The Cellular and Molecular Basis for Planarian Regeneration

Regeneration is one of the great mysteries of biology. Planarians are flatworms capable of dramatic feats of regeneration, which have been studied for over 2 centuries. Recent findings identify key cellular and molecular principles underlying these feats. A stem cell population (neoblasts) generates new cells and is comprised of pluripotent stem cells (cNeoblasts) and fate-specified cells (specialized neoblasts). Positional information is constitutively active and harbored primarily in muscle, where it acts to guide stem cell-mediated tissue turnover and regeneration. I describe here a model in which positional information and stem cells combine to enable regeneration. Regeneration is one of the great mysteries of biology. Planarians are flatworms capable of dramatic feats of regeneration, which have been studied for over 2 centuries. Recent findings identify key cellular and molecular principles underlying these feats. A stem cell population (neoblasts) generates new cells and is comprised of pluripotent stem cells (cNeoblasts) and fate-specified cells (specialized neoblasts). Positional information is constitutively active and harbored primarily in muscle, where it acts to guide stem cell-mediated tissue turnover and regeneration. I describe here a model in which positional information and stem cells combine to enable regeneration. Regeneration involves the replacement of missing organs, appendages, or large body regions. Capacity for regeneration exists in representative species from almost every major animal phylum, including in chordates, platyhelminthes, ctenophores, arthropods, echinoderms, hemichordates, sponges, annelids, arthropods, nermerteans, and acoels. Interest in regeneration is centuries old, stemming from the prominent and widespread existence of regeneration in biology, interest in regenerative medicine, and also because the process easily captures the imagination: the regrowth of limbs, lower jaws, parts of the heart, spinal cord, and complete new heads ignites curiosity. How do highly regenerative animals do it, and why can’t we? Planarians are flatworms (phylum Platyhelminthes) found in freshwater bodies, and their regenerative abilities have been documented for centuries (Pallas, 1766Pallas, P.S. (1766). Miscellanea zoologica: quibus novae imprimis atque obscurae animalium species describuntur et observationibus iconibusque illustrantur. (Hagae Comitum: Apud Pterum van Cleef).Google Scholar, Dalyell, 1814Dalyell J.G. Observations on Some Interesting Phenomena in Animal Physiology, Exhibited by Several Species of Planariae. Illustrated by Coloured Figures of Living Animals, Edinburgh1814Crossref Google Scholar). Planarians can regenerate new heads, tails, sides, or entire organisms from small body fragments in a process taking days to weeks. Because of their ease of culture and robust regeneration, they have been popular subjects. For instance, planarian regeneration has caught the attention over the years (to differing degrees) of diverse investigators, such as Michael Faraday and T.H. Morgan (Faraday, 1833Faraday M. On the Planariae.The Edinburgh New Philosophical Journal. 1833; 14: 183-189Google Scholar, Morgan, 1898Morgan T.H. Experimental studies of the regeneration of Planaria maculata.Arch Entw Mech Org. 1898; 7: 364-397Google Scholar). A razor blade, magnifying glass, and imagination are enough for experimentation. Classical inquiry into planarian regeneration involved diverse injuries and transplantations. A suite of molecular and cellular tools have enabled a recent era of intensive molecular genetic inquiry into planarian regeneration (Umesono et al., 1997Umesono Y. Watanabe K. Agata K. A planarian orthopedia homolog is specifically expressed in the branch region of both the mature and regenerating brain.Dev. Growth Differ. 1997; 39: 723-727Crossref PubMed Google Scholar, Sánchez Alvarado and Newmark, 1999Sánchez Alvarado A. Newmark P.A. Double-stranded RNA specifically disrupts gene expression during planarian regeneration.Proc. Natl. Acad. Sci. USA. 1999; 96: 5049-5054Crossref PubMed Scopus (378) Google Scholar, Newmark and Sánchez Alvarado, 2000Newmark P.A. Sánchez Alvarado A. Bromodeoxyuridine specifically labels the regenerative stem cells of planarians.Dev. Biol. 2000; 220: 142-153Crossref PubMed Scopus (300) Google Scholar, Reddien et al., 2005aReddien P.W. Bermange A.L. Murfitt K.J. Jennings J.R. Sánchez Alvarado A. Identification of genes needed for regeneration, stem cell function, and tissue homeostasis by systematic gene perturbation in planaria.Dev. 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Fujiyama A. et al.Draft genome of Dugesia japonica provides insights into conserved regulatory elements of the brain restriction gene nou-darake in planarians.Zoological Letters. 2018; 4https://doi.org/10.1186/s40851-018-0102-2Crossref PubMed Scopus (1) Google Scholar, Fincher et al., 2018Fincher C.T. Wurtzel O. de Hoog T. Kravarik K.M. Reddien P.W. Cell type transcriptome atlas for the planarian Schmidtea mediterranea.Science. 2018; 360: 874Crossref Scopus (8) Google Scholar, Grohme et al., 2018Grohme M.A. Schloissnig S. Rozanski A. Pippel M. Young G.R. Winkler S. Brandl H. Henry I. Dahl A. Powell S. et al.The genome of Schmidtea mediterranea and the evolution of core cellular mechanisms.Nature. 2018; 554: 56-61Crossref PubMed Scopus (12) Google Scholar, Plass et al., 2018Plass M. Solana J. Wolf F.A. Ayoub S. Misios A. Glažar P. Obermayer B. Theis F.J. Kocks C. Rajewsky N. Cell type atlas and lineage tree of a whole complex animal by single-cell transcriptomics.Science. 2018; 360: 875Crossref Scopus (12) Google Scholar, Zeng et al., 2018Zeng A. Li H. Guo L. Gao X. McKinney S. Wang Y. Yu Z. Park J. Semerad C. Ross E. et al.Prospectively isolated tetraspanin+ neoblasts are adult pluripotent stem cells underlying planaria regeneration.Cell. 2018; 173: 1593-1608.e20Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Much excellent and fascinating work on planarian biology will not be reviewed here, such as the role of myriad molecules that give planarian stem cells (neoblasts) their attributes, the molecular genetics of the planarian germline, organ formation and function, signaling pathway function and evolution, cilia, genome repair and protection, aging, epigenetics, regulatory RNAs, immune biology, and planarian embryogenesis. Instead, I aim to synthesize key recent results into a mechanistic model for planarian regeneration. After introducing planarian biology, there are four sections. First, I describe pluripotent stem cells (cNeoblasts) and fate-specified stem cells (specialized neoblasts) that provide the cellular basis for regeneration. Second, I describe positional information that is harbored in muscle and how it is re-set after injury. Third, I describe how the combination of positional information and its influence on stem cells (neoblasts) can explain the logic of regeneration. I describe how progenitor targeting by extrinsic cues and self-organization combine to determine where regenerative progenitors go. Finally, I synthesize these findings into pillar concepts that promote understanding of regeneration, tissue turnover, and growth. Planarians have a complex anatomy including brain, eyes, musculature, intestine, protonephridia, and epidermis, all arranged in complex patterns (Hyman, 1951Hyman L.H. The Invertebrates: Platyhelminthes and Rhynchocoela The acoelomate bilateria.Volume II. McGraw-Hill Book Company Inc., New York1951Google Scholar). The bilobed planarian brain is comprised of myriad different neuron types and glia and connects to two ventral nerve cords. The body-wall musculature contains longitudinal, circular, and diagonal fibers. The epidermis produces mucous and is heavily ciliated ventrally for locomotion. A ciliated excretory system, the protonephridia, is distributed broadly for waste excretion and osmoregulation. A highly branched intestine distributes nutrients and connects to a muscular pharynx located centrally that serves as both mouth and anus. Surrounding internal organs is a mesenchymal tissue compartment (the parenchyma) that includes the only proliferative cells of the adult soma, the neoblasts. Extensive single-cell sequencing (SCS) has generated a transcriptome atlas for essentially all cell types that comprise planarian anatomy, giving planarians a wealth of molecular resources for their study (Fincher et al., 2018Fincher C.T. Wurtzel O. de Hoog T. Kravarik K.M. Reddien P.W. Cell type transcriptome atlas for the planarian Schmidtea mediterranea.Science. 2018; 360: 874Crossref Scopus (8) Google Scholar, Plass et al., 2018Plass M. Solana J. Wolf F.A. Ayoub S. Misios A. Glažar P. Obermayer B. Theis F.J. Kocks C. Rajewsky N. Cell type atlas and lineage tree of a whole complex animal by single-cell transcriptomics.Science. 2018; 360: 875Crossref Scopus (12) Google Scholar). Because small body fragments can regenerate an entire planarian, there exist mechanisms in the adult for the production of all adult cell types and tissue patterns. Planarian regeneration involves new tissue production in blastemas (Figure 1A). Because a small planarian body fragment cannot eat until suitable anatomy has been regenerated (including pharynx and brain), regeneration must occur with existing resources. Missing tissues thus cannot be regrown at their original scale. Consequently, blastema formation typically only regenerates some of the missing tissues (such as a head) and is coupled with changes in pre-existing body regions for the regeneration of other missing tissues (Figure 1A). Because the consequent animal will be smaller than the original, some tissues will initially be overabundant in the amputated fragment. Such tissues adjust their position and size relative to regenerating tissues (Figure 1B) (Morgan, 1898Morgan T.H. Experimental studies of the regeneration of Planaria maculata.Arch Entw Mech Org. 1898; 7: 364-397Google Scholar, Agata et al., 2003Agata K. Tanaka T. Kobayashi C. Kato K. Saitoh Y. Intercalary regeneration in planarians.Dev. Dyn. 2003; 226: 308-316Crossref PubMed Scopus (0) Google Scholar, Oviedo et al., 2003Oviedo N.J. Newmark P.A. Sánchez Alvarado A. Allometric scaling and proportion regulation in the freshwater planarian Schmidtea mediterranea.Dev. 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Petersen C.P. Positional information specifies the site of organ regeneration and not tissue maintenance in planarians.eLife. 2018; 7: e33680Crossref PubMed Scopus (1) Google Scholar, Atabay et al., 2018Atabay K.D. LoCascio S.A. de Hoog T. Reddien P.W. Self-organization and progenitor targeting generate stable patterns in planarian regeneration.Science. 2018; 360: 404-409Crossref PubMed Scopus (2) Google Scholar). These changes have been referred to as morphallaxis (Morgan, 1898Morgan T.H. Experimental studies of the regeneration of Planaria maculata.Arch Entw Mech Org. 1898; 7: 364-397Google Scholar, Reddien and Sánchez Alvarado, 2004Reddien P.W. Sánchez Alvarado A. Fundamentals of planarian regeneration.Annu. Rev. Cell Dev. Biol. 2004; 20: 725-757Crossref PubMed Scopus (402) Google Scholar). Morphallaxis is largely (if not entirely) the result of new cell production and cell loss rather than changes in the differentiated state of cells. Planarians can also regenerate missing tissues on their medial-lateral (ML) axis, such as following sagittal and parasagittal amputations (Figure 1C). Partial amputations can result in duplicated structures (Johnson, 1825Johnson J.R. Further observations on Planariae.Philos. Trans. R. Soc. Lond. 1825; 115: 247-256Crossref Google Scholar). A sagittal incision between the eyes, for instance, can result in the regeneration of two heads (Figure 1D). Removal of irregularly shaped tissue fragments can result in regenerative intercalation of missing anatomy (intercalary regeneration) between fused wound faces. Similarly, one region of the body can be transplanted next to another and regenerative responses can be triggered, including the formation of outgrowths that appear to intercalate missing coordinates between juxtaposed tissues (Figure 1E) (Santos, 1931Santos F.V. Studies on transplantation in planaria.Physiol. 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Muscle cells provide instructions for planarian regeneration.Cell Rep. 2013; 4: 633-641Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, Oderberg et al., 2017Oderberg I.M. Li D.J. Scimone M.L. Gaviño M.A. Reddien P.W. Landmarks in Existing Tissue at Wounds Are Utilized to Generate Pattern in Regenerating Tissue.Curr. Biol. 2017; 27: 733-742Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). The highly plastic, dramatic body plans that result from these various experiments are the source of endless fascination. Neoblasts are dividing cells that are widespread in the planarian body (Figure 2A). Neoblasts were recognized as simple, embryonic-like cells possessing a large nucleus and little cytoplasm, present in the context of adult tissues (Keller, 1894Keller J. Die ungeschlechtliche Fortpflanzungder Süsswasser-Turbellarien.Jen Zeit Naturw. 1894; 28: 370-407Google Scholar, Baguñà, 2012Baguñà J. The planarian neoblast: the rambling history of its origin and some current black boxes.Int. J. Dev. Biol. 2012; 56: 19-37Crossref PubMed Scopus (57) Google Scholar). Neoblasts are specifically eliminated by irradiation, and irradiated animals cannot generate any new tissues (Bardeen and Baetjer, 1904Bardeen C.R. Baetjer F.H. The inhibitive action of the Roentgen rays on regeneration in planarians.J. Exp. Zool. 1904; 1: 191-195Crossref Google Scholar, Dubois, 1949Dubois F. Contribution á l ’ètude de la migration des cellules de règènèration chez les Planaires dulcicoles.Bull. Biol. Fr. Belg. 1949; 83: 213-283Google Scholar, Reddien et al., 2005bReddien P.W. Oviedo N.J. Jennings J.R. Jenkin J.C. Sánchez Alvarado A. SMEDWI-2 is a PIWI-like protein that regulates planarian stem cells.Science. 2005; 310: 1327-1330Crossref PubMed Scopus (347) Google Scholar). A host of other histological, BrdU, transplantation, and transcript- and protein-labeling experiments demonstrated that all new somatic cells in tissue turnover and regeneration come from neoblasts (Baguñà et al., 1989Baguñà J. Saló E. Auladell C. Regeneration and pattern formation in planarians. III. Evidence that neoblasts are totipotent stem cells and the source of blastema cells.Development. 1989; 107: 77-86Crossref Google Scholar, Newmark and Sánchez Alvarado, 2000Newmark P.A. Sánchez Alvarado A. Bromodeoxyuridine specifically labels the regenerative stem cells of planarians.Dev. Biol. 2000; 220: 142-153Crossref PubMed Scopus (300) Google Scholar, Reddien et al., 2005bReddien P.W. Oviedo N.J. Jennings J.R. Jenkin J.C. Sánchez Alvarado A. SMEDWI-2 is a PIWI-like protein that regulates planarian stem cells.Science. 2005; 310: 1327-1330Crossref PubMed Scopus (347) Google Scholar, Eisenhoffer et al., 2008Eisenhoffer G.T. Kang H. Sánchez Alvarado A. Molecular analysis of stem cells and their descendants during cell turnover and regeneration in the planarian Schmidtea mediterranea.Cell Stem Cell. 2008; 3: 327-339Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, Baguñà, 2012Baguñà J. The planarian neoblast: the rambling history of its origin and some current black boxes.Int. J. Dev. Biol. 2012; 56: 19-37Crossref PubMed Scopus (57) Google Scholar). The neoblasts are therefore collectively pluripotent. Are neoblasts comprised of multiple different populations of renewing stem cells, each with restricted differentiation potential, or do they include cells pluripotent at the single-cell level? The key to addressing this problem involved assessing the potential of individual neoblasts. Irradiation and transplantation experiments demonstrated that some individual neoblasts can produce many more neoblasts (Wagner et al., 2011Wagner D.E. Wang I.E. Reddien P.W. Clonogenic neoblasts are pluripotent adult stem cells that underlie planarian regeneration.Science. 2011; 332: 811-816Crossref PubMed Scopus (286) Google Scholar) (Figure 2B). Upon transplantation, individual neoblasts restored regenerative capacity to lethally irradiated host animals that entirely lacked their own neoblasts, slowly converting a transplant recipient into a genetic clone of the donor (Wagner et al., 2011Wagner D.E. Wang I.E. Reddien P.W. Clonogenic neoblasts are pluripotent adult stem cells that underlie planarian regeneration.Science. 2011; 332: 811-816Crossref PubMed Scopus (286) Google Scholar) (Figure 2B). Furthermore, in animals irradiated with doses that leave sparse surviving neoblasts, neoblast clones are generated that produce neurons, epidermis, and intestine (Wagner et al., 2011Wagner D.E. Wang I.E. Reddien P.W. Clonogenic neoblasts are pluripotent adult stem cells that underlie planarian regeneration.Science. 2011; 332: 811-816Crossref PubMed Scopus (286) Google Scholar). Therefore, at least some neoblasts, the “cNeoblasts,” are pluripotent stem cells that provide the cellular basis for all new tissue production in planarian regeneration (Wagner et al., 2011Wagner D.E. Wang I.E. Reddien P.W. Clonogenic neoblasts are pluripotent adult stem cells that underlie planarian regeneration.Science. 2011; 332: 811-816Crossref PubMed Scopus (286) Google Scholar). An antibody to the protein Tetraspanin-1, which has enriched cell-surface expression in a subset of neoblasts, allows fluorescence-activated cell sorting (FACS)-based enrichment of cells that can be transplanted and have the functional hallmarks of cNeoblasts (Zeng et al., 2018Zeng A. Li H. Guo L. Gao X. McKinney S. Wang Y. Yu Z. Park J. Semerad C. Ross E. et al.Prospectively isolated tetraspanin+ neoblasts are adult pluripotent stem cells underlying planaria regeneration.Cell. 2018; 173: 1593-1608.e20Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The ability to prospectively isolate cells with cNeoblast properties should enable substantial molecular investigation of these pluripotent cells. Neoblasts respond to injuries by increasing their rate of proliferation. An initial peak in the proliferative response is widespread, occurs approximately 6 hr after injury, and is followed by a second phase of sustained proliferation near the wound occurring by approximately 48 hr after injury (Baguñà, 1976Baguñà J. Mitosis in the intact and regenerating planarian Dugesia mediterranea n.sp. I. Mitotic studies during growth, feeding and starvation.J. Exp. Zool. 1976; 195: 53-64Crossref Google Scholar, Saló and Baguñà, 1984Saló E. Baguñà J. Regeneration and pattern formation in planarians. I. The pattern of mitosis in anterior and posterior regeneration in Dugesia (G) tigrina, and a new proposal for blastema formation.J. Embryol. Exp. Morphol. 1984; 83: 63-80PubMed Google Scholar, Wenemoser and Reddien, 2010Wenemoser D. Reddien P.W. Planarian regeneration involves distinct stem cell responses to wounds and tissue absence.Dev. Biol. 2010; 344: 979-991Crossref PubMed Scopus (136) Google Scholar). The initial proliferative response occurs after any injury type, including injuries such as incisions, that do not require substantial production of new tissues (Wenemoser and Reddien, 2010Wenemoser D. Reddien P.W. Planarian regeneration involves distinct stem cell responses to wounds and tissue absence.Dev. Biol. 2010; 344: 979-991Crossref PubMed Scopus (136) Google Scholar). By contrast, the second phase of proliferation only occurs at wounds that result in substantial tissue loss and is referred to as a “missing-tissue” or “regenerative” response (Wenemoser and Reddien, 2010Wenemoser D. Reddien P.W. Planarian regeneration involves distinct stem cell responses to wounds and tissue absence.Dev. Biol. 2010; 344: 979-991Crossref PubMed Scopus (136) Google Scholar). This response is associated with neoblast accumulation at the wound. Additional responses also distinguish wounds that remove substantial tissue from those that do not. A burst in cell death (TUNEL+ cells) occurs proximal to essentially any wound within 4 hr of injury (Pellettieri et al., 2010Pellettieri J. Fitzgerald P. Watanabe S. Mancuso J. Green D.R. Sánchez Alvarado A. Cell death and tissue remodeling in planarian regeneration.Dev. Biol. 2010; 338: 76-85Crossref PubMed Scopus (155) Google Scholar), whereas a second, sustained phase of elevated cell death (involved in morphallaxis) is associated only with injuries that remove substantial tissue (Pellettieri et al., 2010Pellettieri J. Fitzgerald P. Watanabe S. Mancuso J. Green D.R. Sánchez Alvarado A. Cell death and tissue remodeling in planarian regeneration.Dev. Biol. 2010; 338: 76-85Crossref PubMed Scopus (155) Google Scholar). These proliferative and cell-death responses, detectable by 48–72 hr post-amputation and unique to missing-tissue injury contexts, are prominent features of planarian regeneration. How do neoblasts produce progeny cells that ultimately acquire the correct identity to replace missing tissues? Naive- and specialized-neoblast models were considered to address this question (Reddien, 2013Reddien P.W. Specialized progenitors and regeneration.Development. 2013; 140: 951-957Crossref PubMed Scopus (62) Google Scholar). The naive model posits that neoblasts are a largely homogeneous, naive population of cells. Fate specification would occur in their non-dividing, but not yet differentiated, progeny cells, for instance by the position of such cells in a blastema or by neighboring cells. The specialized-neoblast model posits that neoblasts are heterogeneous, with subsets having different fates. Blastema cells would thus have fates predetermined by which specialized-neoblast classes generated them (Figures 2C and 2D). Substantial evidence now supports the specialized-neoblast model (Reddien, 2013Reddien P.W. Specialized progenitors and regeneration.Development. 2013; 140: 951-957Crossref PubMed Scopus (62) Google Scholar). Studies of planarian eye and protonephridia regeneration first uncovered specialized-neoblast classes, demonstrating that fate specification for planarian regenerative lineages occurs in neoblasts (Lapan and Reddien, 2011Lapan S.W. Reddien P.W. dlx and sp6-9 Control optic cup regeneration in a prototypic eye.PLoS Genet. 2011; 7: e1002226Crossref PubMed Scopus (78) Google Scholar, Lapan and Reddien, 2012Lapan S.W. Reddien P.W. Transcriptome analysis of the planarian eye identifies ovo as a specific regulator of eye regeneration.Cell Rep. 2012; 2: 294-307Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, Scimone et al., 2011Scimone M.L. Srivastava M. Bell G.W. Reddien P.W. A regulatory program for excretory system regeneration in planarians.Development. 2011; 138: 4387-4398Crossref PubMed Scopus (83) Google Scholar). Because neoblasts are the only dividing somatic cell population, FACS with DNA labeling can isolate neoblasts in S/G2/M phases, called “X1” cells to reflect their X-ray sensitivity (Hayashi et al., 2006Hayashi T. Asami M. Higuchi S. Shibata N. Agata K. Isolation of planarian X-ray-sensitive stem cells by fluorescence-activated cell sorting.Dev. Growth Differ. 2006; 48: 371-380Crossref PubMed Scopus (141) Google Scholar). Some genes typically associated with differentiated cell function (such as the muscle marker DjMHC-A) were expressed in individual X1 cells, which indicated that potential functional heterogeneity would exist (Hayashi et al., 2010Hayashi T. Shibata N. Okumura R. Kudome T. Nishimura O. Tarui H. Agata K. Single-cell gene profiling of planarian stem cells using fluorescent activated cell sorting and its “index sorting” function for stem cell research.Dev. Growth Differ. 2010; 52: 131-144Crossref PubMed Scopus (68) Google Scholar). Eye-specialized neoblasts express eye-associated transcription factors (TFs), such sp6-9, dlx, otxA, six1/2/-1, eya, and ovo, and are required for eye regeneration (Lapan and Reddien, 2011Lapan S.W. Reddien P.W. dlx and sp6-9 Control optic cup regeneration in a prototypic eye.PLoS Genet. 2011; 7: e1002226Crossref PubMed Scopus (78) Google Scholar, Lapan and Reddien, 2012Lapan S.W. Reddien P.W. Transcriptome analysis of the planarian eye identifies ovo as a specific regulator of eye regeneration.Cell Rep. 2012; 2: 294-307Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Eye-specialized neoblasts have the appearance of all other neoblasts, except for the expression of these eye TF genes. The planarian protonephridia is a waste and osmoregulatory system that is distributed broadly in the planarian body and is comprised of multiple cell types (Hyman, 1951Hyman L.H. The Invertebrates: Platyhelminthes and Rhynchocoela The acoelomate bilateria.Volume II. McGraw-Hill Book Company Inc., New York1951Google Scholar, Rink et al., 2011Rink J.C. Vu H.T. Sánchez Alvarado A. The maintenance and regeneration of the planarian excretory system are regulated by EGFR signaling.Development. 2011; 138: 3769-3780Crossref PubMed Scopus (0) Google Scholar, Scimone et al., 2011Scimone M.L. Srivastava M. Bell G.W. Reddien P.W. A regulatory program for excretory system regeneration in planarians.Development. 2011; 138: 4387-4398Crossref PubMed Scopus (83) Google Scholar, Thi-Kim Vu et al., 2015Thi-Kim Vu H. Rink J.C. McKinney S.A. McClain M. Lakshmanaperumal N. Alexander R. Sánchez Alvarado A. Stem cells and fluid flow drive cyst formation in an invertebrate excretory organ.eLife. 2015; 4: e07405Crossref Google Scholar). Similar to the case of the eye, specialized neoblasts expressing TF-encoding genes associated with protonephridia formation (POU2/3 and six1/2-2) were found (Scimone et al., 2011Scimone M.L. Srivastava M. Bell G.W. Reddien P.W. A regulatory program for excretory system regeneration in planarians.Development. 2011; 138: 4387-4398Crossref PubMed Scopus (83) Google Scholar). RNAi studies demonstrated that POU2/3 and six1/2-2 were required for protonephridia-specialized neoblast formation and protonephridia regeneration (Scimone et al., 2011Scimone M.L. Srivastava M. Bell G.W. Reddien P.W. A regulatory program for excretory system regeneration in planarians.Development. 2011; 138: 4387-4398Crossref PubMed Scopus (83) Google Scholar). Subsequent work identified candidate specialized neoblasts for serotonergic (Currie and Pearson, 2013Currie K.W. Pearson B.J. Transcription factors lhx1/5-1 and pitx are required for the maintenance and regeneration of serotonergic neurons in planarians.Development. 2013; 140: 3577-3588Crossref PubMed Scopus (56) Google Scholar, März et al., 2013März M. Seebeck F. Bartscherer K. A Pitx transcription factor controls the establishment and maintenance of the serotonergic lineage in planarians.Development. 2013; 140: 4499-4509Crossref PubMed Scopus (0) Google Scholar) and other neurons (Wenemoser et al., 2012Wenemoser D. Lapan S.W. Wilkinson A.W. Bell G.W. Reddien P.W. 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