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Multicellularity, stem cells, and the neoblasts of the planarian Schmidtea mediterranea
Abstract
All multicellular organisms depend on stem cells for their survival and perpetuation. Their central role in reproductive, embryonic, and post-embryonic processes, combined with their wide phylogenetic distribution in both the plant and animal kingdoms intimates that the emergence of stem cells may have been a prerequisite in the evolution of multicellular organisms. We present an evolutionary perspective on stem cells and extend this view to ascertain the value of current comparative studies on various invertebrate and vertebrate somatic and germ line stem cells. We suggest that somatic stem cells may be ancestral, with germ line stem cells being derived later in the evolution of multicellular organisms. We also propose that current studies of stem cell biology are likely to benefit from studying the somatic stem cells of simple metazoans. Here, we present the merits of neoblasts, a largely unexplored, yet experimentally accessible population of stem cells found in the planarian Schmidtea mediterranea. We introduce what we know about the neoblasts, and posit some of the questions that will need to be addressed in order to better resolve the relationship between planarian somatic stem cells and those found in other organisms, including humans.
... Müller and Newman (1999) [18] refer to the "pre-multicellular and early multicellular world" as "pre-Mendelian" and, in the case of metazoans, conclude that the establishment of the major body plans was required for the "...increasingly unique matching between genotype and phenotype [that] led ultimately to Mendelian heritability" [19]. Alvarado and Kang (2005) [20] consider the emergence of stem cells as "... a fundamental evolutionary adaptation that allows multicellular organisms to satisfy Darwin's conditions of heritability and variation in fitness". The clearest expression of this view is probably that of Libby and Rainey (2013) [21]: "It is not sufficient to assume that Darwinian properties inherent in the lower level units are simply 'moved up' to the higher level. ...
... Müller and Newman (1999) [18] refer to the "pre-multicellular and early multicellular world" as "pre-Mendelian" and, in the case of metazoans, conclude that the establishment of the major body plans was required for the "...increasingly unique matching between genotype and phenotype [that] led ultimately to Mendelian heritability" [19]. Alvarado and Kang (2005) [20] consider the emergence of stem cells as "... a fundamental evolutionary adaptation that allows multicellular organisms to satisfy Darwin's conditions of heritability and variation in fitness". The clearest expression of this view is probably that of Libby and Rainey (2013) [21]: "It is not sufficient to assume that Darwinian properties inherent in the lower level units are simply 'moved up' to the higher level. ...
The major transitions in evolution include events and processes that result in the emergence of new levels of biological individuality. For collectives to undergo Darwinian evolution, their traits must be heritable, but the emergence of higher-level heritability is poorly understood and has long been considered a stumbling block for nascent evolutionary transitions. Using analytical models, synthetic biology, and biologically-informed simulations, we explored the emergence of trait heritability during the evolution of multicellularity. Prior work on the evolution of multicellularity has asserted that substantial collective-level trait heritability either emerges only late in the transition or requires some evolutionary change subsequent to the formation of clonal multicellular groups. In a prior analytical model, we showed that collective-level heritability not only exists but is usually more heritable than the underlying cell-level trait upon which it is based, as soon as multicellular groups form. Here, we show that key assumptions and predictions of that model are borne out in a real engineered biological system, with important implications for the emergence of collective-level heritability.
... The understanding of this phenomenon begins with the identification of the cells that are involved and the mechanisms that trigger and achieve these regenerative responses. It has been documented that regenerative models either have stem cells or cells that undergo dedifferentiation (Ferrario et al., 2020;Saló et al., 2009;Sánchez Alvarado, 2006;Sánchez Alvarado & Kang, 2005). For instance, regeneration in flatworms depends on the presence of neoblasts, proliferating stem cells that give rise to every cell in the organisms (Saló et al., 2009;Sánchez Alvarado, 2006;Sánchez Alvarado & Kang, 2005). ...
... It has been documented that regenerative models either have stem cells or cells that undergo dedifferentiation (Ferrario et al., 2020;Saló et al., 2009;Sánchez Alvarado, 2006;Sánchez Alvarado & Kang, 2005). For instance, regeneration in flatworms depends on the presence of neoblasts, proliferating stem cells that give rise to every cell in the organisms (Saló et al., 2009;Sánchez Alvarado, 2006;Sánchez Alvarado & Kang, 2005). In contrast, limb regeneration in salamanders depends on the dedifferentiation and proliferation of the cells adjacent to the injury site (Morrison et al., 2006;Tsonis, 2000aTsonis, , 2000b. ...
Regeneration is a key developmental process by which organisms recover vital tissue and organ components following injury or disease. A growing interest is focused on the elucidation and characterization of the molecular mechanisms involved in these regenerative processes. We have now analyzed the possible role of the Wnt/β-catenin pathway on the regeneration of the intestine in the sea cucumber Holothuria glaberrima. For this we have studied the expression in vivo of Wnt-associated genes and have implemented the use of Dicer-substrate interference RNA (DsiRNA) to knockdown the expression of β-catenin transcript on gut rudiment explants. Neither cell dedifferentiation nor apoptosis were affected by the reduction of β-catenin transcripts in the gut rudiment explants. Yet, the number of proliferating cells decreased significantly following the interference, suggesting that the Wnt/β-catenin signaling pathway plays a significant role in cell proliferation, but not in cell dedifferentiation nor apoptosis during the regeneration of the intestine. The development of the in vitro RNAi protocol is a significant step in analyzing specific gene functions involved in echinoderm regeneration.
... It is highly adventurous that during the development of the vertebrates, retinol is abundantly and specifically distributed in the posterior position, which is a niche for cellular differentiation through the transcriptional regulation of several specific genes [39] and functions as a signaling molecule [40]. Similar histological distribution of TsMFABP2 and retinol suggests that certain types of as-yet undefined neoblasts [41] deposited in the canal region might differentiate into diverse cell types during metamorphosis and maturation of the adult worm, during which retinol might mediate essential signaling. This intriguing issue awaits future studies. ...
... Unlike in deuterostomians, divergence of FABP-like proteins into each of the subfamilies seemed not yet occurred in protostomians including parasitic cestodes ( Figure 1B and Figure S2). Considering the fact that all the vertebrate iLBP genes conserve their genomic structures composed of four exons and three intervening introns [13], each gene for the subfamily lineages might have duplicated during an early stage of chordate evolution [41]. Meanwhile, invertebrate homologs display exon-intron structures distinguishable along with their donor organisms [13]. ...
Background:
Fatty acid (FA) binding proteins (FABPs) of helminths are implicated in acquisition and utilization of host-derived hydrophobic substances, as well as in signaling and cellular interactions. We previously demonstrated that secretory hydrophobic ligand binding proteins (HLBPs) of Taenia solium metacestode (TsM), a causative agent of neurocysticercosis (NC), shuttle FAs in the surrounding host tissues and inwardly transport the FAs across the parasite syncytial membrane. However, the protein molecules responsible for the intracellular trafficking and assimilation of FAs have remained elusive.
Methodology/principal findings:
We isolated two novel TsMFABP genes (TsMFABP1 and TsMFABP2), which encoded 133- and 136-amino acid polypeptides with predicted molecular masses of 14.3 and 14.8 kDa, respectively. They shared 45% sequence identity with each other and 15-95% with other related-members. Homology modeling demonstrated a characteristic β-barrel composed of 10 anti-parallel β-strands and two α-helices. TsMFABP2 harbored two additional loops between β-strands two and three, and β-strands six and seven, respectively. TsMFABP1 was secreted into cyst fluid and surrounding environments, whereas TsMFABP2 was intracellularly confined. Partially purified native proteins migrated to 15 kDa with different isoelectric points of 9.2 (TsMFABP1) and 8.4 (TsMFABP2). Both native and recombinant proteins bound to 11-([5-dimethylaminonaphthalene-1-sulfonyl]amino)undecannoic acid, dansyl-DL-α-amino-caprylic acid, cis-parinaric acid and retinol, which were competitively inhibited by oleic acid. TsMFABP1 exhibited high affinity toward FA analogs. TsMFABPs showed weak binding activity to retinol, but TsMFABP2 showed relatively high affinity. Isolation of two distinct genes from an individual genome strongly suggested their paralogous nature. Abundant expression of TsMFABP1 and TsMFABP2 in the canal region of worm matched well with the histological distributions of lipids and retinol.
Conclusions/significance:
The divergent biochemical properties, physiological roles and cellular distributions of the TsMFABPs might be one of the critical mechanisms compensating for inadequate de novo FA synthesis. These proteins might exert harmonized or independent roles on lipid assimilation and intracellular signaling. The specialized distribution of retinol in the canal region further implies that cells in this region might differentiate into diverse cell types during metamorphosis into an adult worm. Identification of bioactive systems pertinent to parasitic homeostasis may provide a valuable target for function-related drug design.
... The neoblasts proliferation can be evaluated also by the flow cytometry technique [19], which offers a broad range of applications in the cell research and provides possibility to investigate the stem cell proliferation. Planarian tissues consist of heterogeneous cell populations, more than 30 cell types [20], therefore adaptation of flow cytometry in the study of stem cells proliferation requires a specific technique for the preparation of cell suspensions from animal tissues. However, using the original method of cell isolation from planarian tissue [19], the recovery of all cell types was not possible. ...
... Thus, in planarian G. tigrina the tetraploid and the hexaploid fractions of the cells were observed, which disappeared 3 days after X-ray irradiation. These subpopulations of the cells were The term of ''heterogenous cell population'' relate in our work to the population of planarian somatic cells for about 30 cell types described based on their morphological organization [4,20]. By our method of cell preparation we can also identify several cell types microscopically, but by cytofluorymetric study only relative values (the level of fluorescence and the light diffraction level) can be measured. ...
The stem cells in freshwater flatworms (planarian) are called neoblasts. Neoblasts are capable of proliferation and differentiation into every cell type, including the gametes. For the investigation of the mechanisms of stem cells proliferation and differentiation the proper evaluation of changes in the cell cycle of neoblasts in different physiological conditions of planarian is necessary. In the present study the possibility of qualitative and quantitative characteristics of the neoblasts population were investigated using flow cytometry. In the cell suspension prepared from planarian tissue proliferating neoblasts have been observed in heterogenic cell population. Quantitative estimation of the cell cycle related changes of planarian stem cells system have been performed in various physiological conditions (intact and regenerating animals) and under the influence of physical (ionizing radiation) and chemical (melatonin and colchicine) factors. The modified protocol for planarian stem cells isolation proved to be effective and reproducible and can be recommended for flow cytometry analyses of human and animal proliferating cells.
... It has further been suggested that the invention of a gametogenic lineage, or at least a pluripotent lineage whose responsibilities included reproduction (see discussion by Sanchez Alvarado and Kang 2005), was not just an added bonus, but in fact a sine qua non of the evolution of multicellular organisms that acted, and were acted on by natural selection, as true individuals (Michod 1999). This is because as long as all cells retain the possibility to contribute to future generations, intra-individual competition among cell lineages is predicted to prevent the fitness gains of the group (that is, of the multicellular organism) from exceeding the fitness gains of the component cells. ...
... Acronym Full name Animal group found Naturally occurring Derivation Differentiation Potential References – Neoblast Planarians Yes Embryo Soma/Gametes (Shibata et al. 1999; Sanchez Alvarado and Kang 2005; Sato et al. 2006) – Archaeocyte Sponges Yes Embryo Soma/Gametes (Pilato 2000; Muller 2006) – Interstitial cell Cnidarians Yes Embryo Soma/Gametes (Littlefield 1985, 1991; Littlefield and Bode 1986; Bode 1996; Pilato 2000; Muller et al. 2004) ...
A key focus of evolutionary developmental biology (evo-devo) in recent years has been to elucidate the evolution of developmental mechanisms as a means of reconstructing the hypothetical last common ancestors of various clades. Prominent among such reconstructions have been proposals as to the nature of the mysterious "Urbilateria," originally defined as the last common ancestor of the extant Bilateria (protostomes and deuterostomes). Indeed, drawings of this animal can now be found, as well as detailed information on the genetics and morphological processes that it used to construct its gut, heart, eyes, appendages, segments, and body regions. Perhaps surprisingly, however, no explanations have yet been offered as to how this animal might have achieved the successful reproduction that must have been necessary for it to give rise to those lineages that are ancestral to today's diverse clades. The present article examines the comparative data available to date on the specification of the only cells containing the genetic hereditary material, the germ cells, and speculates on the possible evolutionary and developmental origin of the Urbilaterian germ line.
... Thus, neoblasts coexist in a microenvironment that is tightly regulated and allows them to respond to signals to self-renew, proliferate, and migrate, giving rise to differentiated progeny that are properly incorporated into demanding tissues. In recent years, an important focus of research in planarians has been centered on understanding, at the genetic and biochemical levels, how neoblasts are regulated in vivo to respond to demanding signals while renewing themselves (Agata 2003;Sánchez Alvarado and Kang 2005;Sánchez Alvarado 2006;Rossi et al. 2007a). ...
... Neoblast populations are mainly recognized by their morphology, spatial distribution, sensitivity to γ-irradiation, ultrastructural composition, and gene expression patterns (Orii et al. 2005;Reddien et al. 2005;Hayashi et al. 2006;Sato et al. 2006;Handberg-Thorsager and Saló 2007;Higuchi et al. 2007;Oviedo and Levin 2007;Rossi et al. 2007a,b;Wang et al. 2007). Study of the molecular conservation of regulatory molecules (e.g., the piwi genes) in neoblasts has expanded our knowledge of the evolution of stem cells (Sánchez Alvarado and Kang 2005). Furthermore, the identification and characterization of stem cell regulatory molecules that are conserved between vertebrates and planarians (but absent in other classical invertebrate model organisms such as Drosophila melanogaster and Caenorhabditis elegans) is now possible in S. mediterranea (Oviedo et al. 2008d). ...
INTRODUCTION
In recent years, planarians have been increasingly recognized as an emerging model organism amenable to molecular genetic techniques aimed at understanding complex biological tasks commonly observed among metazoans. Growing evidence suggests that this model organism is uniquely poised to inform us about the mechanisms of tissue regeneration, stem cell regulation, tissue turnover, pharmacological action of diverse drugs, cancer, and aging. This article provides an overview of the planarian model system with special attention to the species Schmidtea mediterranea . Additionally, information is provided about the most popular use of this organism, together with modern genomic resources and technical approaches.
... Neoblasts are the only cells capable of proliferation in planarians [7], present extreme sensitivity to X-and γ-ray irradiation, and account for 10-20% of the total cells in the organisms [8]. Neoblasts are mainly distributed in parenchymal tissues [9].When planarians are injured, neoblasts migrate and converge rapidly at the wound site, where a 1 blastema is formed [10]. The missing tissues are then gradually regenerated through the cell signal transmission and correct proliferation and differentiation of neoblasts [11][12][13]. ...
Stem cells and regenerative medicine have recently become important research topics. However, the complex stem cell regulatory networks involved in various microRNA (miRNA)-mediated mechanisms have not yet been fully elucidated. Planarians are ideal animal models for studying stem cells owing to their rich stem cell populations (neoblasts) and extremely strong regeneration capacity. The roles of planarian miRNAs in stem cells and regeneration have long attracted attention. However, previous studies have generally provided simple datasets lacking integrative analysis. Here, we have summarized the miRNA family reported in planarians and highlighted conservation in both sequence and function. Furthermore, we summarized miRNA data related to planarian stem cells and regeneration and screened potential involved candidates. Nevertheless, the roles of these miRNAs in planarian regeneration and stem cells remain unclear. The identification of potential stem cell-related miRNAs offers more precise suggestions and references for future investigations of miRNAs in planarians. Furthermore, it provides potential research avenues for understanding the mechanisms of stem cell regulatory networks. Finally, we compiled a summary of the experimental methods employed for studying planarian miRNAs, with the aim of highlighting special considerations in certain procedures and providing more convenient technical support for future research endeavors.
... Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a040931 ry out their whole-body regeneration (Sánchez Alvarado and Kang 2005;Reddy et al. 2019). In hydra, three major cell lineages with varying rates of self-renewal activity (ectodermal, ectodermal, and interstitial cells) are responsible for regeneration in this organism (Govindasamy et al. 2014;Siebert et al. 2019;Vogg et al. 2019). ...
For hundreds of years, the question of why some organisms can regenerate missing body parts while others cannot has remained poorly understood. This has been due in great part to the inability to genetically, molecularly, and cellularly dissect this problem for most of the history of the field. It has only been in the past 20-30 years that important mechanistic advances have been made in methodologies that introduce loss and gain of gene function in animals that can regenerate. However, we still have a very incomplete understanding of how broadly regenerative abilities may be dispersed across species and whether or not such properties share a common evolutionary origin, which may have emerged independently or both. Understanding regeneration, therefore, will require rigorously practiced fundamental, curiosity-driven, discovery research. Expanding the number of research organisms used to study regeneration allows us to uncover aspects of this problem we may not yet know exist and simultaneously increases our chances of solving this long-standing problem of biology.
... Therefore, spatial patterns of morphogens can serve as instructive pre-patterns to induce changes to single cell identity and ultimately specify aspects of the final body-plan [6,[15][16][17][18] (Fig 1). Planaria maintain a population of pluripotent adult stem cells (neoblasts), which migrate to wounds to form a blastema, where subsequent proliferation and differentiation ultimately regenerates lost portions of head, trunk, and tail [19][20][21][22]. Neoblasts respond to morphogenetic gene products [19,20,[22][23][24] and chemical messengers [25][26][27] to produce the needed cell types to transition the blastema at the former anterior portion of a wound into a new head, while creating tails at wound sites facing the original posterior. ...
Control of axial polarity during regeneration is a crucial open question. We developed a quantitative model of regenerating planaria, which elucidates self-assembly mechanisms of morphogen gradients required for robust body-plan control. The computational model has been developed to predict the fraction of heteromorphoses expected in a population of regenerating planaria fragments subjected to different treatments, and for fragments originating from different regions along the anterior-posterior and medio-lateral axis. This allows for a direct comparison between computational and experimental regeneration outcomes. Vector transport of morphogens was identified as a fundamental requirement to account for virtually scale-free self-assembly of the morphogen gradients observed in planarian homeostasis and regeneration. The model correctly describes altered body-plans following many known experimental manipulations, and accurately predicts outcomes of novel cutting scenarios, which we tested. We show that the vector transport field coincides with the alignment of nerve axons distributed throughout the planarian tissue, and demonstrate that the head-tail axis is controlled by the net polarity of neurons in a regenerating fragment. This model provides a comprehensive framework for mechanistically understanding fundamental aspects of body-plan regulation, and sheds new light on the role of the nervous system in directing growth and form.
... Asexual planaria have been a major model system for developmental and regenerative biology for over a century (for reviews, see Durant et al. 2016;Elliott and Sánchez Alvarado 2012;Lobo et al. 2012;Newmark and Sánchez Alvarado 2002). Planaria have a complex anatomy (see Fig. 1) comprising up to 40 distinct cell types (Sánchez Alvarado and Kang 2005). They have well-developed brains with photosensitive eye spots and paired ventral nerve cords (VNCs) that provide dense innervation to the rest of the body (Pagán 2014;Sarnat and Netsky 2002). ...
Freshwater planaria (Platyhelminthes, Turbellaria, Tricladida) pose a challenge to current concepts of biological individuality. We review molecular and developmental evidence suggesting that mature intact planaria are not biological individuals but their totipotent stem cells (neoblasts) are individuals. Neoblasts within a single planarian body are, in particular, genetically heterogeneous, migratory, effectively immortal, and effectively autonomous. They cooperate to maintain the planarian body as an obligate environment but compete to make this environment maximally conducive to the survival of their own neoblast lineages. These results suggest that planaria have not fully completed the transition to multicellularity, but instead represent an intermediate form in which a small number of genetically-heterogeneous, reproductively-competent cells effectively “farm” their reproductively-incompetent offspring.
... Proses selanjutnya adalah mekanisme regenerasi untuk menggantikan bagian tubuh yang hilang atau rusak. Mekanisme regenerasi planaria didukung oleh pembentukan jaringan blastema serta remodeling jaringan yang telah ada sebelumnya (Alvarado 2003;Reddien and Alvarado, 2004;Alvarado and Kang 2005). Proses remodeling jaringan melalui tersedianya neoblast, yaitu sel yang belum terdiferensiasi (undifferentiated cells) yang tersebar di seluruh bagian tubuh planaria, selanjutnya neoblast akan bermigrasi menuju daerah luka untuk memulai regenerasi yang diawali dengan proliferasi neoblast untuk membentuk blastema (Reddien and Alvarado, 2004). ...
Planaria dikenal sebagai cacing pipih yang merupakan hewan dalam Phylum Platyhelminthes dengan kemampuan regenerasi yang tinggi. Kandungan neoblast dalam tubuh planaria merupakan penyebab planaria dapat mengganti bagian tubuh yang hilang atau rusak melalui proses regenerasi. Meskipun demikian, banyak faktor diketahuhi mempengaruhi kemampuan regenerasi, diantaranya nutrisi, fisiologis tubuh serta faktor fisika kimia lingkungan. Penelitian ini bertujuan untuk mengevaluasi pengaruh perbedaan ukuran tubuh terhadap kemampuan regenerasi planaria dari perairan lereng gunung Slamet, Baturraden, Banyumas. Penelitian ini menggunakan metode eksperimental dengan Rancangan Acak Lengkap. Perlakuan meliputi perbedaan ukuran tubuh dengan kategori kecil (5 – 10 mm), sedang (10,1 – 15 mm) dan besar (15,1 – 20 mm), dengan 5 kali ulangan pada setiap perlakuan. Planaria diamputasi bagian anterior pharynk kemudian dipelihara dalam petri dish selama 20 hari pasca amputasi dengan suhu 22 – 24ºC. Setiap 2 hari, planaria diberi makan berupa hati ayam segar. Data pertambahan panjang jaringan regenerat diukur setiap 4 hari sekali. Planaria yang telah diukur difiksasi menggunakan bouin selama 24 jam untuk pembuatan preparat histologis. Data kuantitatif dianalisis menggunakan ANOVA dan data perkembangan jaringan regenerat dianalisis menggunakan analisis deskriptif. Hasil penelitian menunjukkan bahwa kemampuan regenerasi planaria dipengaruhi oleh ukuran tubuh. Hal tersebut dibuktikan dengan perkembangan jaringan regenerat yang lebih cepat pada planaria kelompok kategori besar dibandingkan kelompok lainnya. Sehingga dapat disimpulkan bahwa aktivitas regenerasi planaria meningkat dengan semakin besar ukuran tubuh planaria yang teramputasi.
... Irrespective of the broader evolutionary implications of pan-platyhelminth characteristics, the clade is also widely known for those of its members which have been adopted as models of fundamental zoological concepts. Freshwater planarians such as Schmidtea mediterranea (Tricladida) have a long history of utility in classical zoology, and modern molecular genetic appropriations of this system, as well as the more recently developed model Macrostomum lignano (Macrostomorpha) (Ladurner et al., 2005), have provided insights into especially non-embryonic developmental processes inaccessible in other familiar invertebrate models, such as whole body regeneration (Sánchez Alvarado, 2012), stem-cell maintenance (Sánchez Alvarado and Kang, 2005), tissue homeostasis (Pellettieri and Alvarado, 2007;Reddien, 2011), and aging (Mouton et al., 2011). The marine polyclad flatworms (Polycladida) have also been a subject of perennial study, not least due to their compelling reproductive biology: although they engage in (an often elaborately achieved [Michiels and Newman, 1998]) internal fertilization unlike most other marine macroinvertebrates, their embryos show a clear quartet spiral cleavage and cell fate (Boyer et al., 1998), and many species present a long-lived planktotrophic larva (Rawlinson, 2014) with well-developed ciliary bands and cerebral ganglia, which have been homologized to the trochophora larvae of other Spiralia (Nielsen, 2005). ...
ELife digest
Flatworms are relatively simple invertebrates with soft bodies. They can be found living in nearly every aquatic environment on the planet, are well-known for their ability to regenerate, and some species live as parasites in humans and other animals. Studies of the physical characteristics of flatworms have provided us with clues about how some groups, for example, the parasitic flatworms, have evolved, but the evolutionary origins of other groups of flatworms are less clear.
The genetic studies of flatworm evolution have focused on a single gene that makes a molecule called ribosomal ribonucleic acid, which is required to make all the proteins in flatworms and other animals. By comparing the sequences of this gene in different species of flatworm, it is possible to infer how they are related in evolutionary terms—that is, species with shared gene sequence features are likely to be more closely related than two species with less similar gene sequences. Although this approach has proved to be useful, it has also produced some results that conflict with the conclusions of previous studies.
Here, Laumer et al. studied the evolution of flatworms using an approach called RNA sequencing. This approach made it possible to sequence many hundreds of genes in all major groups of flatworms, and compare these genes in different species. Laumer et al. used the data to build a ‘phylogenetic tree’ that infers the evolutionary relationships between the different groups of flatworms.
This tree provides evidence that supports some of the ideas about flatworm evolution produced by the previous studies based on both physical features and ribosomal ribonucleic acid. It also presents several unexpected evolutionary relationships; for example, it suggests that the parasitic flatworms are most closely related to a group of small flatworms called Bothrioplanida, which are predators of other invertebrates. Bothrioplanida can live in many freshwater environments, and the physical characteristics that allow them to survive might resemble those found in the earliest parasitic flatworms.
The phylogenetic tree made by Laumer et al. represents a guide for researchers seeking clues to the origins of the genetic and developmental innovations that underlie the various physical features found in different flatworms.
DOI: http://dx.doi.org/10.7554/eLife.05503.002
... Planarians owe their regenerative ability and neuronal turnover (homeostasis) to a large population of somatic stem cells, called "neoblasts, " which populate the mesenchyme of the worm and account for ∼20% of all cells in the animal (Baguñà et al., 1989;Sánchez Alvarado and Kang, 2005;Baguñà, 2012;Zhu and Pearson, 2016; Figure 1). As far as has been examined, every tissue in the adult animal undergoes some level of turnover without injury, and every tissue can be regenerated. ...
Powerful genetic tools in classical laboratory models have been fundamental to our understanding of how stem cells give rise to complex neural tissues during embryonic development. In contrast, adult neurogenesis in our model systems, if present, is typically constrained to one or a few zones of the adult brain to produce a limited subset of neurons leading to the dogma that the brain is primarily fixed post-development. The freshwater planarian (flatworm) is an invertebrate model system that challenges this dogma. The planarian possesses a brain containing several thousand neurons with very high rates of cell turnover (homeostasis), which can also be fully regenerated de novo from injury in just 7 days. Both homeostasis and regeneration depend on the activity of a large population of adult stem cells, called neoblasts, throughout the planarian body. Thus, much effort has been put forth to understand how the flatworm can continually give rise to the diversity of cell types found in the adult brain. Here we focus on work using single-cell genomics and functional analyses to unravel the cellular hierarchies from stem cell to neuron. In addition, we will review what is known about how planarians utilize developmental signaling to maintain proper tissue patterning, homeostasis, and cell-type diversity in their brains. Together, planarians are a powerful emerging model system to study the dynamics of adult neurogenesis and regeneration.
... There are a number of different planarian species available for laboratory research. The most widely used is a clonal line of the Schmidtea mediterranea species which was generated via repeated rounds of cutting and regeneration (Alvarado, 2003;Alvarado & Kang, 2005). S. mediterranea are diploid, exist in sexual and asexual strains and their genome has been sequenced and annotated . ...
The full scope of regulatory RNA evolution and function in epigenetic processes is still not well understood. The development of planarian flatworms to be used as a simple model organism for research has shown a great potential to address gaps in the knowledge in this field of study. The genomes of planarians encode a wide array of regulatory RNAs that function in gene regulation. Here, we review planarians as a suitable model organism for the identification and function of regulatory RNAs.
... Neoblasts are the only mitotically active cells in planarians (14). Upon ageing or wounding, neoblasts migrate, proliferate and differentiate to replace lost cells (5,18). They account for 20-30% of all cells in planarians (5,19) and include both true pluripotent cells and already committed cells that retain neoblast-like features (5). ...
... Although traditionally, the terms "self-renewal," "differentiation," and "quiescence" are associated with metazoans and the stem cell concept in multicellular context, and the pluripotent stem cell systems supporting asexual reproduction in sponges was traditionally considered [102] as the origin form of stemness, to reach the roots of stemness it is necessary to analyze the protists, their life cycle, and their relationship with the ecological niche. In fact, if nobody questions the fact that all multicellular organisms depend on stem cells for their survival and perpetuation [103], the idea that somatic stem cells may be ancestral, with germ line stem cells being derived later in the evolution of multicellular organisms, is not yet generally accepted. In most cases the appearance of stemness is associated with the appearance of multicellular organisms. ...
In this chapter we present a comparative analysis of stem cell systems, retro-chronologically, from mammals to the simplest metazoans. In order to understand the roots of stemness—that is, the origin of self-renewal and differentiation phenomena—we also analyzed available data on the life cycle of protists. All these different cell systems were addressed through the prism of anaerobiosis, microaerobiosis, and aerobiosis in the geological context related to the time of appearance of the analyzed animal clades. This approach revealed some interesting analogies and pointed to the first eukaryotic common ancestor (FECA) and to the appearance of multiform life cycles of ancestral protists as the starting points of self-renewal and differentiation (as well as dedifferentiation), respectively. On the basis of this perception (i.e., this evolutionary paradigm), we propose our “integrative model of stemness.”
... The complete structure of planarians can be rebuilt from any piece of their body, a capacity that depends on the pluripotency of the neoblasts [33]. After cutting, the neoblasts adjacent to the wound proliferate, giving rise to the regenerative blastema where new structures will differentiate [34,35]. Our results showed that the appearance of auricles was easier affected by SDS than that of eyespots in the decapitated D. japonica fragments. ...
In this study, the regeneration performance and antioxidant activity effects of Sodium dodecyl sulfate (SDS) on planarian Dugesia japonica were investigated. The planarians were transversely cut just behind their auricles, and the tail fragments were allowed to regenerate at SDS solution with concentrations of 0, 0.5, 1, 1.5, 2, 2.5, or 3 mg/L, respectively. The head regrowth of the D. japonica was traced by daily observation in a Nikon SMZ1500 stereomicroscope during the process of regeneration. The number of planarians with regenerated eyespots and auricles in each sample was recorded. At the same time, superoxide dismutase (SOD) and catalase (CAT) activities were measured during the process of regeneration. The results showed that the appearance of auricles was more susceptible to SDS treatment than that of eyespots in regenerating planarians, and SDS modified the activity of antioxidant enzymes by increasing SOD and CAT during the process of regeneration.
... Even pieces of planaria that do not contain cephalic ganglia or ventral nerve cord can regenerate an intact, fully-functional nervous system (Morgan, 1898;Gentile et al., 2011). This regenerative potential arises from an abundant population of pluripotent adult somatic stem cells known as neoblasts, which comprise approximately 20-35% of cells in a planarian (Sanchez Alvarado and Kang, 2005;Baguñà, 2012;Wagner et al., 2011). The stem cell-specific regulatory programs in planarian neoblasts have been shown to have both unique and highly conserved elements, with genes found to be enriched in stem cells conserved across humans, mice, and planaria ( Labbé et al., 2012;Resch et al., 2012). ...
Prenatal exposure to ethanol affects neurodevelopmental processes, leading to a variety of physical and cognitive impairments collectively termed Fetal Alcohol Spectrum Disorders (FASD). The molecular level ethanol-induced alterations that underlie FASD are poorly understood and are difficult to study in mammals. Ethanol exposure has been shown to affect regulation and differentiation of embryonic stem cells in vitro, suggesting that in vivo effects such as FASD could arise from similar alterations of stem cells. In this study, we hypothesize that ethanol exposure affects head regeneration and neuroregeneration in the Schmidtea mediterranea planarian. S. mediterranea freshwater flatworms have remarkable regenerative abilities arising from an abundant population of pluripotent adult somatic stem cells known as neoblasts. Here, we evaluated the mobility-normalized photophobic behavior of ethanol-exposed planaria as an indicator of cognitive function in intact and head-regenerating worms. Our studies show that exposure to 1% ethanol induces a delay in the reacquisition of behavior during head regeneration that cannot be attributed to the effect of ethanol on intact worms. This suggests that the S. mediterranea planarian could provide insight into conserved neurodevelopmental processes that are affected by ethanol and that lead to FASD in humans.
Copyright © 2015. Published by Elsevier Inc.
... Die Plattwürmer (Plathelminthes) gerieten in der Ära der modernen Molekularbiologie als Forschungsobjekte weitestgehend in Vergessenheit, wurden aber jetzt als Modellorganismus neu entdeckt. Zu den Plattwürmern gehören neben den unangenehm parasitischen Gruppen, wie den Bandwürmern und Leberegeln, auch die frei lebenden Strudel-würmer (Turbellarien), deren bekanntester Vertreter die Planarien sind [2]. Auf den ersten Blick mag es seltsam erscheinen, diese Würmer als Modellorganismen für die Molekularbiologie zu wählen. ...
Flow cytometry is by far the most sophisticated and accurate method for sorting of living stem cells. In general, the target cells need to be labeled for various cell identity markers, which are exposed on the surface of the cells. However, in non-model organisms, we usually lack specific labels for such cell surface markers. Here we describe a method for isolating stem cells from planarians with flow cytometry, based on physiological and morphological properties of these cells.
... It is clear the emergence of stem cells and their development was the prerequisite for the evolution of multicellularity [12], but the evolutionary origin of stem cells and their evolutionary history remain obscure and poorly understood [13]. Unfortunately, microbial eukaryotes remained virtually unstudied. ...
The appearance of stem cells was a major evolutionary advance. It remains however unknown which ancestral biosystem evolved first for stemness and under what conditions. The purpose of this report is to provide an overview of the evolutionary origin of stem cells. It summarizes the current knowledge and dogmas on the origin of stem cell evolution. Understanding stemness in metazoans illustrates the basic principles of lineage organization and stem cell hierarchy in ancient single-celled eukaryotes.
Keywords: Entamoeba; Ancient stem cells (AnSC); Cell differentiation; Physiological hypoxia; Quiescent cells
... The enormous regenerative capacity of the E. multilocularis metacestode is demonstrated by the fact that metacestode tissue can be kept for years to decades in the laboratory through serial passages in the peritoneum of suitable intermediate hosts (e.g., Mongolian jirds) (Siles-Lucas and . Such regenerative capacity is a hallmark of flatworms and has been attributed to a population of totipotent stem cells called 'neoblasts' in the case of free-living flatworms such as planarians or 'germinal cells' in case of the obligately parasitic groups (Reuter and Kreshchenko, 2004;Sanchez-Alvarado and Kang, 2005). Neoblasts (or germinal cells) are regarded to be the only flatworm cell type that is mitotically active (Reuter and Kreshchenko, 2004) and, in E. multilocularis, germinal cells are thought to contribute crucially to metacestode growth via fusion with the tegument as well as to metastasis during chronic infection Mehlhorn et al., 1983). ...
... The freshwater planarian Schmidtea mediterranea (S. med), is a free-living flatworm of the lophotrochozoan superphylum and is well known for its ability to regenerate any missing body part as an adult, including the entire CNS and peripheral nervous system (PNS) (Cebrià, 2007;Cebrià, 2008;Cebrià et al., 2002;Gentile et al., 2011;Reddien and Sánchez Alvarado, 2004). This regenerative ability is made possible by a large population of pluripotent adult stem cells (ASCs) (Sánchez Alvarado and Kang, 2005;Wagner et al., 2011). S. med is a constitutive asexual adult and is amenable to gene-function studies by use of RNA interference (RNAi), which is fed to adult animals and subsequently spreads to all tissues, including neurons (Sánchez Alvarado and Newmark, 1999). ...
In contrast to most adult organisms, freshwater planarians can regenerate any injured body part, including their entire nervous system. This allows for the analysis of genes required for both the maintenance and regeneration of specific neural subtypes. In addition, the loss of specific neural subtypes may uncover previously unknown behavioral roles for that neural population in the context of the adult animal. Here we show that two homeodomain transcription factor homologs, Smed-lhx1/5-1 and Smed-pitx, are required for the maintenance and regeneration of serotonergic neurons in planarians. When either lhx1/5-1 or pitx was knocked down by RNA interference, the expression of multiple canonical markers for serotonergic neurons was lost. Surprisingly, the loss of serotonergic function uncovered a role for these neurons in the coordination of motile cilia on the ventral epidermis of planarians that are required for their nonmuscular gliding locomotion. Finally, we show that in addition to its requirement in serotonergic neurons, Smed-pitx is required for proper midline patterning during regeneration, when it is required for the expression of the midline-organizing molecules Smed-slit in the anterior and Smed-wnt1 in the posterior.
... An interesting topic of study in the developmental biology of flatworms is the population of totipotent somatic stem cells denominated 'neoblasts' in free-living planarians or 'germinal cells' in obligate parasitic trematodes and cestodes (Reuter and Kreshchenko, 2004;Brehm, 2010a). Many studies carried out in planarians have shown that these stem cells possess the capacity to directly differentiate into all somatic cells and are regarded as the only flatworm cell type that is mitotically active (Reuter and Kreshchenko, 2004;Sanchez-Alvarado and Kang, 2005;Rossi et al., 2008;Brehm and Spiliotis, 2008). ...
In vitro culture of parasitic helminths provides an important tool to study cell regeneration and physiology, as well as for molecular biology and genetic engineering studies. In the present study, we established in vitro propagation of cells from Echinococcus granulosus germinal cyst layer. E. granulosus germinal cells grew beyond 100 passages and showed no signs of reduced proliferation capacity. Microscopic analysis revealed that cells grew both attached to the substrate and in suspension, forming three-dimensional structures like mammalian stem cell aggregates. Examination of the chromosome number of attached germinal cells showed a high degree of heteroploidy, suggesting the occurrence of transformation during culture. Monolayer cells survived cryopreservation and were able to proliferate after thawing. Based on the characteristics displayed by E. granulosus germinal cells, we establish a cell line from the E. granulosus germinal layer. Furthermore, we propose that this cell line could be useful for drug screening and for obtaining parasite material.
... The lophotrochozoan phylum Platyhelminthes ('flatworms') represents a diverse grouping of unsegmented, bilaterally symmetrical, triploblastic worm species that hold significance for both basic and medical science (Fig. 1A). The free-living platyhelminths include planarian flatworms, which have long used as a model system for studying regenerative biology [16][17][18][19]. Planarian worms can be cut into small pieces, and each of these pieces retains an intrinsic ability to regenerate the original body plan. ...
Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular Ca2+ channels that elevate cytoplasmic Ca2+ in response to the second messenger IP3. Here, we describe the identification and in vivo functional characterization of the planarian IP3R, the first intracellular Ca2+ channel to be defined in flatworms. A single IP3R gene in Dugesia japonica encoded a 2666 amino acid protein (Dj.IP3R) that shared well conserved structural features with vertebrate IP3R counterparts. Expression of an NH2-terminal Dj.IP3R region (amino acid residues 223-585) recovered high affinity 3H-IP3 binding (0.9±0.1nM) which was abolished by a single point mutation of an arginine residue (R495L) important for IP3 coordination. In situ hybridization revealed that Dj.IP3R mRNA was most strongly expressed in the pharynx and optical nerve system as well as the reproductive system in sexualized planarians. Consistent with this observed tissue distribution, in vivo RNAi of Dj.IP3R resulted in a decreased egg-laying behavior suggesting Dj.IP3R plays an upstream role in planarian reproductive physiology.
... They are part of the lophotrocozoan clade. This clade maintains more genetic similarity with the Deuterostomates than with Ecdisozoa, to which both C. elegans (often used in space experiments) and D. melanogaster pertain (Alvarado and Kang 2005;Adoutte et al. 2000). Planarians are dorso-ventrally flattened bilaterians lacking circulatory, respiratory and skeletal system. ...
Planarians of the species Schmidtea mediter- ranea are a well-established model for regeneration studies. In this paper, we first recall the morphological characters and the molecular mechanisms involved in the regeneration process, especially focussing on the Wnt pathway and the establishment of the antero- posterior axial polarity. Then, after an assessment of a space-experiment (run in 2006 on the Russian Segment of the International Space Station) on planarians of the species Girardia tigrina, we present our experimental program to ascertain the effects that altered-gravity conditions may have on regeneration processes in S. mediterrnea at the molecular and genetic level.
... The space between the pharyngeal muscle layers contains fixed parenchymal, nervous, vacuolar, and ciliary (excretory) cells as well as glandular cells of the pharyngeal glands, whose bodies lie at the base of the pharynx and whose ducts run to its distal part where the gland products are secreted (Ishii, 1964;Bowen and Ryder, 1973;Farnesi and Pascolini, 1973;Jennings, 1977;Gamo and Garsia-Corrales, 1985). Most reports indicate the absence of neoblasts (stem cells) in the planarian pharynx, which are distributed in the body parenchyma in planarians ( Brøndsted, 1969;Bagu ñá, 1976;Kishida and Asai, 1980;Sanchez Alvarado and Kang, 2005). The pharyngeal nervous system is relatively autonomous but anatomically linked to the central nervous system, namely, the major (ventral) nerve cords (Joffe and Reuter, 1993;. ...
The obtained and published data on pharynx regeneration in planarians have been reviewed. Planarians can regenerate from a
small body fragment and restore all missing organs including the pharynx. The pharynx is a relatively autonomous organ with
a differentiated structure and specialized function. Pharynx regeneration has specific features, and its studies are of considerable
theoretical interest. Pharynx regeneration can also be a convenient model to study the molecular mechanisms of regeneration
that remain undisclosed. In addition, this model can be used to test biologically active compounds in order to elucidate their
effect on morphogenesis. This subject of investigation benefits by a simpler and more adequate analysis as well as a possibility
to use large numbers of animals and small quantities of analyzed substances.
... progeny eventually differentiate to replace the missing structures (Handberg-Thorsager et al., 2008;Salvetti et al., 1998;Sánchez Alvarado and Kang, 2005). In most cases, regeneration should be complete within a week to 10 days. ...
In this study, the toxicity, behavioural and regeneration effects of dimethylformamide (DMF) on planarian Dugesia japonica were investigated. One control and six different concentrations of DMF (10 ppm, 100 ppm, 500 ppm, 1000 ppm, 5000 ppm and 10,000 ppm) were used in triplicate. The results showed that the mortality was directly proportional to the DMF concentration and planarian locomotor velocity (pLMV) was significantly reduced by increasing the exposure time and DMF concentration. pLMV of D. japonica was significantly reduced at a lower concentration of 10 ppm after 7 days of continuous exposure to DMF. The recovery of the motility of planarians pretreated with DMF was found to be time- and dose dependent, all planarians had complete recovery in their motility after 48 h. The appearance of auricles in regenerating animals was easily affected by DMF exposure in comparison with the appearance of eyespot. The present results suggest that the intact adult mobility in the aquatic planarian D. japonica is a more sensitive biomarker than mortality, and the appearance of auricles in regenerating animals is a more sensitive biomarker than eyespot.
... The above-mentioned studies indicate that regeneration is possible only when the renewable cells, the stem cells, are present. Pluripotent stem cells called neoblasts [located throughout the body (3,4,201)] are involved in regeneration occurring in invertebrates and some vertebrates while tissue-associated stem/progenitor cells play a crucial role in the regeneration of most mammalian tissues (37). Thus a huge effort has been made in the last few years to characterize intrinsic cellular properties of stem cells, the nature of the niches allowing their survival in adult tissue, and their physiological and reparative regenerative capacities in different animal species (153,236). ...
Regenerative processes occurring under physiological (maintenance) and pathological (reparative) conditions are a fundamental part of life and vary greatly among different species, individuals, and tissues. Physiological regeneration occurs naturally as a consequence of normal cell erosion, or as an inevitable outcome of any biological process aiming at the restoration of homeostasis. Reparative regeneration occurs as a consequence of tissue damage. Although the central nervous system (CNS) has been considered for years as a "perennial" tissue, it has recently become clear that both physiological and reparative regeneration occur also within the CNS to sustain tissue homeostasis and repair. Proliferation and differentiation of neural stem/progenitor cells (NPCs) residing within the healthy CNS, or surviving injury, are considered crucial in sustaining these processes. Thus a large number of experimental stem cell-based transplantation systems for CNS repair have recently been established. The results suggest that transplanted NPCs promote tissue repair not only via cell replacement but also through their local contribution to changes in the diseased tissue milieu. This review focuses on the remarkable plasticity of endogenous and exogenous (transplanted) NPCs in promoting repair. Special attention will be given to the cross-talk existing between NPCs and CNS-resident microglia as well as CNS-infiltrating immune cells from the circulation, as a crucial event sustaining NPC-mediated neuroprotection. Finally, we will propose the concept of the context-dependent potency of transplanted NPCs (therapeutic plasticity) to exert multiple therapeutic actions, such as cell replacement, neurotrophic support, and immunomodulation, in CNS repair.
... On the other hand, it is important to emphasize that ingressing cells in the holothurian are not necessarily equivalent to the "blastemal cells" found in regenerating amphibian limbs [4] or fish fin blastemas [42,46] nor to planarian neoblasts [47]. The main difference, other than their origin, is shown by the limited proliferation activity; there is less BrdU incorporation in these cells and proliferative events last less than in cells of the overlying coelomic epithelium, or the forming luminal epithelium. ...
Determining the type and source of cells involved in regenerative processes has been one of the most important goals of researchers in the field of regeneration biology. We have previously used several cellular markers to characterize the cells involved in the regeneration of the intestine in the sea cucumber Holothuria glaberrima.
We have now obtained a monoclonal antibody that labels the mesothelium; the outer layer of the gut wall composed of peritoneocytes and myocytes. Using this antibody we studied the role of this tissue layer in the early stages of intestinal regeneration. We have now shown that the mesothelial cells of the mesentery, specifically the muscle component, undergo dedifferentiation from very early on in the regeneration process. Cell proliferation, on the other hand, increases much later, and mainly takes place in the mesothelium or coelomic epithelium of the regenerating intestinal rudiment. Moreover, we have found that the formation of the intestinal rudiment involves a novel regenerative mechanism where epithelial cells ingress into the connective tissue and acquire mesenchymal phenotypes.
Our results strongly suggest that the dedifferentiating mesothelium provides the initial source of cells for the formation of the intestinal rudiment. At later stages, cell proliferation supplies additional cells necessary for the increase in size of the regenerate. Our data also shows that the mechanism of epithelial to mesenchymal transition provides many of the connective tissue cells found in the regenerating intestine. These results present some new and important information as to the cellular basis of organ regeneration and in particular to the process of regeneration of visceral organs.
... It is generally recognized that the regenerative ability in invertebrates and some vertebrates is basically granted by a population of pluripotent stem cells, called -neoblasts‖, which can be found throughout the body [36,38,47]. On the other hand, in most mammalian tissues, the cell sources of regeneration are represented by stem/progenitor cells associated with the tissue itself [3]. ...
Regeneration is a strategy to maintain form and function throughout life. Studies carried out on animal models throughout the phylogenetic tree have flourished in the last decades in search of mechanisms underlying the regenerative processes. The development of such studies is strictly linked with stem cell research and both are viewed as one of the most promising outcomes for regenerative medicine; yet, regeneration, stem cells, and tissue repair do not seem to follow a logical path through the different animal species and tissues. As a result, some mammalian organs, e.g., kidney and brain, have lost most of their regenerative capacity. The human nervous system, although harboring neural stem cells, is placed at the extreme of "perennial" tissues. In addition, it is affected by neurodegenerative diseases, whose heavy burden is heightened by enhanced life spans. This review, starting from the basic principles of tissue regeneration viewed in a comparative context, tries to answer this question: To which extent can regenerative medicine be figured out in a mammalian brain equipped with many anatomical/evolutionary constraints?
Background
Regeneration plays a key role in energy recycling and homeostasis maintenance. Planarians, as ideal model animals for studying regeneration, stem cell proliferation, and apoptosis, have the strong regenerative abilities. Considerable evidence suggests that ubiquitin plays an important role in maintaining homeostasis and regulating regeneration, but the function of Ubiquitin specific proteases 7 (Usp7) on regeneration in planarians remains elusive.
Methods
We identified an evolutionarily conserved gene, Usp7, and utilized RNA interference (RNAi), Quantitative real-time PCR (qRT-PCR), Whole-mount immunofluorescence, Tunnel, Whole-mount in situ hybridization (WISH), and western blotting to detect the function of Usp7 during the planarian regeneration.
Results
In this study, we found that the regenerative trunk fragments in the Usp7 RNAi worms could not regenerate missing tails; meanwhile, the level of cell proliferation was decreased, while cell apoptosis was increased. Furthermore, the expression of Islet was inhibited in the Usp7 RNAi worms during planarian regeneration. The hybridization signal of wnt1/P-1 exhibited the dot-like pattern at the posterior of the regenerating planarians after Usp7 RNAi at regenerative 1 day (R 1 d). However, the concentrated expression pattern wnt1/P-1 dramatically declined at regenerative 3 days (R 3 d) and disappeared at regenerative 7 days (R 7 d). In addition, activating the Wnt pathway partially rescued regenerative defects induced by inhibition of Usp7.
Conclusions
Collectively, Usp7 is necessary for tissue regeneration and tail blastema formation partially by regulating the cell proliferation and apoptosis during planarian regeneration. It could also promote the posterior polarity reconstruction of the regenerative planarians via the Islet/Wnt1 axis.
Multicopy sequences evolve adaptations for increasing their copy number within nuclei. The activities of multicopy sequences under constraints imposed by cellular and organismal selection result in a rich intranuclear ecology in germline cells. Mitochondrial and ribosomal DNA are managed as domestic herds subject to selective breeding by the genes of the single-copy genome. Transposable elements lead a peripatetic existence in which they must continually move to new sites to keep ahead of inactivating mutations at old sites and undergo exponential outbreaks when the production of new copies exceeds the rate of inactivation of old copies. Centromeres become populated by repeats that do little harm. Organisms with late sequestration of germ cells tend to evolve more “junk” in their genomes than organisms with early sequestration of germ cells.
Hyaluronic acid plays a crucial role in the skin by increasing hydration and stimulating collagen growth (Papakonstantinou, Roth, & Karakiulakis, 2012). It has a key position in wound healing and tissue repair processes such as healing and the stimulation of growth factors, and cellular components (Gonzalez, Costa, Andrade, & Medrado, 2016). Planaria Schmidtea Mediterranea are flatworms able to regenerate parts of their body if injured within a span of five days. These flatworms contain adult totipotent stem cells called neoblasts allowing them to do so (Wagner, Wang, & Reddien, 2011). Ultraviolet radiation (UV radiation), however, damages the planarian’s abilities to regenerate tissue (Franjevic, Kalafatić, & Kovačević, 2006). In the present work, the effect of hyaluronic acid on two groups of ten planarians was investigated. Experimental populations of the Planarian Schmidtea Mediterranea were placed and physiological changes were monitored daily with a microscope in laboratory conditions at room temperature. Both groups of the planaria received ten seconds of UV radiation at the beginning of the experiment. Hyaluronic acid was only given to only one out of the two groups of planaria for 10 days. Data indicates that the ten deciliters of added hyaluronic acid predominantly slowed down the rate of tissue regeneration in planarians and reduced tissue regeneration rate compared to the other group of planarians. These results illustrate how hyaluronic acid had the opposite influence on planarian tissue regeneration and how it significantly diminished tissue growth.
In this chapter, the evolution of primitive eukaryote organisms before and after appearance of O2 is reviewed in light of the actual hypothesis concerning “Proterozoic ocean chemistry.” The recent geological data suggest atmospheric O2 concentrations during the Great Oxygenation Event substantially lower than believed previously, as well as a delayed oxygenation of oceans. However, the notion of heterogeneity of ecologic niches in Proterozoic oceans and the biological indices showing that extremely low O2 concentrations may have a profound metabolic and physiologic impact, point to O2 as a factor involved in substantial evolutionary impacts. The recent data suggest that the basic processes as appearance of mitochondria, mitosis, and meiosis (i.e., asexual and sexual reproduction) as well as the appearance of the first metazoan took place at the borderline between anaerobiosis and nano/microaerobiosis, which is an important point for the discussion in further chapters.
As freshwater planarians (flatworms) are further developed as a model system, the most valuable tool continues to be in situ hybridization (ISH) analysis of gene expression in whole mount adult animals (WISH). Multiple hurdles have been overcome during the optimization of a standard protocol for colorimetric, single color detections. However, gene function studies on planarians have evolved to the point where virtually all investigations require the analysis of multiple RNAs, simultaneously. Thus, considerable effort by the entire planarian community has been put forth to create adequate methodologies towards this goal. Here we summarize the field, methodological evolution, and describe a new method for more rapid and more sensitive detection of multiple RNAs by multi-fluorescent ISH.
The myosin essential light chain (ELC) is a structure component of the actomyosin cross-bridge, however, the functions in the central nervous system (CNS) development and regeneration remain poorly understood. Planarian Dugesia japonica has revealed fundamental mechanisms and unique aspects of neuroscience and neuroregeneration. In this study, the cDNA DjElc, encoding a planarian essential light chain of myosin, was identified from the planarian Dugesia japonica cDNA library. It encodes a deduced protein with highly conserved functionally domains EF-Hand and Ca(2+) binding sites that shares significant similarity with other members of ELC. Whole mount in situ hybridization studies show that DjElc expressed in CNS during embryonic development and regeneration of adult planarians. Loss of function of DjElc by RNA interference during planarian regeneration inhibits brain lateral branches regeneration completely. In conclusion, these results demonstrated that DjElc is required for maintenance of neurons and neurite outgrowth, particularly for involving the brain later branch regeneration.
Asexual reproduction by division in higher organisms is rare, because a
prerequisite is the ability to regenerate an entire organism from a
piece of the original body. Freshwater planarians are one of the few
animals that can reproduce this way, but little is known about the
regulation of their reproduction cycles or strategies. We have
previously shown that a planarian's reproduction strategy is randomized
to include fragmentations, producing multiple offspring, as well as
binary fissions, and can be partially explained by a maximum relative
entropy principle. In this study we attempt to decompose the factors
controlling their reproduction cycle. Based on recent studies on the
cell cycle of budding yeast, which suggest that molecular noise in gene
expression and cell size at birth together control cell cycle
variability, we investigated whether the variability in planarian
reproduction waiting times could be similarly regulated. We find that
such a model can indeed explain the observed distribution of waiting
times between birth and next reproductive event, suggesting that birth
size and a stochastic noise term govern the reproduction dynamics of
asexual planarians.
Keeping Cells Cooperating
Multicellular organisms have certain advantages over those that are single-celled. To evolve, however, they must surmount a persistent challenge: ensuring that their constituent cells cooperate with one another rather than “cheat” or compete with each other for resources. Dejosez et al. (p. 1511 , published online 12 September) performed a genome-wide screen in induced pluripotent stem cells to search for genes that promote cell cooperation. A number of genes were identified of which knockdown allowed competitive behavior to dominate. These genes formed a network centered on p53, topoisomerase 1, and olfactory receptors. Thus, a genetic mechanism may promote cooperation in the developing embryo.
Objective: Freshwater planarians were used as models for studying metazoan regeneration and stem cell biology. Here a simple, fast and high throughput method for extracting their stem cells (neoblasts) is represented. Materials and Methods: Specimens of the Dugesia sp with an average length of 18 mm were homogenized by a glass Dounce tissue grinder which contained about 1 ml of planarian saline solution. The extracted suspension was serially filtered by 60, 41, 30, 20 and 11 μm nylon meshes. In order to obtain purified neoblasts in the final suspension; this suspension has been compared with a cell suspension from 30 Gy irradiated worms. Hoechst 33342 was used to determine cells from non-cellular particles; methylene blue and propidium iodide were used to detect the number of dead cells in each extraction. Results: About 2.6-3 million cells were extracted from 10-12 worms. Flow cytometry analy-sis showed about 83% of the extracted particles were cells. In suspensions from irradiated animals, about 50% of the cells were absent, the final suspension contained about 62-66% neoblasts and about 17% non-cellular particles. When these extracts were treated with dis-tilled water to destroy the cells, only rabdites and chitinous spines of the parenchyma were observed in the extract. Conclusion: Results show that the purity of neoblasts in the final suspension is about 66%. Non-cellular particles have a carbohydrate nature and, therefore, this extraction method is completely compatible with molecular (e.g. proteomics and transcriptomics) and cellular methods (e.g. neoblast culture).
This chapter deals with the loss of cell replication capacity, or in some cases loss of a stimulus to carry out replication,
that occurs with aging in animals of several species. The effect of dietary conditions, hormonal stimulus, and metabolic status
are considered. The overall conclusion is that an age-related slowing of steady state or stimulated cell replication occurs
in several tissues, whether tested in vivo or in vitro, and that this is also apparent in non-first intention wound healing.
A brief summary of non-mammalian cell, tissue, and body part healing and replacement is included.
KeywordsHuman-Dog-Monkey-Mouse-Rat-Anuran-Froglet-Wound healing-Regeneration-Age-Caloric restriction-Bone-Dermis-Fracture-Cell replication-Telomere
Extracellular matrix (ECM) not only provides a stable framework for maintaining the shape of multicellular organisms under
physical load and during locomotion, but it is also essential for their morphogenesis and regenerative capacity. In this introductory
chapter, we describe the basic features of the major classes of ECM components, namely, collagens, glycoproteins, and proteoglycans.
We emphasize their multidomain structure that allows multiple functions to be combined in one, often large molecule. Of the
many types of protein modules found in ECM components, some are devoted to multimeric assembly, and hence, for their crucial
ability to form extended multimolecular networks or matrices. We argue that ECM components together with integrin receptors
on the cell surface can be viewed as intricate nanodevices that allow cells to physically organize their 3D environment, as
well as to sense and respond to various types of mechanical stress. In addition, ECM functions as part of a cell-controlled
machinery to store and activate growth factors during development. We also make the point that metazoan evolution would not
have been possible without the concomitant expansion of ECM complexity. Using examples of phylogenetically “old” versus “young”
ECM protein families, we review the evidence that today’s incredible diversity of ECM components arose from the recombination
of preexisting protein modules by exon shuffling during evolution.
Can stem cells be defined as legitimate units of selection (UOS)? Here I review traits of hallmark stem cells (many of which are manifested in marine organisms), which collectively reveal that some types of stem cells behave as real UOS entities. The two existing categories of stem cells maintain the soma and the germ line, have infallible high capacity for self-renewal, produce diverse lines of differentiated progenies and exist ad infinitum (within the organism's life span). Because of their primitive, undifferentiated state, stem cells are pliable to adopt totipotentiality, but that is not enough to be considered as genuine UOS agents. The discussion on highly transmitted cancer, germ and somatic stem cells, the autonomous traits of somatic stem cells and their stemness, stem cells consortia in biological chimerism, and parasitism vs. cooperation on the level of stem cells, point to a group of characters that denote some types of stem cells as UOS. Prime features of these stem cells include: (a) efficient motility within an organism and between organisms (conspecifics/different taxa; vertical/horizontal transmission); (b) ability to parasitize effectively host organisms, in the same species or in other species; (c) stemness and unlimited replication; (d) ability to blend with other cell lineages, blurring the line between distinct somatic and germ cell lineages; (e) ability to designate a variety of different soma as hosts; (f) ability to co-opt and manage somatic cells to collaborate in parasitic agenda; (g) lack of a distinct niche where they reside; and (h) manifestation of the power of holism. Events in which stem cells acquire the traits of legitimate UOS as real Darwinian individuals, are widespread in multicellular organisms, even when lacking structural cohesion; a phenomenon that confounded accepted dogma for UOS entity. It is therefore concluded that some stem cell lines foster the traits of UOS, as efficiently as whole organisms or genes.
Planarians possess amazing abilities to regulate tissue homeostasis and regenerate missing body parts. These features reside
on the presence of a population of pluripotent/totipotent stem cells, the neoblasts, which are considered as the only planarian
cells able to proliferate in the asexual strains. Neoblast distribution has been identified by mapping the cells incorporating
bromodeoxyuridine, analyzing mitotic figures and using cell proliferation markers. Recently identified molecular markers specifically
label subgroups of neoblasts, revealing thus the heterogeneity of the planarian stem cell population. Therefore, the apparent
totipotency of neoblasts probably reflects the composite activities of multiple stem cell types. First steps have been undertaken
to understand how neoblasts and differentiated cells communicate with each other to adapt the self-renewal and differentiation
rates of neoblasts to the demands of the body. Moreover, the introduction of molecular resource database on planarians now
paves the way to renewed strategies to understand planarian regeneration and stem cell-related issues. (Part of a Multi-author
Review)
In this study, the toxicity, behavioral and antioxidant activity effects of dimethylsulfoxide (DMSO) on planarian Dugesia japonica were investigated. The results showed that the mortality was directly proportional to the DMSO concentration, and planarian locomotor velocity decreased as the concentration of DMSO increased. The recovery of the motility for planarians pre-exposed to DMSO was found to be time- and dose-dependent, and only those pre-exposed to 0.1-3% DMSO resulted in full recovery. The antioxidant enzymes of planarians in response to long-term DMSO stress was also altered in a time- and dose-dependent manner. Planarians revealed more tolerance to DMSO toxicity at low DMSO (0.1%) level in short- and long-term DMSO stress, in which an efficient antioxidant system was involved and the motility was not affected.
We examined the spatial and temporal expression of the planarian Dugesia japonica STAG-related gene (DjStag), in both intact and regenerating planarians, by whole-mount in situ hybridization and relative quantitative real-time PCR. The first localized transcripts of DjStag were detected in the blastemas three days after amputation, in all regenerates including those from head, tail and trunk pieces. The maximum level of expression of DjStag transcripts occurred at five days after cutting. After regeneration for seven days, DjStag was weakly expressed. A similar decrease occurs regardless of the orientation of the cut. The expression pattern did not differ significantly in the different types of regeneration. Relative quantitative real-time PCR analysis of DjStag mRNA indicated that the expression of DjStag mRNA was increased after amputation compared to that in normal intact planarians, and the maximum level of expression of DjStag transcripts occurred at five days after amputation. All results suggest that DjStag, implicated in planarian regeneration, plays a role in maintaining the ability of pluripotent stem cells to regenerate lost tissue in planarians.
The therapeutic effects elicited by photobiostimulation in the near infrared range may be associated with increased proliferation rate of particular cell-types. The present study utilized commercial light emitting diodes to investigate the effects of low-level near-infrared radiation on the proliferation rate of stem cells in amputated planarian. Whole and amputated animals were exposed to either ambient diurnal lighting, darkness, white light, red light, or near-infrared (880 nm) light. Irradiation was consistent for the duration of the experiments and was provided using commercial 5mm light emitting diodes (∼1.0 mW/m(2) in power density and ∼0.01 J/cm(2) in radiant exposure). Compared to other groups amputated planarian exposed to near-infrared displayed increased mobility by the 3rd day of exposure (F((4,26))=4.31, p<0.04, η(2)=41%). Higher densities of stem cells were measured in these worms 84 h post injury (F((4,72))=4.78, p<0.01, η(2)=21%). These findings suggest that non-coherent light sources with power-densities about 1000 times lower than contemporary low-power laser settings remain effective in generating photobiostimulation effects and warrants further investigation on stem-cell proliferation induced by near-infrared light emitting diodes.
In most regenerating systems, blastema cells arise by dedifferentiation of functional tissue cells. In planarians, though, it is still debatable whether dedifferentiated cells or a population of undifferentiated cells, the neoblasts, are the main source of blastema cells. Moreover, it is unclear whether in the intact organisms neoblasts are quiescent cells 'reserved' for regeneration or if they serve as functional stem cells of all differentiated cell types. Both uncertainties partly stem from the failure to distinguish by conventional labelling methods neoblasts from differentiated cells. Here we describe a new approach to these problems based on testing the regenerative and stem cell capabilities of purified neoblasts and differentiated cells when introduced, separately, into irradiated hosts. Introduction of neoblasts led to resumed mitotic activity, blas-tema formation, and extended or complete survival of the host; differentiated cells, in contrast, never did so. Therefore, planarian neoblasts can be qualified as totipotent stem cells and the main source of blastema cells, while dedifferentiation does not seem to operate either in intact or regenerating organisms. In addition, these results strengthen the idea that different types of regeneration and blastema formation, linked to the tissular complexity of the organisms, are present in the animal kingdom.
DNA sequence analysis dictates new interpretation of phylogenic
trees. Taxa that were once thought to represent successive grades of
complexity at the base of the metazoan tree are being displaced to much
higher positions inside the tree. This leaves no evolutionary
“intermediates” and forces us to rethink the genesis of
bilaterian complexity.
Ramalho-Santos et al . ([ 1 ][1]) and Ivanova et al. ([ 2 ][2]), comparing the same three “stem cells”— embryonic stem cells (ESCs); neural stem cells (NSCs), referred to as neural progenitor/stem cells (NPCs) in the present study; and hematopoietic stem cells (HSCs)—with their
Mitotic activity during regeneration in the planarian Dugesia (G) tigrina shows a biphasic pattern, with a first maximum at 4-12 h, a second and higher maximum at 2-4 days, and a relative minimum in between. The first peak is mainly due to pre-existing G2 cells entering mitosis shortly after cutting, whereas the second maximum is due to cells that divide after going through the S period from the onset of regeneration. From a spatial point of view, the highest mitotic values are found in stump (postblastema) regions near the wound (0-300 micron), though regions far from it also show increased mitotic values but always lower overall values. As regeneration continues the postblastema maximum shifts slightly to more proximal regions. In contrast, no mitosis has been found within the blastema, even though the number of blastema cells increases steadily during regeneration. The results suggest that blastema in planarians forms through an early accumulation of undifferentiated cells from the stump to the base of blastema. The results obtained demonstrate that blastema formation in planarians occurs through mechanisms somewhat different to those shown to occur in the classical epimorphic models of regeneration (Annelida, Insecta, Amphibia), and suggest that planarian regeneration could represent an intermediate stage between morphallactic and epimorphic modalities of regeneration.
Whether in sexual or asexual organisms, selection among cell lineages during development is an effective way of eliminating deleterious mutations. Using a mathematical analysis, we find that relatively small differences in cell replication rates during development can translate into large differences in the proportion of mutant cells within the adult, especially when development involves a large number of cell divisions. Consequently, intraorganismal selection can substantially reduce the deleterious mutation rate observed among offspring as well as the mutation load within a population, because cells rather than individuals provide the selective "deaths" necessary to stem the tide of deleterious mutations. The reduction in mutation rate among offspring is more pronounced in organisms with plastic development than in those with structured development. It is also more pronounced in asexual organisms that produce multicellular rather than unicellular offspring. By effecting the mutation rate, intraorganismal selection may have broad evolutionary implications; as an example, we consider its influence on the evolution of ploidy levels, finding that cell-lineage selection is more effective in haploids and tends to favor their evolution.
Structural studies of fixed cells have revealed that interphase chromosomes are highly organized into specific arrangements in the nucleus, and have led to a picture of the nucleus as a static structure with immobile chromosomes held in fixed positions, an impression apparently confirmed by recent photobleaching studies. Functional studies of chromosome behavior, however, suggest that many essential processes, such as recombination, require interphase chromosomes to move around within the nucleus.
To reconcile these contradictory views, we exploited methods for tagging specific chromosome sites in living cells of Saccharomyces cerevisiae with green fluorescent protein and in Drosophila melanogaster with fluorescently labeled topoisomerase ll. Combining these techniques with submicrometer single-particle tracking, we directly measured the motion of interphase chromatin, at high resolution and in three dimensions. We found that chromatin does indeed undergo significant diffusive motion within the nucleus, but this motion is constrained such that a given chromatin segment is free to move within only a limited subregion of the nucleus. Chromatin diffusion was found to be insensitive to metabolic inhibitors, suggesting that it results from classical Brownian motion rather than from active motility. Nocodazole greatly reduced chromatin confinement, suggesting a role for the cytoskeleton in the maintenance of nuclear architecture.
We conclude that chromatin is free to undergo substantial Brownian motion, but that a given chromatin segment is confined to a subregion of the nucleus. This constrained diffusion is consistent with a highly defined nuclear architecture, but also allows enough motion for processes requiring chromosome motility to take place. These results lead to a model for the regulation of chromosome interactions by nuclear architecture.
A recent study ([1][1]) coauthored by several of us and discussed by Elizabeth Pennisi (Research News, 5 Sept. 1997, [p. 1435][2]) examined inaccuracies in embryo drawings published last century by Ernst Haeckel. Our work has been used in a nationally televised debate to attack evolutionary theory,
Metazoan regeneration is one of the least understood fundamental problems of biology. The lack of progress in understanding this phenomenon at the molecular level has been due to the poor regenerative abilities of the genetic organisms used for developmental studies, as well as the difficulties encountered with molecular and genetic manipulations of the commonly studied vertebrate models (the urodele amphibians). Here, we demonstrate that introduction of double-stranded RNA selectively abrogates gene function in planarians, a classic model of regeneration. The ability to eliminate gene function in a regenerating organism such as the planarian overcomes previous experimental limitations and opens the study of animal regeneration to unprecedented levels of molecular detail.
During mammalian development, the Cdx1 homeobox gene exhibits an early period of expression when the embryonic body axis is established, and a later period where expression is restricted to the embryonic intestinal endoderm. Cdx1 expression is maintained throughout adulthood in the proliferative cell compartment of the continuously renewed intestinal epithelium, the crypts. In this study, we provide evidence in vitro and in vivo that Cdx1 is a direct transcriptional target of the Wnt/(beta)-catenin signaling pathway. Upon Wnt stimulation, expression of Cdx1 can be induced in mouse embryonic stem (ES) cells as well as in undifferentiated rat embryonic endoderm. Tcf4-deficient mouse embryos show abrogation of Cdx1 protein in the small intestinal epithelium, making Tcf4 the likely candidate to transduce Wnt signal in this part of gut. The promoter region of the Cdx1 gene contains several Tcf-binding motifs, and these bind Tcf/Lef1/(beta)-catenin complexes and mediate (beta)-catenin-dependent transactivation. The transcriptional regulation of the homeobox gene Cdx1 in the intestinal epithelium by Wnt/(beta)-catenin signaling underlines the importance of this signaling pathway in mammalian endoderm development.
A pool of stem cells that arise from the mesoderm during embryogenesis initiates hematopoiesis. However, factors that regulate the expansion of blood stem cells are poorly understood. We show here that cytokine-induced proliferation of primitive human hematopoietic cells could be inhibited with antibodies to hedgehog (Hh). Conversely, Sonic hedgehog (Shh) treatment induced the expansion of pluripotent human hematopoietic repopulating cells detected in immunodeficient mice. Noggin, a specific inhibitor of bone morphogenetic protein 4 (BMP-4), was capable of inhibiting Shh-induced proliferation in a similar manner to anti-Hh; however, anti-Hh had no effect on BMP-4-induced proliferation. Our study shows that Shh functions as a regulator of primitive hematopoietic cells via mechanisms that are dependent on downstream BMP signals.
Multicellular organisms probably originated as groups of cells formed in several ways, including cell proliferation from a group of founder cells and aggregation. Cooperation among cells benefits the group, but may be costly (altruistic) or beneficial (synergistic) to individual cooperating cells. In this paper, we study conflict mediation, the process by which genetic modifiers evolve that enhance cooperation by altering the parameters of development or rules of formation of cell groups. We are particularly interested in the conditions under which these modifiers lead to a new higher-level unit of selection with increased cooperation among group members and heritable variation in fitness at the group level. By sculpting the fitness variation and opportunity for selection at the two levels, conflict modifiers create new functions at the organism level. An organism is more than a group of cooperating cells related by common descent; organisms require adaptations that regulate conflict within. Otherwise their continued evolution is frustrated by the creation of within-organism variation and conflict between levels of selection. The evolution of conflict modifiers is a necessary prerequisite to the emergence of individuality and the continued well being of the organism. Conflict leads--through the evolution of adaptations that reduce i--to greater individuality and harmony for the organism.
In skin, multipotent stem cells generate the keratinocytes of the epidermis, sebaceous gland, and hair follicles. In this paper, we show that Tcf3 and Lef1 control these differentiation lineages. In contrast to Lef1, which requires Wnt signaling and stabilized beta-catenin to express the hair-specific keratin genes and control hair differentiation, Tcf3 can act independently of its beta-catenin interacting domain to suppress features of epidermal terminal differentiation, in which Tcf3 is normally shut off, and promote features of the follicle outer root sheath (ORS) and multipotent stem cells (bulge), the compartments which naturally express Tcf3. These aspects of Tcf3's action are dependent on its DNA binding and Groucho repressor-binding domains. In the absence of its beta-catenin interacting domain, Lef1's behavior (Delta NLef1) seems to be markedly distinct from that of Delta NTcf3. Delta NLef1 does not suppress epidermal differentiation and promote ORS/bulge differentiation, but rather suppresses hair differentiation and gives rise to sebocyte differentiation. Taken together, these findings provide powerful evidence that the status of Tcf3/Lef complexes has a key role in controlling cell fate lineages in multipotent skin stem cells.
During the growth of Drosophila imaginal discs a process called 'cell competition' eliminates slow-proliferating but otherwise viable cells. We report here that cell competition requires the function of the brinker (brk) gene, whose expression is normally repressed by Decapentaplegic (Dpp) signalling but is upregulated in slow-growing Minute/+ cells. Excess brk expression activates the c-Jun amino-terminal kinase pathway, which in turn triggers apoptosis in these cells. We propose that slow-proliferating cells upregulate Brk levels owing to a disadvantage in competing for, or in transducing, the Dpp survival signal. This sequence of events might represent a general mechanism by which weaker cells are eliminated from a growing population, and might serve as a method of controlling cell number and optimizing tissue fitness and hence organ function.
The hypothesis that individuality is a derived trait in animals (Buss, '87, The Evolution of Individuality, Princeton, NJ: Princeton University Press; Michod, '99, Darwinian Dynamics, Princeton, NJ: Princeton University Press) can be further tested by a "tree-based" analysis utilizing a comparative methodology and recent phylogenies. We conducted a maximum parsimony analysis in which we mapped character states for clonality, coloniality, and mode of germline development onto four recent phylogenetic hypotheses (Peterson and Eernisse, 2001, Evol Dev 3:170-205). Clonality appears to be a shared primitive character for metazoans. Coloniality, on the other hand, is a derived trait found in relatively few phyla. The germline appears to have been derived at or near the origin of the first bilaterians. The stem-lineage metazoan thus appears to have been a clonal, acolonial organism that exhibited somatic embryogenesis. The stem-lineage bilaterian also was likely clonal and acolonial. Nevertheless, this lineage likely exhibited preformation, i.e., its germline was determined during embryonic development. In addition to supporting the hypothesis that the germline is a derived feature in animals, this analysis is relevant to current debates concerning the nature of the latest common ancestor of the bilaterians. (C) 2003 Wiley-Liss, Inc.
Chk2 is a protein kinase that is activated in response to DNA damage and may regulate cell cycle arrest. We generated Chk2-deficient mouse cells by gene targeting. Chk2−/− embryonic stem cells failed to maintain γ-irradiation–induced arrest in the G2 phase of the cell cycle. Chk2−/−thymocytes were resistant to DNA damage–induced apoptosis. Chk2−/− cells were defective for p53 stabilization and for induction of p53-dependent transcripts such as p21 in response to γ irradiation. Reintroduction of the Chk2 gene restored p53-dependent transcription in response to γ irradiation. Chk2 directly phosphorylated p53 on serine 20, which is known to interfere with Mdm2 binding. This provides a mechanism for increased stability of p53 by prevention of ubiquitination in response to DNA damage.
In planarian regeneration mitosis occurs in both anterior and posterior blastemas. The mitotic increase is visible as early as one hour after amputation, reaches a rapid first maximum at 5–12 hours, a relative minimum at 24–30 hours, and a higher and longer second maximum at two to four days depending on body size, level of amputation, and on whether blastema is anterior or posterior. The spatial dynamics of mitosis show a rapid and high mitotic increase in the distal regions but not in the proximal regions. Later, the proliferative zone shifts in sucessive mitotic waves to more proximal regions, paralleling the formation of the head ganglia and nerve cords.
These results do not agree with theories of blastema formation through neoblast migration or through cell dedifferentiation, in as much as the local proliferation of neoblasts encountered is sufficiently high and early to account for the number of blastema cells found during period of regeneration. Therefore, we suggest that blastema formation during planarian regeneration occurs mainly through local neoblast proliferation.
These results and the data obtained on regeneration rates enable us to suggest that nervous tissue may be one of the factors responsible for the differing mitotic increases found. Since neoblasts are the only planarian cells capable of mitosis, we suggest that neoblast proliferation could be under nervous tissue control through some stimulatory substance (s) released from the nerve terminals. The implications of this hypothesis for the maintenance of axial polarity are also discussed.
Planarian neoblasts, the only cell type in this organism endowed with mitotic power, decrease in density as total body volume, total cell number, and length increase. Studies on mitosis show that the neoblast mitotic rate is a function of body length, being lower as the organism increases in length. The cephalo-caudal distribution of mitotic figures in animals of different sizes shows a change towards a more even distribution of mitosis jointly with a mitotic increase in the caudal regions as length increases. However, in the sagittal and dorso-ventral axes, the distribution of mitosis is always nonuniform, probably due to the uneven distribution of parenchymal tissue along them.
The pharynx of animals in all size groups examined shows no mitotic figures except some near its base. This may suggest for this organ a proximal type of growth with some cell migration.
Feeding elicits a very rapid increase in mitosis which lasts for almost a week, whereas long periods of starvation do not cause a significant increase or decrease in the basal mitotic rate. All these results stress the need of looking at the planarian neoblast mainly as a stem or replacement cell of most differentiated cell types and not as a mere “regeneration cell.” The implications of the results obtained for the mechanisms of growth and shrinkage are discussed.
A method of isolating neoblasts from Dugesia dorotocephala and the process of their aggregation in vitro are described. Morphology and behavior of gastrodermal cells and fixed parenchyma cells in vitro are also reported. Basically the neoblast is an oval cell with a large primary process and a finer secondary process at the opposite pole. Neoblasts have strong mutual affinity and very little affinity with other types of cells. The only observed method of locomotion of neoblasts in vitro was the contraction of the firmly attached primary processes between two or more neoblasts, which ends in their aggregation. Gastrodermal cells and fixed parenchyma cells are the most active migratory cells in vitro. With the observation of the cell movement both in vitro and in the fragments of the body, it was concluded that the primary accumulation of neoblasts at the open wound is not due to the active migration of neoblasts from the intact region as surmised by some other authors but due to the aggregation of local neoblasts. The migration of gastrodermal cells from the wound area causes the condensation of the parenchyma at the wound; the cytolyses of cells other than neoblasts in the parenchyma under a hypotonic condition evoke the aggregation of neoblasts. Active mitosis increases the number of neoblasts in the regeneration blastema. The “Parenchymal movement” or the mobility of the fixed parenchyma cells which must be responsible for the cellular transportation in the parenchyma was discussed.
The paper is a study of the cytology of the regeneration cells (neoblasts) in Planaria vitta.
The morphology of the living cells has first been examined to provide a reference for an investigation of the fixed neoblasts as studied by ordinary cytological, cytochemical and electron microscopical technics.
A rather selective staining method has been devised based on the strong basophilic properties of the scanty cytoplasm. The morphology of the fixed neoblasts and their distribution in the intact animal have been described, using this method.
The marked cytoplasmic basophilia was found to be exclusively due to ribonucleic acid, and not to desoxyribonucleic acid or acid mucopolysaccharides.
The cytoplasm contains moderate to considerable amounts of basic proteins. Tyrosine, cysteine/cystin, arginine, lysine and perhaps histidine were present, while tryptophan could not be demonstrated.
No enzymes could be demonstrated apart perhaps from cytochrome oxidase.
The mitochondria are small and inconspicuous and more or less evenly distributed throughout the cytoplasm. A Golgi apparatus could not be demonstrated.
The electron microscopic picture is very characteristic, because of the high electron density of the cytoplasm. This density is the result of the presence of a great number of ribonucleoprotein granules. Most of the granules are free and only a minor part bound to the membranes of the endoplasmatic reticulum. The interesting features of the cell membrane are discussed in relation to the structure of the parenchyma.
The cytochemical properties of the neoblast (RNA and sulfhydryl-groupcontaining protein) and the fine structure as revealed in the electron microscope characterize the neoblast as a morphogenetically active cell.
In Platyhelminthes, totipotent stem cells (neoblasts) are supposed to be the only dividing cells. They are responsible for the renewal of all cell types during development, growth, and regeneration, a unique situation in the animal kingdom. In order to further characterize these cells, we have applied two immunocytochemical markers to detect neoblasts in different stages of the cell cycle in the acoel flatworm Convolutriloba longifissura: (1) the thymidine analog 5'-bromo-2'-deoxyuridine (BrdU) to identify cells in S-phase, and (2) an antibody to phosphorylated histone H3 to locate mitosis. BrdU pulse-chase experiments were carried out to follow differentiation of neoblasts. We demonstrate the differentation into four labeled, differentiated cell types. S-phase cells and mitotic cells showed a homogenous distribution pattern throughout the body of C. longifissura. Two different types of S-phase cells could be distinguished immunocytochemically by their pattern of incorporated BrdU in the nuclei. Transmission electron microscopy was used to study ultrastructural characters of neoblasts and revealed two different stages in maturation of neoblasts, each with a characteristic organization of heterochromatin. The stem-cell pool of C. longifissura is an important prerequisite for the extraordinary mode of asexual reproduction and the high capacity of regeneration. A comparison of the stem-cell pool in Acoela and higher platyhelminth species can provide evidence for the phylogenetic relationships of these taxa.
In many organisms, early germline development takes place within cysts of interconnected cells that form by incomplete cytokinesis and later undergo programmed breakdown. We recently identified similar cell clusters within the fetal mouse ovary, but the fate and functional significance of these germ cell cysts remained unclear. Here, we show that mouse cysts undergo programmed breakdown between 20.5–22.5 dpc, during which approximately 33% of the oocytes survive to form primordial follicles. This process accounts for most of the perinatal reduction in germ cell numbers and germ cell apoptosis reported by previous authors, and suggests that perinatal germ cell loss is a developmentally regulated process that is distinct from the follicular atresia that occurs during adult life. Our observations also suggest a novel function for a transient cyst stage of germ cell development. Prior to breakdown, mitochondria and ER reorganize into perinuclear aggregates, and can be seen within the ring canals joining adjacent germ cells. Cysts may ensure that oocytes destined to form primordial follicles acquire populations of functional mitochondria, through an active process that has been evolutionarily conserved.
The Evolution of Individuality cites Rensch: "by far most phylogenetic changes in form arise by heterochrony"
MicroRNAs (miRNAs), an abundant class of ~22 nucleotide non-coding RNAs, are thought to play an important regulatory role in animal and plant development at the posttranscriptional level. Many miRNAs cloned from mouse bone marrow cells are differentially regulated in various hematopoietic lineages, suggesting that they might influence hematopoietic lineage differentiation. Some human miRNAs are linked to leukemias: the miR-15a/miR-16 locus is frequently deleted or down-regulated in patients with B-cell chronic lymphocytic leukemia and miR-142 is at a translocation site found in a case of aggressive B-cell leukemia. miR-181, a miRNA upregulated only in the B cell lineage of mouse bone marrow cells, promotes B cell differentiation and inhibits production of CD8⁺ T cells when expressed in hematopoietic stem/progenitor cells. In contrast miR-142s inhibits production of both CD4⁺ and CD8⁺ T cells and does not affect B cells. Collectively, these results indicate that microRNAs are components of the molecular circuitry controlling mouse hematopoiesis and suggest that other microRNAs have similar regulatory roles during other facets of vertebrate development. Singapore-MIT Alliance (SMA)
Using a new technique to stain and count planarian mitotic cells, a very rapid increase in neoblast mitotic activity has been found following feeding. The increase becomes visible 1 hour after feeding, reaches a maximum in 3–8 hours, and lasts for almost a week. These results suggest that a substantial proportion of neoblasts in the intact worm must be in the G2 phase ready to enter mitosis. The fact that the mitotic figures occur throughout the parenchyma also suggests that the stimulus may be of a neurohumoral kind.
The term apoptosis is proposed for a hitherto little recognized mechanism of controlled cell deletion, which appears to play a complementary but opposite role to mitosis in the regulation of animal cell populations. Its morphological features suggest that it is an active, inherently programmed phenomenon, and it has been shown that it can be initiated or inhibited by a variety of environmental stimuli, both physiological and pathological.
The structural changes take place in two discrete stages. The first comprises nuclear and cytoplasmic condensation and breaking up of the cell into a number of membrane-bound, ultrastructurally well-preserved fragments. In the second stage these apoptotic bodies are shed from epithelial-lined surfaces or are taken up by other cells, where they undergo a series of changes resembling in vitro autolysis within phagosomes, and are rapidly degraded by lysosomal enzymes derived from the ingesting cells.
Apoptosis seems to be involved in cell turnover in many healthy adult tissues and is responsible for focal elimination of cells during normal embryonic development. It occurs spontaneously in untreated malignant neoplasms, and participates in at least some types of therapeutically induced tumour regression. It is implicated in both physiological involution and atrophy of various tissues and organs. It can also be triggered by noxious agents, both in the embryo and adult animal.
ImagesFig. 8-10Fig. 1Fig. 2Fig. 3Fig. 4Fig. 6Fig. 7Fig. 11-14Fig. 15-18Fig. 19Fig. 20-22Fig. 23 and 24
Cell cycle checkpoints are regulatory pathways that control the order and timing of cell cycle transitions and ensure that
critical events such as DNA replication and chromosome segregation are completed with high fidelity. In addition, checkpoints
respond to damage by arresting the cell cycle to provide time for repair and by inducing transcription of genes that facilitate
repair. Checkpoint loss results in genomic instability and has been implicated in the evolution of normal cells into cancer
cells. Recent advances have revealed signal transduction pathways that transmit checkpoint signals in response to DNA damage,
replication blocks, and spindle damage. Checkpoint pathways have components shared among all eukaryotes, underscoring the
conservation of cell cycle regulatory machinery.
Multipotent haemopoietic progenitor cells appear to be 'primed' for commitment by co-expression of a multiplicity of genes characteristic of different lineages. Lineage commitment proceeds as the consolidation of a distinct pattern of gene expression out of this milieu.
Understanding how the ageing process is regulated is a fascinating and fundamental problem in biology. Here we demonstrate that signals from the reproductive system influence the lifespan of the nematode Caenorhabditis elegans. If the cells that give rise to the germ line are killed with a laser microbeam, the lifespan of the animal is extended. Our findings suggest that germline signals act by modulating the activity of an insulin/IGF-1 (insulin-like growth factor) pathway that is known to regulate the ageing of this organism. Mutants with reduced activity of the insulin/IGF-1-receptor homologue DAF-2 have been shown to live twice as long as normal, and their longevity requires the activity of DAF- 16, a member of the forkhead/winged-helix family of transcriptional regulators. We find that, in order for germline ablation to extend lifespan, DAF-16 is required, as well as a putative nuclear hormone receptor, DAF-12. In addition, our findings suggest that signals from the somatic gonad also influence ageing, and that this effect requires DAF-2 activity. Together, our findings imply that the C. elegans insulin/IGF-1 system integrates multiple signals to define the animal's rate of ageing. This study demonstrates an inherent relationship between the reproductive state of this animal and its lifespan, and may have implications for the co-evolution of reproductive capability and longevity.
Chk2 is a protein kinase that is activated in response to DNA damage and may regulate cell cycle arrest. We generated Chk2-deficient mouse cells by gene targeting. Chk2-/- embryonic stem cells failed to maintain gamma-irradiation-induced arrest in the G2 phase of the cell cycle. Chk2-/- thymocytes were resistant to DNA damage-induced apoptosis. Chk2-/- cells were defective for p53 stabilization and for induction of p53-dependent transcripts such as p21 in response to gamma irradiation. Reintroduction of the Chk2 gene restored p53-dependent transcription in response to gamma irradiation. Chk2 directly phosphorylated p53 on serine 20, which is known to interfere with Mdm2 binding. This provides a mechanism for increased stability of p53 by prevention of ubiquitination in response to DNA damage.
The singular regenerative abilities of planarians require a population of stem cells known as neoblasts. In response to wounding, or during the course of cell turnover, neoblasts are signaled to divide and/or differentiate, thereby replacing lost cell types. The study of these pluripotent stem cells and their role in planarian regeneration has been severely hampered by the reported inability of planarians to incorporate exogenous DNA precursors; thus, very little is known about the mechanisms that control proliferation and differentiation of this stem cell population within the planarian. Here we show that planarians are, in fact, capable of incorporating the thymidine analogue bromodeoxyuridine (BrdU), allowing neoblasts to be labeled specifically during the S phase of the cell cycle. We have used BrdU labeling to study the distribution of neoblasts in the intact animal, as well as to directly demonstrate the migration and differentiation of neoblasts. We have examined the proposal that a subset of neoblasts is arrested in the G2 phase of the cell cycle by double-labeling with BrdU and a mitosis-specific marker; we find that the median length of G2 (approximately 6 h) is sufficient to account for the initial mitotic burst observed after feeding or amputation. Continuous BrdU-labeling experiments also suggest that there is not a large, slow-cycling population of neoblasts in the intact animal. The ability to label specifically the regenerative stem cells, combined with the recently described use of double-stranded RNA to inhibit gene expression in the planarian, should serve to reignite interest in the flatworm as an experimental model for studying the problems of metazoan regeneration and the control of stem cell proliferation.
Why does regeneration occur? And why, when it manifests itself, does it do so in some but not all metazoan species? Hence, what are the permissive or inhibitory factors operating behind this phenomenon? When it comes to regeneration, many questions, such as these, remain unanswered. In fact, the problem of animal regeneration has withstood the probing of scientific inquiry for over 250 years and still awaits a satisfactory mechanistic explanation. In this essay, I will review the distribution and the modes of regeneration that are found in the different metazoan phyla. Also, I will re-examine ideas on its evolutionary origins, and discuss its possible relationship to both asexual reproduction and embryogenesis. This endeavor has two objectives. First, to bring forward an interpretation of regeneration which integrates evolutionary and developmental considerations into its discussion. And second, to suggest a comparative experimental approach to this problem that may bring us closer to understanding the molecular basis of this long-standing biological problem. BioEssays 22:578-590, 2000.
Stem cells (neoblasts) in Platyhelminthes are pluripotent, and likely totipotent, undifferentiated cells which retain throughout adult life the capacity to proliferate and from which all somatic cells as well as the germ cells derive. However, basic data on the pool and heterogeneity of neoblasts, their rates of differentiation into sets and subsets of differentiated cells, and their migration to different body regions are still lacking. To fill this gap, S-phase cells in the macrostomid Macrostomum sp. were labeled with the thymidine analog 5-bromo-2'-deoxyuridine (BrdU). S-phase cells were found to be neoblasts and to be distributed in two bands along the lateral sides of the body leaving unlabeled the median axis of the body and the region anterior to the eyes. This distribution is parallel to that of mitotic cells demonstrated using an antibody to phosphorylated histone H3. At different chase times, clusters of BrdU-labeled cells appear, labeled cells migrate to formerly unlabeled areas, and they differentiate into several somatic cell types and into germ cells. Finally, continuous exposure to BrdU shows an extensive renewal of the epithelial cells. Altogether, these results strengthen the idea of platyhelminth neoblasts as an unparalleled stem-cell system within the Animal Kingdom calling for further investigation.
Primordial germ cells (PGCs) are the founders of the gametes. They arise at the earliest stages of embryonic development and migrate to the gonadal ridges, where they differentiate into oogonia/oocytes in the ovary, and prospermatogonia in the testis. The present article is a review of the main studies undertaken by the author with the aim of clarifying the mechanisms underlying the development of primordial germ cells. Methods for the isolation and purification of migratory and post-migratory mouse PGCs devised in the author's laboratory are first briefly reviewed. Such methods, together with the primary culture of PGCs onto suitable cell feeder layers, have allowed the analysis of important aspects of the control of their development, concerning in particular survival, proliferation and migration of mouse PGCs. Compounds and growth factors affecting PGC numbers in culture have been identified. These include survival anti-apoptotic factors (SCF, LIF) and positive regulators of proliferation (cAMP, PACAPs, RA). Evidence has been provided that the motility of migrating PGCs relies on integrated signals from extracellular matrix molecules and the surrounding somatic cells. Moreover, homotypic PGC-PGC interaction has been evidenced that might play a role in PGC migration and in regulating their development. Several molecules (i.e. integrins, specific types of oligosaccharides, E-cadherin, the tyrosine kinase receptor c-kit) have been found to be expressed on the surface of PGCs and to mediate adhesive interactions of PGCs with the extracellular matrix, somatic cells and neighbouring PGCs.
Understanding how stem-cell proliferation is controlled to maintain adult tissues is of fundamental importance. Drosophila oogenesis provides an attractive system to study this issue since cell production in the ovary depends on small populations of observable germ-line and somatic stem cells. By controlling the amount of protein-rich nutrients in the diet, we established conditions under which the rate of egg production varied 60-fold. Using a cell-lineage labeling system, we found that both germ-line and somatic stem cells, as well as their progeny, adjust their proliferation rates in response to nutrition. However, the number of active stem cells does not appear to change. Proliferation rates varied fourfold; the remaining 15-fold difference in egg production resulted from different frequencies of cell death at two precise developmental points: (1) the region 2a/2b transition within the germarium, and (2) stage 8 egg chambers that are entering vitellogenesis. To initiate a genetic analysis of these changes in cell proliferation and apoptosis, we show that ovarian cells require an intact insulin pathway to fully upregulate their rate of cycling in response to a protein-rich diet and to enter vitellogenesis.
In Platyhelminthes, totipotent stem cells (neoblasts) are supposed to be the only dividing cells. They are responsible for the renewal of all cell types during development, growth, and regeneration, a unique situation in the animal kingdom. In order to further characterize these cells, we have applied two immunocytochemical markers to detect neoblasts in different stages of the cell cycle in the acoel flatworm Convolutriloba longifissura: (1) the thymidine analog 5'-bromo-2'-deoxyuridine (BrdU) to identify cells in S-phase, and (2) an antibody to phosphorylated histone H3 to locate mitosis. BrdU pulse-chase experiments were carried out to follow differentiation of neoblasts. We demonstrate the differentation into four labeled, differentiated cell types. S-phase cells and mitotic cells showed a homogenous distribution pattern throughout the body of C. longifissura. Two different types of S-phase cells could be distinguished immunocytochemically by their pattern of incorporated BrdU in the nuclei. Transmission electron microscopy was used to study ultrastructural characters of neoblasts and revealed two different stages in maturation of neoblasts, each with a characteristic organization of heterochromatin. The stem-cell pool of C. longifissura is an important prerequisite for the extraordinary mode of asexual reproduction and the high capacity of regeneration. A comparison of the stem-cell pool in Acoela and higher platyhelminth species can provide evidence for the phylogenetic relationships of these taxa.