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An insight into planarian regeneration

Abstract Background Planarian has attracted increasing attentions in the regeneration field for its usefulness as an important biological model organism attributing to its strong regeneration ability. Both the complexity of multiple regulatory networks and their coordinate functions contribute to the maintenance of normal cellular homeostasis and the process of regeneration in planarian. The polarity, size, location and number of regeneration tissues are regulated by diverse mechanisms. In this review we summarize the recent advances about the importance genetic and molecular mechanisms for regeneration control on various tissues in planarian. Methods A comprehensive literature search of original articles published in recent years was performed in regards to the molecular mechanism of each cell types during the planarian regeneration, including neoblast, nerve system, eye spot, excretory system and epidermal. Results Available molecular mechanisms gave us an overview of regeneration process in every tissue. The sense of injuries and initiation of regeneration is regulated by diverse genes like follistatin and ERK signaling. The Neoblasts differentiate into tissue progenitors under the regulation of genes such as egfr‐3. The regeneration polarity is controlled by Wnt pathway, BMP pathway and bioelectric signals. The neoblast within the blastema differentiate into desired cell types and regenerate the missing tissues. Those tissue specific genes regulate the tissue progenitor cells to differentiate into desired cell types to complete the regeneration process. Conclusion All tissue types in planarian participate in the regeneration process regulated by distinct molecular factors and cellular signaling pathways. The neoblasts play vital roles in tissue regeneration and morphology maintenance. These studies provide new insights into the molecular mechanisms for regulating planarian regeneration.


| INTRODUCTION
Considering the great threat to animal survival posed by injuryinduced loss of organs, regeneration is always an attracting topic in the field of medicine. According to the previous studies, the regeneration process occurs extensively in certain kinds of animals. For example, researchers have uncovered the regeneration ability of limb (axolotl), heart (zebrafish) and liver (human) after injury. [1][2][3] However, the exact biological mechanisms underlying regeneration are currently poorly understood and need to be further elucidated.
Planarians are flatworms capable of regenerating any missing body region, making it an ideal model to study the in-depth mechanisms of regeneration. The powerful regenerative ability of planarians benefits from a proliferative cell population that contains pluripotent stem cells, also called neoblast. Neoblasts widely distributed throughout the body, and account for 25%-30% of the body cells in the whole worms. 4 When injury happened, neoblasts near the wound migrate to the injury site and differentiate into tissue specific progenitor cells, then form a transparent tissue called blastema. [5][6][7] The blastema will express PCGs (positional control genes) for polarity determination. 8 The specification of the blastema and the subsequent formation of local signalling centres allow for the growth and patterning of the blastema, leading to the highly organized regeneration of lost body parts. 9,10 The migration, oriented differentiation and self-renewal of neoblast during planaria regeneration are regulated by a series of biological processes like cell cycle, apoptosis, proliferation and so on. Recent studies have shown that planaria can regenerate all tissue parts. Various signalling pathways and molecular mechanisms play important roles in the regeneration process of planarians. It is these organized specific regulation that enables planarians to accurately regenerate appropriate tissues after injury.
In this review, we first discuss the biological background of planarians, and then summarize the most recent findings about the critical regulatory factors and the underlying genetic and molecular mechanisms for the process of regeneration within every tissues of planarian.

| NEOBLAST: THE FOUNTAIN OF REGENERATION
The regenerative ability of planarians is mainly related to neoblasts. Therefore, it is of great significance to reveal how this cell type is involved in the regeneration process. As neoblasts are distributed throughout the body, the key to the above puzzle is to clarify the mechanisms for their directional differentiation into other cell types.
First, planarian stem cells are considered to be heterogenous cells with a high nucleo-cytoplasmic ratio, with a cell diameter of about 5-10 μm. At the same time, a commonly recognized characteristic of planarian stem cells is that the cytoplasm contains more chromatoid bodies, which enable the neoblasts to respond quickly to external stimulis. 11 In planarians, all dividing cells except sperm and eggs are considered as neoblasts. 4 Because of this characteristic, stem cells are sensitive to X-rays and gamma rays 12,13 which can stall cell division. The application of labelling and staining techniques also enables us to identify these stem cells. By staining the dividing cells with BrdU, the spatial and temporal distribution of stem cells can be clearly unvealed, 14 and In situ hybridization can locate the neoblasts with specific marker genes like smedwi-1, tgs-1 and tspan-1. [15][16][17] Neoblasts with dividing capability have significant role in regeneration, but not all neoblasts have the ability to differentiate into other cell types. Wagner et al. identified a group of neoblasts called cNeoblast (Clonogenic Neoblasts), cNeoblast can differentiate into all other tissues. If a cNeoblast was transplanted into a planarian that had been irradiated with X-rays to ablate all existing neoblast, the planarian could survive and regenerate neurons, muscles, protonephridia, intestines and all other tissues. 18 cNeoblast can be isolated by fluorescence-activated cell sorting using surface protein marker TSPAN-1. 16 By single-cell sequencing analysis, the researchers divided the planarian neoblasts into 12 different subpopulations, in which NB2 was the most pluripotent subpopulation. NB2 can differentiate into various progenitors including epidermal progenitor (NB3), muscle progenitor (NB4), gut progenitor (NB5), pharynx progenitor (NB7), protonephridia (NB9) and neural progenitor (NB11), which then differentiate into specific cell types under the regulation of different genes. 16 Upon injury, neoblasts in planarians can response immediately with two peaks of mitotic cell numbers (6 and 48 h post-amputation) in mitotic cell in numbers. The first peak of neoblasts proliferation occurs at 6 h, involving a wide range of neoblasts distributed throughout the body and being responsible for initiating the wound healing and regeneration process. The second peak occurs specifically following major injuries (amputation) and is biased towards wound sites. 19 Following the tissue loss, there are two peaks of apoptosis for tissue remodelling. Cell death (TUNEL + cells) occurred proximally at any wound within 4 h of injury, while a second sustained phase of elevated cell death involved in morphallaxis is specifically associated with tissue loss injury. These proliferative and cell death responses specific to missing tissue injury at 48-72 h after amputation, are specific to missing tissue injury, a striking features of planarian regeneration. [20][21][22] Neoblast repopulation can be achieved from even a single pluripotent stem cell in planarians. Proliferation of neoblasts involves symmetric and asymmetric division. 23,24 In the asymmetric division, neoblast divides into one self-renewing cell and one differentiated cell. This process is regulated by the Egf signalling pathway. EGFR-3 as the membrane surface of neoblasts is asymmetrically distributed when division initiate. Cell with more EGFR-3 protein turn out to be self-renewing cells, while the other cells without EGFR-3 protein differentiates into other cell types under the regulation of different signalling pathways. Interestingly, inhibition of egfr-3 results in the failure of cell division suggesting that egfr-3 regulates asymmetric cell division in neoblast repopulation. 25 Neoblasts is essential for planarians, but when and where the neoblasts differentiate in the process of regeneration and the mechanisms of regulating the termination of neoblasts differentiation are still a blur. Accordingly, the spatial technologies and other aspects of investigations is needed to elucidate these problems.

| GENERIC WOUND RESPONSE: HEALING OR REGENERATION?
How does the planarian distinguish the severity of injuries and determine the destinations of repair process, only requires wound healing or need to further regenerate missing tissue? What are the molecular mechanisms differentially controlling these process? Irrespective of healing or regeneration, more than 200 genes are activated in planarians after injury, but only amputation will result in the induction of missing-tissue response (MTR). 26 Gavino  targeted tail fragments will not be able to regenerate the lost tissue and no longer express PCGs, so the anterior-posterior (A/P) axis cannot be re-established. The follistatin negatively regulates wnt1, thus affecting head regeneration. 27 Owlarn et al. showed that virtually all injuries have the ability to induce regeneration, but regeneration only occurs when planarian tissues are lost or PCGs dysfunction, such as the Wnt signalling pathway. 28 They found that the expression of ERK signalling pathway increased significantly in a short time after injury, and the regeneration of planarians could not be carried out after disturbing any components of ERK signalling pathway. When the inhibition of ERK signalling pathway was removed, the regeneration of planarians can restart, indicating crucial roles of ERK signalling in initiating the regeneration process. 29

| ENVIRONMENTAL FACTORS INFLUENCE REGENERATION
The regeneration of planarians is also affected by environmental factors, such as temperature, gravity, reactive oxygen species and other factors. Researchers found that the optimal culturing temperature of planaria is from 19 to 25 C. 30 The study by Hammoudi et al. showed that the regeneration speed of planarian was the highest at 26 C. The planarian Schmidtea mediterranea would not survive between 30 and 32 C. However, there was no significant difference in migration and feeding behaviour of planarians at temperatures between 19 and 28 C. They also demonstrated that RNAi remained effective at 26 and 28 C by feeding dsRNA. Interestingly, the planarians reared at 19 C were able to completely clear Staphylococcus aureus within 6 days, while this clearing duration was shortened to less than 3 days cultured between 26 and 28 C, indicating that the immune response was intensified during 26-28 C. 31 Therefore, there also exist commensal bacteria in planarians, previous studies have shown that one planarian can harbour 42 distinct bacterial operational taxonomic units (OTUs), and the bacteria will delay the regeneration process of planarians by producing indole. 32 Michael Levin lab found that Aquitalea sp. FJL05 is an endogenous commensal bacterium of Dugesia japonica planarian. Aquitalea sp. FJL05 can influence A/P axial and positional patterning during regeneration, which will result in twoheaded animals. 33 Since there are many bacteria found in planarians, but not all the functions of these bacteria have been studies. In this case, it would be interesting to explore the possible effect of these bacteria on the planarian regeneration.
Gravity could also be an important factor regulating regeneration. on the regeneration of the Schmidtea mediterranea, and claimed that ROS is an early signal to induce tissue regeneration. They truncated planarian into three fragments at the pre-and post-pharynx levels to study the effect of ROS on regeneration of different body parts. They found that ROS bursts within minutes after amputation, and its production was independent of wound localization, while signals that initiate the regeneration process will only be induced at least 24 h after amputation. Inhibiting ROS production resulted in the failure of three segments to regenerate lost parts, and meanwhile, reduced ROS restricted regeneration of cephalic ganglia and ectopic neuronal cells.
Interfering with ROS production did not affect neoblasts proliferation, but constrained the differentiation of neoblasts into desired cell types. 36 These suggests that environmental factors are crucial in the process of regeneration, and missing any of these factors will result in the failure of regeneration. Investigations of environment factors will be an indicator for human living conditions. For example, studies on the impacts of different kind of pollution on regeneration will help us understand a lot diseases and give us a hint on environment protection.

| IMPACTS OF PHYSIOLOGICAL FACTORS ON REGENERATION
Cilia, which controls the movement of planarians, is an important tissue of planarians. They located on the ventral side of epidermis, and ciliary rootlets are globally polarized. Levin et al. established a framework for the alignment of cellular polarity vectors relative to planarian body plan landmarks. In their article, they found that ciliary rootlets can be regard as the indicator of epidermal polarity, which is defined by head, tail and the body margin. 37 Cellular polarity is also a crucial factor for regulating planarian regeneration. Levin et al. created double head planarians to demonstrate the flexibility of the body polarity.
By visualizing the orientation of cilia beat and cilia-driven flow in living planarians, they found that ciliary beat reorientated within several weeks to match the new axial body plan, and the progresses is independent from cilia beating. 38 Calcium (Ca 2+ ) channel play an important role in depolarizationinduced contractions of muscle, 39 which is indispensable to flatworm neuromuscular physiology. Knockdown of the subunit of Ca 2+ channel will lead to loss of mobility. 40 PCGs are mainly expressed in muscle, indicating the potential correlation between Ca 2+ channel and A/P axis. Praziquantel, which targets Ca 2+ channel, will lead to ectopic head in flatworms, suggesting the role of Ca 2+ channel in regulating stem cell differentiation and A/P axis patterning during regeneration. 41 Understanding the function of bioelectric, neurotransmitter and cell polarity during regeneration will link the physiological factors with molecular mechanism, which will help us to better understand the complex network underlying regeneration.

| ANTERIOR AND POSTERIOR POLARITY DICTATES PLANARIAN PATTERNING DURING REGENERATION
When planarian is injured, such as the head or tail amputation, signalling pathways guiding the error-free regeneration, that is the right tissues and organs in the right place, remains to be elucidated. Studies have shown that the Wnt signalling pathway is the key regulator of the polarity in planarians. β-catenin-1 RNAi will cause planarians to generate heads at any wounded site, resulting ectopic heads. [42][43][44] Knockdown of wntless, wnt1, teashirt and dishevelle genes will result in the planarian to regenerate its head at the tail place and grow multiple heads throughout the body. [44][45][46] Conversely, silencing the genes that are known as the inhibitor of the Wnt signalling pathway produces ectopic tail regeneration. Additionally, the Wnt-related genes are all distributed dependently along the A/P axis of planarian, and are specifically expressed in different regions. For example, genes in the Wnt signalling pathway, including wntP-2 (wnt11-5), wnt11-1, wnt11-2, fz4-1 and wnt1 are genes specifically expressed in the tail, 47 whereas the Wnt pathway inhibiting genes, such as sFRP-1, sFRP-2, foxD, fz5/8-3 and notum, are only expressed in the head region of planarian. 48,49 These findings illustrate the critical roles of the Wnt signalling pathway in regulating the polarity of planarians, pointing to the significance of Wnt functions in other species. Meanwhile, the expression of Wnt is regulating by hedgehog signalling, indicating the hh signalling is indispensable for A/P axis. 50 Interestingly, studies have shown that bioelectric signals in the very early stage of regeneration modulate the re-establishment of planarian A/P axis. Exploring the function of bioelectric signalling during regeneration will help us to understand the relationship between complex physiological patterns and molecular pathways. As early as 3 h post amputation (3 hpa), bioelectric signalling will participates in the A/P axis formation in planarians. In this case, depolarized the endogenous bioelectric at 3 hpa will create a double-headed worm. 51 Furthermore, the abnormality of A/P axis has is permanent effect even after briefly disturbed the endogenous bioelectrical networks is permanently, 52 indicating that bioelectric physiology is essential to animal morphology during planarian regeneration. RNAi screening to validate that the Ft/Ds pathway oriented ciliary rootlets towards the body margins, which will affect the D/V axis. 37 Reddien et al. found that when eliminating neoblast with irradiation, dramatic change of some PCGs still existed during regeneration, indicating that PCGs are not expressed in neoblasts. Several studies showed that the expression of most PCGs is restricted to a layer of subcutaneous cells around the neoblasts. 47,57,58 Subsequently, singlecell RNA sequencing on muscle cells from multiple regions along the planarian AP axis revealed that many additional genes exhibited regional expression in certain muscle tissues. 59,60 wnt1 is activated and expressed at approximately 6 h after injury, and its knockdown causes posterior-facing wounds to regenerate ectopic heads or fail to regenerate, suggesting that wnt1 promotes posterior regeneration and inhibits anterior regeneration. 58 wntP-2 is specifically expressed in the tail fragment, and has no expression in the head region after

| Eye spot
Planarian eyes connect to the brain via axon bundles, thus are true brain eyes with high expression of homologous genes like Otx, Sine oculis, ovo and eyes absence.

| Central nervous system
The nervous system is distributed throughout the planarian whose  66 Planaria head regeneration is a complex and intense process, which is initiated by the wound signalling after injury involving stem cells proliferation and migration to the head region to form a transparent tissue called blastema. The neoblast differentiates into neural system and eventually to regrow the head tissue. To further explain the molecular mechanism underly the CNS regeneration, we review some of newly discovered genes that essential to CNS regeneration and maintenance.
Recent studies showed that the CNS regeneration is negatively regulated by tec-1, while the maintenance of CNS is related to SoxB1.
The gene tec-1 is a negative regulator of neuron regeneration in planaria, with inhibitory role in the neurons regeneration of the brain. It was found that inhibition of tec-1 increased GABAergic, serotonergic, There are also many signalling pathways that regulate neural regeneration. Smed-egfr-4 is the downstream target of Smed-egfr-3 and Smed-egfr-4 is mainly expressed in the brain and necessary for brain regeneration. Smed-nrg-7 is a ligand of Smed-egfr-3. Knockdown of Smed-nrg-7 shows the same phenotype as semd-egfr-3(RNAi).

Reddien et al. found that hedgehog (hh) is expressed in medial
cephalic ganglia neurons and its inhibition led to the expression of filament-1 (if-1) and calamari (cali) express in unidentified non-neural CNS cell type (Figure 2). This type of cells expresses homologues of astrocyterelated genes involved in neurotransmitter uptake and metabolism, as well as extended processes covering regions of high synaptic concentration. It has been shown that the state of the glia is regulated by hh signalling. 72 As neuron regeneration is an intricated process involving many regulatory processes. One single gene or pathway alone cannot direct the whole process. Transcriptomic sequencing analysis by Roberts et al.
on the post-pharyngeal amputated planarians, found that about 45% of the transcripts were up-regulated during regeneration, including the aforementioned list of genes. RNAi screening on 326 up-regulated genes reveal that 9.2% (30/326) of genes could result in defective neuron regeneration after knockdown. These genes regulate nerve regeneration through multiple pathways, for example specification of neuronal progenitors, and anterior polarity and neural patterning. 73

| Excretory system
The excretory system of planarians consists of the protonephridia, which are branched organs widely distributed throughout the body.
Protonephridia are composed of ciliated "flame cells" attached to long tubules or cilia that end on the surface of the planarians. 71 Researchers have identified all genes from the planarian genome, which encode solute carrier (slc) transporter proteins being capable of transporting molecules in and out of protonephridia cells. A total of about 300 slc proteins were identified and their expression patterns were mapped in planarians by in situ hybridization. About 15% of these transporter genes were found to be expressed in the protonephridia. 74 As discussed above, 6 members of EGFR family exist in planarians, and egfr-3 and egfr-4 regulates neoblast repopulation and eye spot regeneration, respectively. In support, egfr-5 was demonstrated to be involved in the morphological maintenance and regeneration of the excretory system of planarians. Its depletion

| Epidermal
Epidermal is regulated by a novel cytoplasmic polyA-binding protein- and clarify its regulatory role of myb-1 in the differentiation process of early epidermal progenitors. 79 Also, Tubgcp3 is a homologue gene in planarian Dugesia japonica, which regulates the planarian epidermal progenitor cells differentiation. Knockdown of Tubgcp3 will result in lysis of amputated planarians. In the homeostatic Tubgcp3(RNAi) planarians, darker region of epidermis will appear and lead to body lysis in the end. 80

| Intestine
As the digestive organ of planarians, the intestine can be fully

Central nervous system
Ectodermal lineage progenitors can differentiate into epidermal and neuronal cells in the regulation of soxB1. During the regeneration process, soxB1, lhx1/5-1, pitx, egfr-4 and so on are important. pitx, slit, wnt1 and wnt11-2 will construct the midline of CNS.

Excretory system
The maintenance and regeneration of the planarian excretory system are mainly regulated by EGFR signalling.

Muscle
In the regeneration of process of muscle, myoD is required for the formation of longitudinal fibres, while nkx1-1 is required for the formation of circular fibres. myoD RNAi planarians cannot regenerate, while nkx1-1 RNAi will cause midline bifurcation.

Epidermal
During epidermal regeneration, myb-1 plays an important role in the specification of the first post-mitotic progenitor phase of epidermal. pabpc2/cdc24 RNAi will result in the abnormal of epidermal.

Intestine
The EGFR signalling pathway controls gut progenitor differentia- Many genes will control the regeneration of tissues, still we do not know how does regeneration know when to stop? And there is little researches on the morphology control of regeneration. We hope more and more researchers will be interested in these questions and focus on the field and make enormous contribution.
Planarian provides a valuable model system to dissect the underlying mechanisms for cell differentiation and tissue/organ regeneration in mammalian. Based on this, accumulating data about the regulatory molecular events have been uncovered and many regeneration models have been proposed. However, the detailed molecular mechanisms for planarian regeneration are still unclear, such as how the newly differentiated cells are directed to the wound site, and what are the underlying molecular network in coordinate regulation of neoblast differentiation. Since then, researchers have developed lots of approaches to investigate the regeneration. With the development of technology, we will have a clear understanding of the regeneration mechanism of the planarian, such as in suit hybridization, 15 RNAi, 89 single cell sequencing and other technology. 16,90 Though we can routinely use RNAi to reduce the expression of desired genes, there is no way to knockout the genes. Single cell technology is becoming more and more common, and the cost is gradually reduced. Moreover, the newly developed spatial transcriptome technology can be used at the same time. Spatial transcriptome can not only explore the detailed transcriptome information during regeneration, but also the spatial information, which will help us to understand the spatial information during regeneration. Levin et al. based on biochemical and biomechanical approaches, providing a brand new physical insight and useful biochemical approaches. The directed self-assembly of multicellular processes requires the understanding and regulation of the emerging structures, so this is of great significant for synthetic morphology. 91 And the computationally modelling of planarian regeneration is rapidly developing, which will provide new angles to understand the planarian regeneration. The comprehensive use of multiple technologies is of great help to solve the mysteries on planarian regeneration. 92 The recent advances in the RNA methylation have provided possible epigenetic mechanisms for the regeneration process. For instance, m 6 A play an important role in the maintenance and development of haematopoietic stem cells regeneration in vertebrate, [93][94][95] pointing to its essential roles in the process of tissue regeneration.
Although, there are a variety of databases providing the genome and transcriptome information needed for analysis, there is no complete genome data available due to high complexity of the genome and the difficulty of assembling. Also, the annotation information of the planarian is incomplete, and many specific genes of the planarian has not been defined by functional characterization. These genes are likely to play crucial roles in the regeneration process, but functional validation of these genes needs the joint efforts of many laboratories. At present, more and more technical means have been developed to analyse biological problems, which will be of great help to the study of the regeneration of the planarian. It is highly expected that, with the development of computing resources and technical approaches, the regeneration mechanism for the planarian will be eventually clarified. ORCID Guan-Shen Cui https://orcid.org/0000-0003-1352-7664