Development 107, 77-86 (1989) 77 Printed in Great Britain © The Company of Biologists Limited 1989 Regeneration and pattern formation in planarians III. Evidence that neoblasts are totipotent stem cells and the source of blastema cells JAUME BAGUNA1, EMILI SAL61 and CARME AULADELL2 1 Departament de Cenetica, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08028 Barcelona, Spain 2Unitat de Biologia Cellular, Departament de Bioqulmica i Fisiologia, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08028 Barcelona, Spain Summary In most regenerating systems, blastema cells arise by tion of neoblasts led to resumed mitotic activity, blas- dedifferentiation of functional tissue cells. In planarians, tema formation, and extended or complete survival of though, it is still debatable whether dedifferentiated cells the host; differentiated cells, in contrast, never did so. or a population of undifferentiated cells, the neoblasts, Therefore, planarian neoblasts can be qualified as are the main source of blastema cells. Moreover, it is totipotent stem cells and the main source of blastema unclear whether in the intact organisms neoblasts are cells, while dedifferentiation does not seem to operate quiescent cells 'reserved' for regeneration or if they either in intact or regenerating organisms. In addition, serve as functional stem cells of all differentiated cell these results strengthen the idea that different types of types. Both uncertainties partly stem from the failure to regeneration and blastema formation, linked to the distinguish by conventional labelling methods neoblasts tissular complexity of the organisms, are present in the from differentiated cells. animal kingdom. Here we describe a new approach to these problems based on testing the regenerative and stem cell capabili- ties of purified neoblasts and differentiated cells when Key words: neoblast, planarians, regeneration, blastema, introduced, separately, into irradiated hosts. Introduc- pattern formation, stem cells, totipotency. Introduction migratory undifferentiated cells (neoblasts) that had migrated throughout the irradiated region from the Although many studies have been published on the unirradiated area. Hence, neoblasts were qualified as mechanisms of blastema formation and differentiation totipotent and migratory cells capable of forming all the in planarians and the cell types that engage in it or do tissues of the regenerate (Dubois, 1949; Lender, 1962; not (see Br0nsted, 1969, and Chandebois, 1976, for Gabriel, 1970). general references) a main question remains still un- The 'dedifferentiation theory', mainly based on histo- answered: do blastema cells arise from a stock of logical and electron microscopic data (Flickinger, 1964; undifferentiated cells (neoblasts) already present in the Woodruff & Burnett, 1965; Hay, 1966; Rose & Shostak, intact organism or by dedifferentiation of differentiated 1968; Coward & Hay, 1972; Chandebois, 1976), functional cells? In the last 50 years, this issue has been suggests that neoblasts do not exist at all, or at least are polarized into what is called the 'neoblast versus dedif- not involved in regeneration, and that dedifferentiation ferentiation' controversy, confronting the so-called of specific cells near the wound is the main way of 'neoblast theory' and the 'dedifferentiation theory' recruiting cells to form the blastema. This is analogous (Slack, 1980; Baguna, 1981). to the situation found in regeneration of vertebrates The 'neoblast theory' stems from Wolff and Dubois and higher invertebrates. demonstration that the lack of regeneration after As presently stated, both theories are incomplete and X-irradiation in planarians was caused by the progress- open to criticism. The neoblast theory does not exclude ive disappearance of neoblasts and lack of cell turnover, the possibility that the migratory neoblasts making the and from the observation that an organism with an blastema may have resulted from the dedifferentiation unirradiated region (either a shielded region or an of differentiated cells in the unirradiated region before unirradiated graft) formed a blastema after a delay their migration to the wound. Moreover, it is also proportional to the distance between the wound and the possible that in normal regeneration differentiated cells healthy unirradiated tissue (Wolff & Dubois, 1948). near the wound can dedifferentiate and contribute, This suggested that a blastema can be formed by albeit slightly, to making the blastema. Finally, it has 78 /. Baguna, E. Said and C. Auladell been shown that the so-called migration of neoblasts is only known mitotic cells in planarians, several attempts not a true cell migration but the result of the slow and have been made to label them with tritiated thymidine progressive spreading of neoblasts mainly caused by to determine whether blastema cells are made of random movements linked to cell proliferation (Sal6 & labelled cells (coming from neoblasts) or unlabelled Baguna, 1985). Therefore, the idea of neoblasts as cells (coming from dedifferentiated cells). However, totipotent and migratory cells making exclusively the except for the reported incorporation of this precursor blastema is still unsettled. into neoblasts in the small turbellarian Microstomum The evidence for dedifferentiation in planarian re- lineare (Palmberg & Reuter, 1983) and Convoluta sp. generation is also not well founded. Three main criti- (Drobysheva, 1986), nobody has succeeded so far in cisms can be raised against it. First, evidence for labelling planarian neoblasts with thymidine or other dedifferentiation of gastrodermal cells to neoblasts precursor (for a discussion, see Coward etal. 1970). On claimed using,vital dyes as markers (Rose & Shostak, the other hand, the very few attempts made to label 1968) is hardly acceptable as the markers used diffuse differentiated cells specifically have also been unsuc- from cell to cell and proper controls were not done. cessful so far (Sal6, 1984). Second, recent EM studies have not found, despite a To overcome these difficulties, we describe here a careful search, any evidence of, or role for, dedifferen- new approach based on purifying, from total cell tiation in the formation of the regenerative blastema in suspensions, neoblasts and differentiated cells, and several species of planarians (Bowen etal. 1982; Hori, testing their regenerative and stem cell capabilities 1983, 1986; Morita & Best, 1984). Finally, mitotic when introduced, separately, into the parenchyma of studies have shown that the early blastema is formed irradiated hosts which have neither functional neoblasts from very early dividing (1-8 h of regeneration) G2 nor mitotic activity. The rationale of this experimental neoblasts, a period too short to make dedifferentiation approach is that if neoblasts are the stem cells of all (or likely (Sal6 & Baguna, 1984). most) differentiated cell types and the main cell type A case for dedifferentiation in planarians was making the blastema, their introduction into a dying brought up some years ago by Gremigni and co-workers nonregenerating irradiated host will lead to its survival in a very interesting series of experiments (Gremigni & and regeneration, whereas introduction of differen- Miceli, 1980; Gremigni etal. 1980, 1982). Using a strain tiated cells will neither rescue the host nor make it of planarians that are naturally occurring mosaics: the regenerate. Conversely, if dedifferentiation does occur, somatic cells are triploids, the male germ diploids, and introduction of differentiated cells will, eventually, the female germ hexaploid; they showed that regener- result in their dedifferentiation and, therefore, in the ation from a cut surface through the gonadal region rescue of the host and/or in its regeneration. gave rise to blastemas and regenerates that contained Whatever the outcome, the results obtained will be of mainly triploid cells, but also diploid and/or hexaploid general significance as they would support either the cells from which somatic cells (e.g. pharyngeal muscle existence in all animal groups of a general mechanism to cells) originated. This suggested that dedifferentiation obtain undifferentiated regenerative cells (e.g. dedif- and transdifferentiation (and hence, metaplasia), how- ferentiation), or the presence, in different animal ever limited, occurred during planarian regeneration. groups, of different mechanisms that may be linked to These results, held as conclusive evidence for dedif- certain structural or functional characteristics. ferentiation in planarians and, hence, for similar mech- anisms of blastema formation in most animal groups (Slack, 1980), can be criticized on the grounds that they Materials and methods do not demonstrate the occurrence of dedifferentiation and metaplasia but, at the most, suggest the existence of Species dedetermination (or transdetermination) (Baguna, Planarians used in this work belong to the fissiparous strain of the species Dugesia(G)tigrina, and to the sexual and asexual 1981). This is because the loss of a haploid complement races of Dugesia(S)mediterranea (Sal<5 & Baguna, 1985). during spermiogenesis and its doubling during They were maintained in Petri dishes in the dark at 17 ± 1°C oogenesis, though one of the first steps from neoblasts in planarian saline (PS; Sal6,1984) and fed with Tubifex sp. In to germ cells, is only a small step in cell determination all experiments, one-week-starved organisms were used and and occurs in undifferentiated