropeptide-like protein) and the CNC (caenacin) families (Engelmann and Pujol, 2010). While some AMPs are evolutionarily conserved (Braff et al., 2005;Mangoni et al., 2016;Pujol et al., 2008b), others show extensive lineage-specific diversification (Pujol et al., 2012). Different pathways, partially convergent, have been shown to be necessary for the increased production of the AMPs in the epidermis and are detailed below.
The nlp-29 and nlp-31 AMP genes are up-regulated in the worm epidermis following damage to the cuticle, either by physical wounding during a laboratory procedure or during a fungal infection involving piercing of the epidermis by the pathogen .
For production of these NLPs during wounding, the G-protein coupled receptor DCAR-1 (DihydroCaffeic Acid Receptor) is first activated by the endogenous ligand HPLA (4hydroxyphenyllactic acid, a tyrosine-derivative) . This activates the Gα protein GPA-12 and RACK-1 and the PLC-3 C-type phospholipase that act upstream of two proteins kinases C: TPA-1 and PKC-3 ( Fig. 1) Zugasti et al., 2014).
The signal transduction then requires the TIR (TOLL-interleukin 1 receptor) domain protein, TIR-1, orthologue of the human SARM (sterile α and armadillo motifs) that activates a p38 MAP kinase cascade Pujol et al., 2008a;Pujol et al., 2008b). The sodium-neurotransmitter symporter SNF-12 and its interactor the STA-2 transcription factor like protein have been shown to act downstream of the p38 MAPK pathway ( Fig. 1; Dierking et al., 2011). Injury also induces the expression of cnc genes, especially a subgroup constituted of cnc-1, cnc-5 and cnc-11 partially via the p38 MAP kinase pathway (Zugasti and Ewbank, 2009).
Sterile wounding also induces AMPs production in worms and mammals Sorensen et al., 2006). Additional roles of the AMPs in wound healing have been described in mammalian and non-mammalian model systems, for example in the stimulation of collagen synthesis or the inactivation of bacteria LPS (lipopolysacchararide) which reduces inflammation (Mangoni et al., 2016). AMP production occurs normally in mutants with defects in Ca 2+ signalling, but is negatively regulated by the Death Associated Protein Kinase DAPK-1 (Xu and Chisholm, 2011;Tong et al., 2009). This DAPK-1 calciumcalmodulin kinase is a negative regulator of the innate immune response, acting upstream of the TIR-1/p38 signalling cascade; upregulation of several epidermal AMP genes is observed in a dapk-1 mutant . DAPK-1 also negatively regulates the wound closure pathway (Xu and Chisholm, 2011). Therefore, DAPK-1 seems to be a general repressor of the epidermal wound healing process in C. elegans. Mutants for the nematode-specific gene sydn-1 suppress the morphological phenotypes of dapk-1, without affecting the elevated AMP gene expression provoked by loss of dapk-1 . dapk-1 also genetically interacts with patronin (PTRN-1), a regulator of microtubule stability that can antagonise DAPK-1 in the process of wound closure, and, in contrast to SYDN-1 is required for the elevated AMP gene expression seen in dapk-1 mutants (Chuang et al., 2016).
Interestingly, the homolog of DAPK-1 in mammals (DAPK) is also implicated in the immune response, with several roles in the regulation of the inflammatory response (Lai and Chen, 2014). Study of the DAPK-1 family in C. elegans is expected to provide insights on the necessary control of the coordinated responses to injury.

Conclusion and perspectives, wound healing studies in C. elegans
Even though there are differences between the mammalian and C. elegans models, like the skin structure and the presence in mammals of cellular immunity, it is now clear that most of the molecular players and the main pathways are conserved. Using the C. elegans model, the early role of the Ca 2+ release in wound healing has been shown at the organismal level for the first time. The worm allowed the characterization of the channel involved in this first Ca 2+ wave, namely GTL-2 (see above). A key aspect to elucidate in the future is to understand how the initial wounding signal is sensed. Mechanical properties of the damaged area during wounding are probably altered (Taffoni and Pujol, 2015;Dodd et al., 2018), and these could conceivably be sensed by TRPM channels and initiate the Ca 2+ release (Enyedi and Niethammer, 2015). This hypothesis remains to be tested and the worm would be a suitable model for such studies, which could then be extended to the mammalian epithelial TRP channels. The studies described above in the worm model have also shed light on the molecular pathways involved in ROS production during wound healing, in particular a newly described Ca 2+ -mediated burst in mtROS. It highlighted the beneficial consequences of ROS production on the regulation of innate immunity gene expression and actin-mediated wound closure at the protein level.
It is interesting to note that the majority of the proteins found to be involved in the response to injury in C. elegans have mammalian counterparts that are suspected to be involved in wound healing as well. Considering the complexity of the epithelial wound repair response in mammalians, C. elegans proves to be an interesting model system in which individual components of the epithelial wound response can be teased apart and their interactions analysed. The elucidation of these pathways might lead to improved therapies for wound treatment.

Axotomy reveals the cellular dynamics and the molecular aspects of axon regeneration in C. elegans
In mammals, axons of the central nervous system (CNS) regenerate poorly, whereas neurites of the peripheral nervous system (PNS) show better regenerative capacities (Chen et al., 2007). Why can axons in the PNS but not in the CNS regrow? Using models to study extrinsic and intrinsic factors crucial for axon regeneration is essential to answer this question.
The simplicity as well as the cellular and synaptic predetermination of the nervous system in C. elegans is a key advantage for axon regeneration's study. Hermaphrodites have 302 nerve cells and males have 385 neurons (www.wormatlas.org; White et al., 1986). Fifty-six glial- like cells are also observed, they are essential for axon guidance and good neuronal connectivity; however, axons are not myelinised in C. elegans (Oikonomou and Shaham, 2011). C. elegans neurons have unipolar or bipolar axon trajectories and can form synapses « en passant » meaning that cells can connect elsewhere than at the axon end, along the neurite or close to the cell body (Hobert, 2013). C. elegans produces all the main neurostransmitters found in mammals including acetylcholine, glutamate, γ-amino butyric acid (GABA), dopamine, and serotonin as well as their receptors (Hobert, 2013).
In 2004, Yanik et al.,2004), developed a technique of femto-second laser surgery in C. elegans for laser axotomy of single axons. Axon regrowth generally occurs within 6 to 24 hrs (Wu et al., 2007;Yanik et al., 2004). The combination of laser axotomy, with accurate single cell fluorescent marking, powerful genetics and phenotype analysis such as restoration of locomotion, have made of C. elegans an extremely powerful model to test axon regeneration molecularly and functionally at the single axon level. Another noninvasive method of axon severing has taken advantage of mutants in the β-spectrin coding gene unc-70 that cause a movement-induced breaking of axons in newly hatched larvae which triggers regeneration before the adult stage (Hammarlund et al., 2007).

Localisation of injury, age, as well as the neuron type are parameters that can influence efficiency and functional axon regeneration in C. elegans
Not all neurons have regenerative abilities in C. elegans but when they do, the process of axon regeneration per se is characterised by an error-prone regrowth as in mammals. Axons of the GABAergic neurons extend from the ventral side to the dorsal side of the worm, they are usually severed at a lateral point of the commissures to test regrowth dorsally (Yanik et al., 2004). Regrowth has been studied after laser axotomy or in the β-spectrin fragile axon model such as in Hammarlund et al., 2007. Results are not directly comparable but together they show that regrowth is observed in around 70% of the injured GABAergic neurons (Hammarlund et al., 2007;Wu et al., 2007;Yanik et al., 2004), but only 10% reconnect with dorsal side and re-establish motor functions (Byrne and Hammarlund, 2017;Wu et al., 2007). Gabel et al., (2008) also observed different rates of successful regrowth for the mechanosensory neurons, depending on the localisation of the damage (Gabel et al., 2008).
The bilaterally symmetric pair of posterior and anterior lateral touch neurons, PLM and ALM, have one long (300 μm in L4) and large axon that can be imaged in one focal plan which is a great advantage for dynamic imaging. The axon regrew when the laser surgery was performed proximal to the collateral synaptic branch whereas it did not regrow when the axon was injured distally to the branch point. Removal of the distal axon allowed regrowth suggesting that the synaptic branch point may be a sensor and a regulator with a positive or a negative effect on regrowth (Gabel et al., 2008). Age was also found to diminish axon regenerative capacities (Gabel et al., 2008).
Globally, axon regeneration follows the same dynamics in C. elegans as in mammals ( Fig.  2A). Below, we examine the recent advances of axonal regeneration in C. elegans: microtubule (MT) dynamics, axon fusion and key signalling pathways.

Injury-dependent disruption of microtubules and calcium transient release activate the DLK-1 and cAMP pathways required for axon regeneration in C. elegans
Axotomy disrupts the cell morphology by interrupting the cell plasma membrane and cutting the axon in two parts. What are the cellular consequences of this insult? The injurydependent disruption and changes in regulation of the cytoskeleton's dynamic is a pivotal parameter of axon regeneration cellular's response notably via formation of an injurydependent structure at the bulge of the distal axon, the growth cone. MT are architects of growth cone formation and their role in axon regeneration in C. elegans is reviewed in Chisholm (2013). In addition, MT disruption is interpreted as a cellular signal for activation of signalling pathways. The importance of MT's dynamics for axon regeneration has been highlighted by the identification in C. elegans of several mutants encoding inhibitors and activators of MT's dynamics. Three MT dependent phases can be described for regeneration: 1) An injury-dependent increase of MT depolymerisation/polymerisation resulting in injurydependent activation of signalling pathways. 2) Remodelling of the cytoskeleton associated with reduction of MT catastrophes which allows growth cone to build up. 3) Axon extension; at this final stage, stabilisation of assembled MT does not appear to prevent regeneration.
What are the signals triggered by axon severing? Several forward and reverse RNAi genetic screens have been performed to identify candidate activators and inhibitors of axon regeneration in C. elegans Hisamoto and Matsumoto, 2017;Nix et al., 2014;Nix et al., 2011) and the main factors are reviewed in Hisamoto and Matsumoto (2017). Calcium transient influx is a conserved key first signal triggered by injury, as revealed by the use of genetically encoded Ca 2+ sensors. This calcium transient increase, associated to IP3 increase, activates cAMP/PKA and the conserved DLK-1 regenerative pathways. Altogether these signalling pathways play multiple roles, ensuring remodelling and regulation of microtubule's dynamics for growth cone formation and extension. The process of growth cone formation and the inter-regulation within these pathways in C. elegans is summarised on Fig. 2B. Many of the key players and steps of axon regeneration are similar in C. elegans and mammals (mouse or rat), and Table 3 compares their role in the two species (Ghosh-Roy et al., 2012;Ghosh-Roy et al., 2010;Tang and Chisholm, 2016;Yan et al., 2009).
Fusion to the distal fragment allows robust functional regeneration. In C. elegans, the distal fragment of mechanosensory neurons, named ALM and PLM, can fuse with the growing axon in a process that is also promoted by calcium and cAMP (Ghosh-Roy et al., 2010;Neumann et al., 2015). Axotomy causes relocalisation of proteins at the distal and proximal membranes for fusion, as summarised in Fig. 2C (Neumann et al., 2015). Oren-Suissa et al., (2017) showed that the secreted factor AFF-1 mediates dendrite fusion with the epithelial fusion failure 1 (EFF-1) in cell non-autonomous manner (Oren-Suissa et al., 2017).

Understanding the effects of environment and developmental stage on axon regeneration
Diverse parameters can influence and reduce axon regeneration capacities. In C. elegans, the role of the niche, of stress and age have been investigated and progresses are described below. What are the key pro-regenerative molecular features setting a permissive environment for axon regeneration? Chen et al., 2007 recapitulates the factors favouring regrow in mammals.
Notably, in the PNS, macrophages and Schwann cells clear the fragment debris permitting the establishment of a permissive regenerative environment (Chen et al., 2007). The favourable niche for axon regrowth is also a combination of attractant and repellent molecules which allow axon guidance during axon extension. A number of them have been identified in C. elegans. svh-1 and svh-2 genes encode a growth factor and its receptor tyrosine kinase (RTK) respectively. SVH-1 is secreted from a pair of ADL sensory neurons in the head and acts on its receptor SVH-2 present in motor neurons and mechanosensory neurons to promote axon regrowth (Li et al., 2012). In addition, Gabel et al., (2008) elucidate the combined action of SLT-1/slit, which is secreted by dorsal muscle and acts as a repellent molecule, whereas UNC-6/netrin expressed by the axons acts as an attractant molecule, on AVM axon trajectories after laser surgery. Mutations in the netrin or SLT-1 led to 30-40% defects in AVM ventral guidance, whereas mutations in both resulted in 90% defects (Hao et al., 2001). Finally, Torpe et al., 2017 observed guidance defects in ret-1 mutants. ret-1 codes for the sole C. elegans member of the Nogo-A family. These defects depended on the activity of the ephrin ligand VAB-2 via the Eph receptor VAB-1 (Torpe et al., 2017). RET-1 appears to be involved in setting a « non-ephrin responsive » environment as in mammals, via a yet to be elucidated mechanism (Torpe et al., 2017).
Could axotomy/injury dependent axon regeneration activate a sum of stress-dependent pathways? Work in C. elegans highlighted a role for the stress Hypoxia induced (HIF) pathway, as found in spinal cord injury, and further studies identified relevant targets of HIF-1 in this context and showed that the HIF1 pathway is differentially used depending on the neuronal type (Alam et al., 2016). The current understanding of HIF-1 involvement in axon regeneration is summarised in Table 3. In addition, C. elegans studies suggested a role for genes ensuring mitochondrial functions, such as eat-3/Opa1, and of iron-sulfur proteins such as ISP-1. Interestingly, ISP-1 could act downstream of DLK-1 and Ca 2+ for axon extension (Knowlton et al., 2017). Finally, while increased protein synthesis is also observed during regeneration, the metabolic variations triggered by injury remain poorly explored and may be the focus of future investigations. Better characterising the injury-dependent molecular pathways related to stress as well as their cellular outcomes for axon regeneration is an interesting perspective that can be pursued in C. elegans.
What happens with age? Axon regeneration has been observed to decrease both in mammals and in C. elegans (Byrne et al., 2014;Gabel et al., 2008;Hammarlund et al., 2009;Nix et al., 2011.;Verdfi et al., 1995;Wu et al., 2007;Zou et al., 2013b). In C. elegans, age triggers a general dramatic decrease of growth response and extension and increased axon guidance errors after insult (Byrne et al., 2014;Wu et al., 2007). However, this may need to be examined at the individual neuron level, as Wu et al., (2007) established that the PLM, AVM and AWB neurites can regrow at adult stage. In the mechanosensory neurons PLM and ALM, functional regeneration efficiency is larval stage dependent. GABAergic motor neurons robustly regenerate in L1/L2 (nearly 100%); however, in L4, even if axons initiate injury response and formation/extension of growth cone, the nerve cord is not reliably reached, and cone mobility is reduced. The levels of MLK-1 seem somewhat predictive of success, since when MLK-1 is overexpressed then growth up to 40% cone initiation (against 12% in controls) is observed in adult and initiation is more than twice faster (Nix et al., 2011). MLK-1 can initiate and accelerate cone formation in a rather non-permissive, age dependent context. Byrne et al., (2014), suggested that the insulin receptor, DAF-2, could be responsible for inhibiting aging GABA motor neuron axon regeneration (Byrne et al., 2014).
They show that this effect is intracellularly mediated by the forkhead transcription factor DAF-16/FOXO, suggesting that aging effects are cell-autonomous (Byrne et al., 2014).
The impact of age is rather negative on axon regeneration and easy to assess in C. elegans.
However, it remains difficult to described the molecular mechanisms behind the changes caused by age. Altogether, more work in C. elegans could help progresses in deciphering how the niche, stress and age affect axon regeneration.

Cellular plasticity in C. elegans
During regeneration, cells undergo several transformations, at the morphological, physiological, transcriptional and translational levels, and they can also migrate. Many of these changes have been suggested to require a certain level of cellular plasticity. In fact, cellular plasticity appears to be a process broadly used during regeneration. One cellular phenomenon involved in such cellular plasticity is transdifferentiation (Td). Td is a process by which a differentiated cell changes its cellular identity to stably adopt another specialised one (Eguchi and Kodama, 1993;Selman and Kafatos, 1974). Studying Td is of major interest for two reasons: 1) It occurs in a wide range of species and settings and addresses fundamental cellular properties -yet little is known about the mechanisms at play; and 2) Experimentally induced Td bears great promises as a method to produce replacement cells in damaged organs.

Current picture and limitations to induced transdifferentiation in vertebrates
In mammals, natural conversion of venous cells to coronary arteries has been observed (Red-Horse et al., 2010). Uesaka et al., (2016) also reviewed neurogenesis from Schwann cell precursors in the enteric nervous system. However, owing to the difficulty of studying such process in vivo, natural Td remains marginally described in mammals, and little is known about its implication in organ regeneration in human. More recently, a host of studies have explored how to obtain various cell types through in vitro induced transdifferentiation, following the TFs cocktail logic first used by Takahashi and Yamanaka for induced pluripotent reprogramming (Takahashi and Yamanaka, 2006), but this time using developmental cell fate determinants TFs involved in the differentiation of the target tissue (Takahashi and Yamanaka, 2006;Xu et al., 2015;Zhou et al., 2008). These studies have been useful to investigate the activities of the transcription factors used to reprogram cell identities, and have suggested that a combination of pioneer transcription factors and of repressor TFs that repress the initial identity expression programme, may be desirable. In addition, they have highlighted the importance of epigenetic factors (Becker and Jarriault, 2016;Mall and Wernig, 2017;Patel et al., 2012;Tursun et al., 2011). Besides a more indepth understanding of the process, several milestones remain to be cleared for the study and potential therapeutic use of in vitro induced transdifferentiation in vertebrates: 1) On the technical side, and as emphasised earlier, a battery of cellular markers and reliable lineage- Europe PMC Funders Author Manuscripts tracing methods are critically needed. Strategies to increase the transparency of poorly accessible organs will also help to follow induced transdifferentiation in vivo in vertebrates.
2) Determine which cells are best to use as starting cells. Answering this question requires understanding of whether the initial identity of the starting cells impacts on the final one, and whether and how it impacts on the efficiency of the transdifferentiation process. 3) Induce efficiently Td, and safely. The current relatively poor efficiency of induced transdifferentiation begs the question of whether barriers to reprogramming exist. If so, understanding the nature of these barriers and whether their strength varies in different cell types will likely allow to design more efficient inducing protocols. In addition, the type of inducing cue and its mode of delivery (and removal) is paramount for potential future therapeutic applications. 4) Enable complete cell type conversion, at the genetic and epigenetic level. This will require sensitive techniques at the single cell level to ensure the selection of complete conversions. 5) Ensure stable cell type conversion over time, and in particular after the inducing reprogramming cue has been removed, a necessity for patients use. 6) Implement functional incorporation into existing tissue/cellular network.
One way to improve the efficiency and completeness of the conversion and obtention of a stable functional final cell is to learn from the mechanisms that naturally ensure these important aspects in vivo, during natural Td events (Hajduskova et al., 2012;Zuryn et al., 2012). Natural Td is by essence efficient and robust, as demonstrated by the perfect regeneration of a lens in newts after 19 successive lensectomies (Sousounis et al., 2015). We will thus focus here on natural reprogramming events in C. elegans. For an overview of TFinduced reprogramming experiments in C. elegans, we refer the readers to recent reviews covering the topic (e.g. Spickard et al., 2018;Becker and Jarriault, 2016).

Natural transdifferentiation occurs during C. elegans development and is accessible at the much needed single-cell level
Owing to the description of the cellular lineage (Sulston and Horvitz, 1977), a putative Td event was postulated and confirmed during the normal development of C. elegans (Jarriault et al., 2008). Notably, a rectal epithelial cell called "Y" transdifferentiates into a motoneuron called "PDA" (Fig. 3). We will also briefly review the conversion of differentiated glial cells (AMso) that give rise to male specific-interneurons through a cell division (Sammut et al., 2015), as it may represent another natural transdifferentiation event in the male. Below, we summarise the conceptual advances that the use of these models has brought.

Y-to-PDA Td: a natural rectal-to-neuron transdifferentiation that involves discrete steps and dedifferentiation in the absence of cell division
The Y cell is born during embryogenesis at around 290 minutes of development after fertilisation (Sulston et al., 1983) (Fig. 3A). Y is a rectal epithelial cell, expressing both epithelial and rectal markers (Jarriault et al., 2008). However, from the L2 to the L3 larval stages, Y retracts from the rectum and migrates anteriorly and dorsally (Fig. 3 A-D and see the timeline in Fig. 3F) and transdifferentiates into a motoneuron with a precise subtype identity: PDA (Fig. 3E) (Jarriault et al., 2008;White et al., 1986). In a forerunner study, Yto-PDA Td was shown to proceed through several steps even in absence of a cell division. This process first involves the erasure of the initial rectal identity, a sensu strictu dedifferentiation, before a step-wise redifferentiation that may mimic developmental differentiation (Richard et al., 2011). The first erasure of initial identity leads to a unipotent dedifferentiated intermediate (Y.0) rather than a multipotent one (Richard et al., 2011). No mixed identity stage is observed. These features (no mixed stage identity and sensu strictu dedifferentiation, uncoupled to increased cellular potential and reversion to stemness) appear to represent general principles, as preB-to-macrophage induced transdifferentiation proceeds similarly by first erasing the initial identity and expression programme before expressing the one of the final identity (Di Tullio et al., 2011). This occurs without retrodifferentiation, i.e no reversion to a previous and more potent precursor state (Di Tullio et al., 2011). In addition, lens regeneration in chicken and amphibians also involves transition through a dedifferentiated unipotent proliferative state (Dumont and Yamada, 1992;Sanchez Alvarado and Tsonis, 2006). In light of Td's therapeutic relevance, this is an exciting prospect as transition through unipotent cells might reduce the risk of tumorigenesis associated with loss of control of the cell's differentiation state.

AMso to MCM Td: generation of new neurons in C. elegans males after one round of division of glial cells
In males C. elegans a pair of glial cells (called the AMso cells for amphid socket cells) divide to give rise to two daughters with asymmetric fate: one is the adult amphid socket glial cell while the other adopts a neuronal identity (Sammut et al., 2015). These two new neurons made in males's head, called mystery cells of the male or MCM, express the neuropeptide pdf-1 and are involved in regulating sex-specific learning. MCM neurons are made in L4 at the time of sexual maturation. The use of a cell cycle marker together with ablation experiments conclusively showed that the AMso glial cells were the MCM progenitors. While further characterisation of the process will be required to fully characterise this event as Td as opposed to asymmetric cell division, it suggests that different transdifferentiation events, involving different initial identities, occur in the worm. Furthermore, it suggests that Td can proceed via a cell division or not in the same model organism. What does cell division add to the process? It is possible that DNA replication could facilitate Td, for example through the erasure of certain epigenetic marks, or by forcing DNA-bound factors to be released. It can also provide a way to enable Td in only one of the daughter cells by asymmetrically segregating Td -or conversely cell identity maintenance -factors.

The initiation of the process, materialised by a dedifferentiation, requires homologues of pluripotency factors that may define a conserved plasticity cassette
Unbiased screening for mutants affected in Y-to-PDA Td identified four critical players in this process: SOX-2 the C. elegans HMG box containing SOX2 factor; the POU-containing CEH-6, the closest C. elegans POU factor to OCT4; EGL-27, the worm counterpart of MTA1; and SEM-4, a SALL4 orthologue (Kagias et al., 2012). Each of these factors is absolutely critical for the initiation of the Td, corresponding to the dedifferentiation phase, to occur. Furthermore, EGL-27, SOX-2 and CEH-6 interact and conversely CEH-6/OCT interacts with SOX-2 and with the two isoforms of SEM-4/SALL suggesting that they could form a multiproteic complex (Kagias et al., 2012). Individually, the mammalian homologues of these factors are known for their key properties in inducing (for the OCT-4 and SOX-2 transcription factors), or maintaining (MTA1/2, a member of nuclear complexes with chromatin remodelling activities) pluripotency, or in enhancing the efficiency of pluripotent reprogramming (SALL4, a zinc finger transcription factor) (Chambers and Tomlinson, 2009;Liang et al., 2008;Wong et al., 2007). Furthermore, not only are these factors conserved but also the complex they form. In mammals, this complex, called NODE, is also found in Embryonic Stem (ES) cells where it is key to maintain their pluripotency (Liang et al., 2008). Altogether, these data suggest that these factors constitute a conserved plasticity cassette. It is possible that their primary or ancestral activity is to allow cells to reach a dedifferentiated state, a property that is combined with endowing cells with pluripotency in mammals. Interestingly, several recent studies have used pulses of SOX2 and OCT4 in direct reprogramming processes and found that it increased the reprogramming efficiency (Efe et al., 2011;Han et al., 2015;Kim et al., 2008;Kurian et al., 2013;Li et al., 2014;Margariti et al., 2012;Thier et al., 2012;Zhu et al., 2014). In fact, of these factors, it is suspected that CEH-6/Oct4 and SOX-2 play an early pivotal role, and maybe successive roles, together with a Notch signal, to make the Y cell competent to change its identity (Jarriault et al., 2008; and A. Ahier, T. Daniele, L.V. and S.J., personal communication). Thus, mimickingat least in part -the mechanisms that exist naturally during transdifferentiation will likely improve our ability to induce it experimentally.

Epigenetic factors such as histone methylase SET-1 and demethylase JMJD.3.1 are required to ensure robustness of dedifferentiation and redifferentiation during transdifferentiation
Zuryn et al., 2014 identified mechanisms and factors involved in Td efficiency and robustness in vivo during C. elegans Y-to-PDA Td (Zuryn et al., 2014). Two complexes involved in histone methylation have been identified, that are not only important to ensure a perfect Td every time, but also to ensure its robustness against environmental variations: jmjd-3.1 which encodes an orthologue of the human di-and tri-demethylase (H3K27me3/me2 demethylase) Jmjd3; and the SET1A/SET1B orthologue, set-2. The SET2 protein is the catalytic subunit of SET-1 for H3K4 methylation. Timed rescue experiments showed that SET1 acts mediating both dedifferentiation and redifferentiation whereas JMJD-3.1 promotes only redifferentiation. Partitioning of these histone-modifying activities is achieved via timely degradation of nuclear JMJD-3.1 during dedifferentiation and interaction with step-specific transcription factors: the SET1 complex subunit WDR-5.1 interacts with factors of the NODE-like complex for dedifferentiation whereas a WDR-5.1, JMJD-3.1 complex associates with UNC-3 for redifferentiation (Zuryn et al., 2014).
Altogether, these results suggest that dynamic regulation of the K27 and K4 methylation state are critical to ensure perfect and robust Td. Again, these interactions and roles are likely to underlie conserved general principles of cellular plasticity. Previous studies in mice showed that Set1 potentiates Oct4 role during iPSCs reprogramming through interaction with Wdr5, while Jmjd3 inhibits the initial phases of pluripotent reprogramming, in line with its detrimental activity if it is not removed during the dedifferentiation phase in C. elegans (Ang et al., 2011;Mansour et al., 2012;Zhao et al., 2013).

Conclusion
In this review, we have focused on the advances in the understanding of wound healing and regeneration obtained in C. elegans. Interestingly, tissular and cellular repair might share some mechanistic similarities. For instances, common molecular players such as the kinases of the MAPK pathway, or signals such as calcium, seem to be important for both wound healing and axon regeneration in C. elegans. By contrast, Td activation during regeneration or normal development seems to be depending on other complex intrinsic or extrinsic mechanisms. How are these strategies encoded at the molecular level remains obscur. In mammals, few organs are able to regenerate large portions following acute injury, and wound healing is predominantly associated with the formation of scar tissue. The poor regenerative abilities of humans have led to therapeutic strategies such as the graft of tissues and organs to replace the damaged ones in patients. In parallel to grafts from donors, tissue engineering using embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) has recently been developed. Nevertheless, protocols to obtain mature cells and promote their functional integration to the endogenous tissue are still lacking. Furthermore, in addition to the potential persistence of pluripotent intermediates, safety issues are raised both for ESCs and iPSCs by possible chromosome abnormalities and by the use of viral vectors to induce reprogramming and directed differentiation, and ethical questions regarding the use of ESCs exist. Transdifferentiation offers another alternative to provide replacements cells, one that avoids the transition through a potentially dangerous pluripotent stage, and that can be carried on either by inducing endogenous cells close to the wound, or by grafting additional cells engineered at the bench. Model organisms have thus much to contribute to move us closer towards understanding the fundamental mechanisms naturally at play during tissue repair and regeneration. It is hoped that these studies will lead to the combined improvement of our abilities to stimulate the endogenous regenerative, limit scar formation and to provide replacement cells. Left: epidermal wounding initiates a first influx of Ca 2+ within the cells through the TRPM channel Gtl-2. A subsequent molecular cascade involving the phospholipase Cβ Egl-8 and its regulatory Gαq protein Egl-30 leads to the release of Ca 2+ stored in the endoplasmic reticulum through the IP3 receptor Itr-1, increasing the intracellular concentration in Ca 2+ . The Cdc-42 GTPase could be activated by the increase in intracellular Ca 2+ concentration and promotes wound closure by actin polymerization through the regulation of actin nucleation factors (Wsp-1/WASP and Arx-2/Arp2/3). On the contrary, Ca 2+ mediated Rho-1 GTPase activation could inhibit F-actin polymerization, either by directly inactivating Cdc-42, or by promoting actomyosin cable formation by the intermediary of non-muscle myosin regulation. The increase in intracellular Ca 2+ level also activates ROS production by the Bli-3 dual oxydase. The increase in ROS and the Cst-1/MST1 protein are responsible for the translocation of the Daf-16/FOXO transcription factor in the nucleus where it will regulate the expression of innate immunity genes. The released Ca 2+ also enters the mitochondria via the mitochondrial Ca 2+ uniporter Mcu-1 and triggers the mtROS release by opening the mitochondrial permeability transition pore mPTP. The released mtROS inhibits the Rho-1 GTPase, thereby promoting the F-actin mediated wound closure. Right: Following damage to the cuticle, an increase in the HPLA ligand triggers the activation of the Gprotein coupled receptor Dcar-1. The Gα protein Gap-12 and Rack-1 and the Egl-8 and Plc-3 C-type phospholipases subsequently activate a signal transduction pathway consisting of the two protein kinases C Tpa-1 and Pkc-3, the Tir-1/SARM protein, and the Pmk-1/p38 MAP kinase cascade. The Snf-12 and Sta-2 transcription factors act downstream of this pathway to activate the AMPs genes. The Dapk-1 calcium-calmodulin kinase is an inhibitor of the molecular response to epidermal wounding, acting upstream of the Tir-1/p38 signaling (A) Injury first causes release of calcium (Ca 2+ ) via 1) membrane release (orange), 2) voltage-gated calcium channel opening (EGL-19 (VGCC a1)) causing a bidirectional membrane propagation (orange arrow) of Ca 2+ (blue), 3) Activation of Ca 2+ -dependent Ca 2+ release from internal storages leading to transient Ca 2+ waves (blue). Ca 2+ dependent activation of cAMP and DLK-1 regeneration-dependent pathway lead to three key steps of axon regeneration: 1) growth cone formation, 2) axon extension and guidance and 3) fusion to target when it occurs. (B) Growth cone formation: Increased calcium levels activate 1)  Europe PMC Funders Author Manuscripts expressed and required in the Y cell to promote its dedifferentiation; the LIN-12/NOTCH receptor is activated and is required until embryonic 2.2-fold for Y formation and transdifferentiation and sem-4/SALL4 is expressed from the embryonic 3-fold stage on (Kagias et al., 2012;T. Daniele & S. Jarriault personal communication). Redifferentiation requires the UNC-3/EBF transcription factor and the histone modifier JMJD-3.1. Perfect efficiency and robustness of the process is ensured via step-wise histone modifications involving H3K4 and H3K27 methylation (see WDR-5.1, JMJD-3.1, Zuryn et al., 2014). Vibert

C. elegans References
Simple culture conditions, little space requirement Petri-dishes containing agar and layer of the bacterium Escherichia coli Stiernagle, 2006 Compact annotated genome Sequenced: a list of C. elegans genes with human orthologues has been published (≈38% of C. elegans genes)

Shaye and Greenwald, 2011
Short life cycle 3 days at 25°C Four larval stages interrupted by molts Stiernagle, 2006 General Anatomical advantages 1 mm long at adult stage Transparency somatic lineage known Sulston and Horvitz, 1977;Sulston et al., 1983;Corsi et al., 2015 Statistics Powerful statistical analyses : one hermaphrodite produces 300 isogenic embryos Fay and Gerow, 2013 Analysis level Tissue and behavioural phenotypes Physiology Cellular (single cell level and precise cell identification) Molecular Huang and Sternberg, 2006;Yochem, 2006 Genetics tools Powerful genetic analysis, a library of loss-or gain-of-function mutants Amenable to forward and reverse systematic genetic screens Ahringer, 2006;Fay, 2013 Biotechnology available Imaging at single cell level Protein and mRNA traceability Transgenic animals, fluorescent reporters, CRISPR-assisted genome editing RNAi "Omic" approaches developed