Germline Formation Research Articles

Origin and development of the germ line in sea stars.

This review summarizes and integrates our current understanding of how sea stars make gametes. Although little is known of the mechanism of germ line formation in these animals, recent results point to specific cells and to cohorts of molecules in the embryos and larvae that may lay the ground work for future research efforts. A coelomic outpocketing forms in the posterior of the gut in larvae, referred to as the posterior enterocoel (PE), that when removed, significantly reduces the number of germ cell later in larval growth. This same PE structure also selectively accumulates several germ-line associated factors-vasa, nanos, piwi-and excludes factors involved in somatic cell fate. Since its formation is relatively late in development, these germ cells may form by inductive mechanisms. When integrated into the morphological observations of germ cells and gonad development in larvae, juveniles, and adults, the field of germ line determination appears to have a good model system to study inductive germ line determination to complement the recent work on the molecular mechanisms in mice. We hope this review will also guide investigators interested in germ line determination and regulation of the germ line into how these animals can help in this research field. The review is not intended to be comprehensive-sea star reproduction has been studied for over 100 years and many reviews are comprehensive in their coverage of, for example, seasonal growth of the gonads in response to light, nutrient, and temperature. Rather the intent of this review is to help the reader focus on new experimental results attached to the historical underpinnings of how the germ cell functions in sea stars with particular emphasis to clarify the important areas of priority for future research.

Selective accumulation of germ-line associated gene products in early development of the sea star and distinct differences from germ-line development in the sea urchin.

Echinodermata is a diverse phylum, a sister group to chordates, and contains diverse organisms that may be useful to understand varied mechanisms of germ-line specification. We tested 23 genes in development of the sea star Patiria miniata that fall into five categories: (1) Conserved germ-line factors; (2) Genes involved in the inductive mechanism of germ-line specification; (3) Germ-line associated genes; (4) Molecules involved in left-right asymmetry; and (5) Genes involved in regulation and maintenance of the genome during early embryogenesis. Overall, our results support the contention that the posterior enterocoel is a source of the germ line in the sea star P. miniata. The germ line in this organism appears to be specified late in embryogenesis, and in a pattern more consistent with inductive interactions amongst cells. This is distinct from the mechanism seen in sea urchins, a close relative of the sea star clad. We propose that P. miniata may serve as a valuable model to study inductive mechanisms of germ-cell specification and when compared with germ-line formation in the sea urchin S. purpuratus may reveal developmental transitions that occur in the evolution of inherited and inductive mechanisms of germ-line specification.

Meeting report – Oocyte maturation and fertilization: lessons from canonical and emerging models

The EMBO workshop 'Oocyte maturation and fertilization: lessons from canonical and emerging models' was held at the Oceanologic Observatory of Banyuls in France in June 2013 and was organized by Anne-Marie Geneviere, Olivier Haccard, Peter Lenart and Alex McDougall. A total of 78 participants shared their research on germline formation, oocyte development, sperm, fertilization and early development. Here, we report the highlights of this meeting.

Mitochondrial DNA Transcription Levels During Spermatogenesis and Early Development in Doubly Uniparental Inheritance of the Mitochondrial DNA System of the Blue MusselMytilus galloprovincialis

In some species of bivalve, there are two highly diverged mitochondrial genomes, one found in all individuals (F type) and the other normally in males only (M type). In Mytilus, a maternally-dependent sex ratio of the progeny has been reported. Some females almost exclusively produce daughters, while others produce a high proportion of sons. We previously reported that in M. galloprovincialis, M type mtDNA copy number may be maintained during spermatogenesis and the development of larvae of male-biased mothers to sustain the doubly uniparental inheritance system. In this study, we investigated transcription levels of M type mtDNA before and after fertilization to understand its function in the germ line. First, we quantified transcription levels of M type mtDNA in testicular cells dissected using laser-capture micro-dissection. The transcription levels of M type mtDNA were not significantly different between spermatogonia and spermatocytes versus spermatids and spermatozoa. Next, we examined differences in transcription levels of M type mtDNA between larvae from male-biased and female-biased mothers. The transcription levels of M type mtDNA significantly increased 24 and 48 h after fertilization in male-biased crosses. By contrast, transcription levels significantly decreased in female-biased crosses. These results suggest M type mtDNA may play a role in early germ line formation.

Structure, transcription, and variability of metazoan mitochondrial genome: perspectives from an unusual mitochondrial inheritance system.

Despite its functional conservation, the mitochondrial genome (mtDNA) presents strikingly different features among eukaryotes, such as size, rearrangements, and amount of intergenic regions. Nonadaptive processes such as random genetic drift and mutation rate play a fundamental role in shaping mtDNA: the mitochondrial bottleneck and the number of germ line replications are critical factors, and different patterns of germ line differentiation could be responsible for the mtDNA diversity observed in eukaryotes. Among metazoan, bivalve mollusc mtDNAs show unusual features, like hypervariable gene arrangements, high mutation rates, large amount of intergenic regions, and, in some species, an unique inheritance system, the doubly uniparental inheritance (DUI). The DUI system offers the possibility to study the evolutionary dynamics of mtDNAs that, despite being in the same organism, experience different genetic drift and selective pressures. We used the DUI species Ruditapes philippinarum to study intergenic mtDNA functions, mitochondrial transcription, and polymorphism in gonads. We observed: 1) the presence of conserved functional elements and novel open reading frames (ORFs) that could explain the evolutionary persistence of intergenic regions and may be involved in DUI-specific features; 2) that mtDNA transcription is lineage-specific and independent from the nuclear background; and 3) that male-transmitted and female-transmitted mtDNAs have a similar amount of polymorphism but of different kinds, due to different population size and selection efficiency. Our results are consistent with the hypotheses that mtDNA evolution is strongly dependent on the dynamics of germ line formation, and that the establishment of a male-transmitted mtDNA lineage can increase male fitness through selection on sperm function.

Expression of vasa and nanos3 during primordial germ cell formation and migration in Atlantic cod (Gadus morhua L.)

Primordial germ cells (PGCs), progenitors of gametes, are specified very early in embryonic development and undergo an active migration to the site where the future gonads will form. While the developmental pattern of PGCs during embryogenesis has been documented in few model teleost fishes, there is currently no information available for any representative of Superorder Paracanthopterygii. This includes Atlantic cod (Gadus morhua), which is a historically important food fish in both fisheries and aquaculture industries. In the present study, we cloned and characterized vasa and nanos3 and used them as germ cell markers in Atlantic cod. Sequencing results showed prospective vasa and nanos3 mRNA contained the domains used to describe their respective protein family. Furthermore, phylogenetic analysis using the amino acid sequence placed Atlantic cod Vasa distinct from representatives of three other taxonomic Superorders. Atlantic cod Nanos3 was placed with other homologues from the Nanos3 subfamily. Expression of both genes was detected from the first cleavage division; both were specifically expressed in Atlantic cod PGCs from the 32-cell stage. While nanos3 expression ceased during early somitogenesis, vasa was strongly expressed throughout embryonic development. Using vasa as a marker, we described the Atlantic cod PGC migration pattern. We demonstrated that Atlantic cod PGCs migrate ventral to the trunk mesoderm. With the exception of Pacific herring (Clupea pallasii), PGCs in other described teleost fishes migrate lateral to the trunk. The results from this study are the first step toward understanding germ line formation in Atlantic cod.

Drosophila Syncrip binds the gurken mRNA localisation signal and regulates localised transcripts during axis specification

SummaryIn the Drosophila oocyte, mRNA transport and localised translation play a fundamental role in axis determination and germline formation of the future embryo. gurken mRNA encodes a secreted TGF-α signal that specifies dorsal structures, and is localised to the dorso-anterior corner of the oocyte via a cis-acting 64 nucleotide gurken localisation signal. Using GRNA chromatography, we characterised the biochemical composition of the ribonucleoprotein complexes that form around the gurken mRNA localisation signal in the oocyte. We identified a number of the factors already known to be involved in gurken localisation and translational regulation, such as Squid and Imp, in addition to a number of factors with known links to mRNA localisation, such as Me31B and Exu. We also identified previously uncharacterised Drosophila proteins, including the fly homologue of mammalian SYNCRIP/hnRNPQ, a component of RNA transport granules in the dendrites of mammalian hippocampal neurons. We show that Drosophila Syncrip binds specifically to gurken and oskar, but not bicoid transcripts. The loss-of-function and overexpression phenotypes of syncrip in Drosophila egg chambers show that the protein is required for correct grk and osk mRNA localisation and translational regulation. We conclude that Drosophila Syncrip is a new factor required for localisation and translational regulation of oskar and gurken mRNA in the oocyte. We propose that Syncrip/SYNCRIP is part of a conserved complex associated with localised transcripts and required for their correct translational regulation in flies and mammals.

Divergent RNA‐binding Proteins, DAZL and VASA, Induce Meiotic Progression in Human Germ Cells Derived in Vitro

Our understanding of human germ cell development is limited in large part due to inaccessibility of early human development to molecular genetic analysis. Pluripotent human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have been shown to differentiate to cells of all three embryonic germ layers, as well as germ cells in vitro, and thus may provide a model for the study of the genetics and epigenetics of human germline. Here, we examined whether intrinsic germ cell translational, rather than transcriptional, factors might drive germline formation and/or differentiation from human pluripotent stem cells in vitro. We observed that, with overexpression of VASA (DDX4) and/or DAZL (Deleted in Azoospermia Like), both hESCs and iPSCs differentiated to primordial germ cells, and maturation and progression through meiosis was enhanced. These results demonstrate that evolutionarily unrelated and divergent RNA-binding proteins can promote meiotic progression of human-derived germ cells in vitro. These studies describe an in vitro model for exploring specifics of human meiosis, a process that is remarkably susceptible to errors that lead to different infertility-related diseases.

GPI-anchor synthesis is indispensable for the germline development of the nematode Caenorhabditis elegans.

Glycosylphosphatidylinositol (GPI)-anchor attachment is one of the most common posttranslational protein modifications. Using the nematode Caenorhabditis elegans, we determined that GPI-anchored proteins are present in germline cells and distal tip cells, which are essential for the maintenance of the germline stem cell niche. We identified 24 C. elegans genes involved in GPI-anchor synthesis. Inhibition of various steps of GPI-anchor synthesis by RNA interference or gene knockout resulted in abnormal development of oocytes and early embryos, and both lethal and sterile phenotypes were observed. The piga-1 gene (orthologue of human PIGA) codes for the catalytic subunit of the phosphatidylinositol N-acetylglucosaminyltransferase complex, which catalyzes the first step of GPI-anchor synthesis. We isolated piga-1-knockout worms and found that GPI-anchor synthesis is indispensable for the maintenance of mitotic germline cell number. The knockout worms displayed 100% lethality, with decreased mitotic germline cells and abnormal eggshell formation. Using cell-specific rescue of the null allele, we showed that expression of piga-1 in somatic gonads and/or in germline is sufficient for normal embryonic development and the maintenance of the germline mitotic cells. These results clearly demonstrate that GPI-anchor synthesis is indispensable for germline formation and for normal development of oocytes and eggs.

Doubly Uniparental Inheritance of Mitochondria As a Model System for Studying Germ Line Formation

BackgroundDoubly Uniparental Inheritance (DUI) of mitochondria occurs when both mothers and fathers are capable of transmitting mitochondria to their offspring, in contrast to the typical Strictly Maternal Inheritance (SMI). DUI was found in some bivalve molluscs, in which two mitochondrial genomes are inherited, one through eggs, the other through sperm. During male embryo development, spermatozoon mitochondria aggregate in proximity of the first cleavage furrow and end up in the primordial germ cells, while they are dispersed in female embryos.Methodology/Principal FindingsWe used MitoTracker, microtubule staining and transmission electron microscopy to examine the mechanisms of this unusual distribution of sperm mitochondria in the DUI species Ruditapes philippinarum. Our results suggest that in male embryos the midbody deriving from the mitotic spindle of the first division concurs in positioning the aggregate of sperm mitochondria. Furthermore, an immunocytochemical analysis showed that the germ line determinant Vasa segregates close to the first cleavage furrow.Conclusions/SignificanceIn DUI male embryos, spermatozoon mitochondria aggregate in a stable area on the animal-vegetal axis: in organisms with spiral segmentation this zone is not involved in cleavage, so the aggregation is maintained. Moreover, sperm mitochondria reach the same embryonic area in which also germ plasm is transferred. In 2-blastomere embryos, the segregation of sperm mitochondria in the same region with Vasa suggests their contribution in male germ line formation. In DUI male embryos, M-type mitochondria must be recognized by egg factors to be actively transferred in the germ line, where they become dominant replacing the Balbiani body mitochondria. The typical features of germ line assembly point to a common biological mechanism shared by DUI and SMI organisms. Although the molecular dynamics of the segregation of sperm mitochondria in DUI species are unknown, they could be a variation of the mechanism regulating the mitochondrial bottleneck in all metazoans.

Drpiwi-1 is essential for germline cell formation during sexualization of the planarian Dugesia ryukyuensis

A piwi homolog is required for the regulation of stem cells, formation and maintenance of germline stem cells, and gametogenesis in many metazoans. Planarians can change their reproductive mode seasonally, both asexually and sexually, and develop and maintain germ cells and sexual organs. They have many pluripotent stem cells (neoblasts) that can differentiate into both somatic and germline stem cells. Thus, we searched for a piwi subfamily in the planarian Dugesia ryukyuensis. Four piwi homologs, identified as Drpiwi-1, -2, -3, and -4, were expressed in sexually reproductive worms. We then selectively destroyed the neoblasts by irradiating the worms with X-rays. In such worms, Drpiwi-1, -2, and -3 were not expressed at all, whereas Drpiwi-4 was expressed to the same degree as that in non-irradiated controls, indicating that Drpiwi-1, -2, and -3, but not Drpiwi-4, are expressed in neoblasts. During the regeneration process, Drpiwi-2(RNAi) and -3(RNAi) worms failed to regenerate after ablation, but Drpiwi-1 and -4(RNAi) worms regenerated. During the sexualizing process, Drpiwi-1(RNAi) worms failed to develop ovaries and testes, but somatic sexual organs were unaffected. Germ cell development was normal in Drpiwi-4(RNAi) worms. Therefore, Drpiwi-2 and -3 may be related to the regulation of neoblasts important for maintaining homeostasis, and Drpiwi-1 is essential for the development of germ cells but not somatic sexual organs. DrPiwi-1 is localized in the cytoplasm of stem cells and germline cells and may be involved in regulating some gene expression. We suggest that planarian Piwi controls germline formation via RNA silencing mechanisms.

Prokaryotic expression,antibody preparation and immunological characterization of VASA protein in the prawn <I>Macrobrachium nipponense</I>

Vasa gene is specifically expressed in gonads and plays an important role in the germline formation and differentiation in most animals.In this study,a full open reading frame of vasa gene was amplified from the testis cDNA pool of the prawn Macrobrachium nipponense,and ligated with expression plasmid pET32a with 6×histine tag.After the recombinant plasmid was transferred into host bacteria Escherichia coli(BL21),the recombinant VASA protein was expressed under the induction of IPTG.The optimum concentration of IPTG was 0.2 mmol/L with a duration of 6 h at 30 ℃.SDS-PAGE analysis showed that the fusion protein was highly expressed as inclusion body and the yield accounted for 48.7% of the total bacterial protein.Western-blotting analysis demonstrated the target protein with the molecular weight of 85 ku.The recombinant protein was subsequently purified by Ni2+-NTA affinity chromatography.To further prepare the prawn VASA antibody,New Zealand white rabbits were immuned by the purified protein.The highest titer of the antiserums was up to 1∶160 000 as revealed by ELISA assay.The specificity of the antibodies against VASA protein was verified by Western-blotting.Immunohistochemical analyses indicated that,in oocyte VASA protein was mainly concentrated at the perinuclear region at vitellogenic oocyte;in testis,VASA protein was mainly distributed in spermatogonia.These data suggest that the VASA could play a key role in the development and differentiation of the germ cells in the prawn.

Recent progress in the epigenetics and chromatin field

Recent progress in the epigenetics and chromatin field

The double face of the histone variant H3.3

Histone proteins wrap DNA to form nucleosome particles that compact eukaryotic genomes while still allowing access for cellular processes such as transcription, replication and DNA repair. Histones exist as different variants that have evolved crucial roles in specialized functions in addition to their fundamental role in packaging DNA. H3.3--a conserved histone variant that is structurally very close to the canonical histone H3--has been associated with active transcription. Furthermore, its role in histone replacement at active genes and promoters is highly conserved and has been proposed to participate in the epigenetic transmission of active chromatin states. Unexpectedly, recent data have revealed accumulation of this specific variant at silent loci in pericentric heterochromatin and telomeres, raising questions concerning the actual function of H3.3. In this review, we describe the known properties of H3.3 and the current view concerning its incorporation modes involving particular histone chaperones. Finally, we discuss the functional significance of the use of this H3 variant, in particular during germline formation and early development in different species.

Targeting and Anchoring Tudor in the Pole Plasm of the Drosophila Oocyte

BackgroundGermline formation is a highly regulated process in all organisms. In Drosophila embryos germ cells are specified by the pole plasm, a specialized cytoplasmic region containing polar granules. Components of these granules are also present in the perinuclear ring surrounding nurse cells, the nuage. Two such molecules are the Vasa and Tudor proteins. How Tudor localizes and is maintained in the pole plasm is, however, not known.Methodology/Principal FindingsHere, the process of Tudor localization in nuage and pole plasm was analyzed. The initial positioning of Tudor at the posterior pole of stage 9 oocytes was found to occur in the absence of a structurally detectable nuage. However, in mutants for genes encoding components of the nuage, including vasa, aubergine, maelstrom, and krimper, Tudor was detached from the posterior cortex in stage 10 oocytes, suggesting a prior passage in the nuage for its stability in the pole plasm. Further studies indicated that Valois, which was previously shown to bind in vitro to Tudor, mediates the localization of Tudor in the pole plasm by physically interacting with Oskar, the polar granule organizer. An association between Tudor and Vasa mediated by RNA was also detected in ovarian extracts.Conclusions/SignificanceThe present data challenge the view that the assembly of the polar granules occurs in a stepwise and hierarchical manner and, consequently, a revised model of polar granule assembly is proposed. In this model Oskar recruits two downstream components of the polar granules, Vasa and Tudor, independently from each other: Vasa directly interacts with Oskar while Valois mediates the recruitment of Tudor by interacting with Oskar and Tudor.

Human germ cell differentiation from fetal- and adult-derived induced pluripotent stem cells

Historically, our understanding of molecular genetic aspects of human germ cell development has been limited, at least in part due to inaccessibility of early stages of human development to experimentation. However, the derivation of pluripotent stem cells may provide the necessary human genetic system to study germ cell development. In this study, we compared the potential of human induced pluripotent stem cells (iPSCs), derived from adult and fetal somatic cells to form primordial and meiotic germ cells, relative to human embryonic stem cells. We found that ∼5% of human iPSCs differentiated to primordial germ cells (PGCs) following induction with bone morphogenetic proteins. Furthermore, we observed that PGCs expressed green fluorescent protein from a germ cell-specific reporter and were enriched for the expression of endogenous germ cell-specific proteins and mRNAs. In response to the overexpression of intrinsic regulators, we also observed that iPSCs formed meiotic cells with extensive synaptonemal complexes and post-meiotic haploid cells with a similar pattern of ACROSIN staining as observed in human spermatids. These results indicate that human iPSCs derived from reprogramming of adult somatic cells can form germline cells. This system may provide a useful model for molecular genetic studies of human germline formation and pathology and a novel platform for clinical studies and potential therapeutical applications.

The Caenorhabditis elegans Ste20 kinase, GCK-3, is essential for postembryonic developmental timing and regulates meiotic chromosome segregation

Ste20 kinases constitute a large family of serine/threonine kinases with a plethora of biological functions. Members of the GCK-VI subfamily have been identified as important regulators of osmohomeostasis across species functioning upstream of ion channels. Although the expression of the two highly similar mammalian GCK-VI kinases is eminent in a wide variety of tissues, which includes also the testis, their potential roles in development remain elusive. Caenorhabditis elegans contains a single ancestral ortholog termed GCK-3. Here, we report a comprehensive analysis of gck-3 function and demonstrate its requirement for several developmental processes independent of ion homeostasis, i.e., larval progression, vulva, and germ line formation. Consistent with a wide range of gck-3 function we find that endogenous GCK-3 is expressed ubiquitously. The serine/threonine kinase activity of GCK-3, but not its presumed C-terminal substrate interaction domain, is essential for gck-3 gene function. Although expressed in female germ cells, we find GCK-3 progressively accumulating during spermatogenesis where it promotes the first meiotic cell division and facilitates faithful chromosome segregation. In particular, we find that different levels of gck-3 activity appear to be important for various aspects of germ line development. Taken together, our findings suggest that members of the GCK-VI kinase subfamily may act as key regulators of many developmental processes and that this newly described role in meiotic progression might be conserved and an important part of sexual reproduction.

The tobacco MAP215/Dis1-family protein TMBP200 is required for the functional organization of microtubule arrays during male germline establishment

The haploid microspore division during pollen development in flowering plants is an intrinsically asymmetric division which establishes the male germline for sexual reproduction. Arabidopsis gem1 mutants lack the male germline as a result of disturbed microspore polarity, division asymmetry, and cytokinesis and represent loss-of-function mutants in MOR1/GEM1, a plant orthologue of the conserved MAP215/Dis1 microtubule associated protein (MAP) family. This provides genetic evidence for the role of MAP215/Dis1 in the organization of gametophytic microtubule arrays, but it has remained unknown how microtubule arrays are affected in gem1 mutant microspores. Here, novel male gametophytic microtubule-reporter Nicotiana tabacum plants were constructed, expressing a green fluorescent protein-α-TUBULIN fusion protein (GFP-TUA6) under the control of a microspore-specific promoter. These plants allow effective visualization of all major male gametophytic microtubule arrays and provide useful tools to study the regulation of microtubule arrays by MAPs and other effectors. Depletion of TMBP200, a tobacco homologue of MOR1/GEM1 in gametophytic microtubule-reporter plants using microspore-targeted RNA interference, induced defects in microspore polarity, division asymmetry and cytokinesis that were associated with striking defects in phragmoplast position, orientation, and structure. Our observations further reveal a requirement for TMBP200 in gametophytic spindle organization and a novel role in spindle position and orientation in polarized microspores. These results provide direct evidence for the function of MAP215/Dis1 family protein TMBP200 in the organization of microtubule arrays critical for male germline formation in plants.

Early lineage specification of long-lived germline precursors in the colonial ascidian Botryllus schlosseri.

In many taxa, germline precursors segregate from somatic lineages during embryonic development and are irreversibly committed to gametogenesis. However, in animals that can propagate asexually, germline precursors can originate in adults. Botryllus schlosseri is a colonial ascidian that grows by asexual reproduction, and on a weekly basis regenerates all somatic and germline tissues. Embryonic development in solitary ascidians is the classic example of determinative specification, and we are interested in both the origins and the persistence of stem cells responsible for asexual development in colonial ascidians. In this study, we characterized vasa as a putative marker of germline precursors. We found that maternally deposited vasa mRNA segregates early in development to a posterior lineage of cells, suggesting that germline formation is determinative in colonial ascidians. In adults, vasa expression was observed in the gonads, as well as in a population of mobile cells scattered throughout the open circulatory system, consistent with previous transplantation/reconstitution results. vasa expression was dynamic during asexual development in both fertile and infertile adults, and was also enriched in a population of stem cells. Germline precursors in juveniles could contribute to gamete formation immediately upon transplantation into fertile adults, thus vasa expression is correlated with the potential for gamete formation, which suggests that it is a marker for embryonically specified, long-lived germline progenitors. Transient vasa knockdown did not have obvious effects on germline or somatic development in adult colonies, although it did result in a profound heterochrony, suggesting that vasa might play a homeostatic role in asexual development.

Stem cells are differentially regulated during development, regeneration and homeostasis in flatworms

The flatworm stem cell system is exceptional within the animal kingdom, as totipotent stem cells (neoblasts) are the only dividing cells within the organism. In contrast to most organisms, piwi-like gene expression in flatworms is extended from germ cells to somatic stem cells. We describe the isolation and characterization of the piwi homologue macpiwi in the flatworm Macrostomum lignano. We use in situ hybridization, antibody staining and RNA interference to study macpiwi expression and function in adults, during postembryonic development, regeneration and upon starvation. We found novelties regarding piwi function and observed differences to current piwi functions in flatworms. First, macpiwi was essential for the maintenance of somatic stem cells in adult animals. A knock-down of macpiwi led to a complete elimination of stem cells and death of the animals. Second, the regulation of stem cells was different in adults and regenerates compared to postembryonic development. Third, sexual reproduction of M. lignano allowed to follow germline formation during postembryonic development, regeneration, and starvation. Fourth, piwi expression in hatchlings further supports an embryonic formation of the germline in M. lignano. Our findings address new questions in flatworm stem cell research and provide a basis for comparison with higher organisms.