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INTRODUCTION: Accurate quantification of the BCR::ABL1 fusion gene in whole blood is pivotal for the clinical management of chronic myeloid leukemia (CML) patients. The fusion protein encoded by BCR::ABL1 can vary in size, depending on the BCR and/or ABL1 gene breakpoint. The vast majority of CML patients have a p210 BCR::ABL1 fusion gene (M-BCR), which can be attributed to the presence of either e14a2 (b3a2) or e13a2 (b2a2) mRNA transcript junctions. METHODS: Twenty-five CML samples were analyzed in two different ISO15189-accredited centers that both use an Europe Against Cancer-based quantitative polymerase chain reaction (qPCR) protocol. Reanalysis of the sample set with transcript-specific standard curves and digital droplet PCR (ddPCR) were performed. RESULTS: qPCR quantification revealed a significant (up to 1 log) difference specifically for the e13a2 transcript variant in contrast to e14a2 transcripts (Hodges-Lehman 4.29; p < 0.001). Reanalysis of the sample set with transcript-specific standard curves abolishes the initial transcript-specific difference (Hodges-Lehman 0.003; p = 0.8192). Comparison of transcript-specific qPCR results of both centers with ddPCR, an absolute quantification method, showed a statically significant association, especially in the lower range, indicating the clinical utility of transcript-specific or absolute quantification methods. CONCLUSION: Our data show that differences between transcript-specific quantification might exist between centers, leading to potential clinical impact on the follow-up of CML patients. The use of transcript-specific standard curves for qPCR quantification, or absolute quantification, can significantly reduce these differences. Specific attention should be applied to the interpretation of quantification differences of CML patients that switch between diagnostic centers.
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Natural killer (NK) cells are important in the immune defense against tumor cells and pathogens, and they regulate other immune cells by cytokine secretion. Although murine NK cell biology has been extensively studied, knowledge about transcriptional circuitries controlling human NK cell development and maturation is limited. By generating ETS1-deficient human embryonic stem cells and by expressing the dominant-negative ETS1 p27 isoform in cord blood hematopoietic progenitor cells, we show that the transcription factor ETS1 is critically required for human NK cell differentiation. Genome-wide transcriptome analysis determined by RNA-sequencing combined with chromatin immunoprecipitation-sequencing analysis reveals that human ETS1 directly induces expression of key transcription factors that control NK cell differentiation (ie, E4BP4, TXNIP, TBET, GATA3, HOBIT, BLIMP1). In addition, ETS1 regulates expression of genes involved in apoptosis and NK cell activation. Our study provides important molecular insights into the role of ETS1 as an important regulator of human NK cell development and terminal differentiation.
Assuntos
Diferenciação Celular/imunologia , Regulação da Expressão Gênica/imunologia , Células-Tronco Embrionárias Humanas/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Proteína Proto-Oncogênica c-ets-1/imunologia , Apoptose/genética , Apoptose/imunologia , Diferenciação Celular/genética , Linhagem Celular , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células Matadoras Naturais/citologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Proteína Proto-Oncogênica c-ets-1/genéticaRESUMO
γδ and αß T cells have unique roles in immunity and both originate in the thymus from T-lineage committed precursors through distinct but unclear mechanisms. Here, we show that Notch1 activation is more stringently required for human γδ development compared to αß-lineage differentiation and performed paired mRNA and miRNA profiling across 11 discrete developmental stages of human T cell development in an effort to identify the potential Notch1 downstream mechanism. Our data suggest that the miR-17-92 cluster is a Notch1 target in immature thymocytes and that miR-17 can restrict BCL11B expression in these Notch-dependent T cell precursors. We show that enforced miR-17 expression promotes human γδ T cell development and, consistently, that BCL11B is absolutely required for αß but less for γδ T cell development. This study suggests that human γδ T cell development is mediated by a stage-specific Notch-driven negative feedback loop through which miR-17 temporally restricts BCL11B expression and provides functional insights into the developmental role of the disease-associated genes BCL11B and the miR-17-92 cluster in a human context.
Assuntos
Receptores de Antígenos de Linfócitos T alfa-beta , Receptores de Antígenos de Linfócitos T gama-delta , Diferenciação Celular , Linhagem da Célula/genética , Humanos , Receptor Notch1/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Proteínas Repressoras , Transdução de Sinais , Timo , Proteínas Supressoras de TumorRESUMO
Regeneration-capable flatworms are informative research models to study the mechanisms of stem cell regulation, regeneration, and tissue patterning. However, the lack of transgenesis methods considerably hampers their wider use. Here we report development of a transgenesis method for Macrostomum lignano, a basal flatworm with excellent regeneration capacity. We demonstrate that microinjection of DNA constructs into fertilized one-cell stage eggs, followed by a low dose of irradiation, frequently results in random integration of the transgene in the genome and its stable transmission through the germline. To facilitate selection of promoter regions for transgenic reporters, we assembled and annotated the M. lignano genome, including genome-wide mapping of transcription start regions, and show its utility by generating multiple stable transgenic lines expressing fluorescent proteins under several tissue-specific promoters. The reported transgenesis method and annotated genome sequence will permit sophisticated genetic studies on stem cells and regeneration using M. lignano as a model organism.
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Técnicas de Transferência de Genes , Genoma Helmíntico/genética , Platelmintos/genética , Regeneração/genética , Animais , Animais Geneticamente Modificados , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Especificidade de Órgãos/genética , Ovário/metabolismo , Platelmintos/embriologia , Platelmintos/fisiologia , Regiões Promotoras Genéticas/genética , Testículo/metabolismo , Transgenes/genéticaRESUMO
The regeneration-capable flatworm Macrostomum lignano is a powerful model organism to study the biology of stem cells in vivo. As a flatworm amenable to transgenesis, it complements the historically used planarian flatworm models, such as Schmidtea mediterranea. However, information on the transcriptome and markers of stem cells in M. lignano is limited. We generated a de novo transcriptome assembly and performed the first comprehensive characterization of gene expression in the proliferating cells of M. lignano, represented by somatic stem cells, called neoblasts, and germline cells. Knockdown of a selected set of neoblast genes, including Mlig-ddx39, Mlig-rrm1, Mlig-rpa3, Mlig-cdk1, and Mlig-h2a, confirmed their crucial role for the functionality of somatic neoblasts during homeostasis and regeneration. The generated M. lignano transcriptome assembly and gene expression signatures of somatic neoblasts and germline cells will be a valuable resource for future molecular studies in M. lignano.
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Células Germinativas/fisiologia , Platelmintos/citologia , Platelmintos/genética , Células-Tronco/fisiologia , Transcriptoma , Animais , Perfilação da Expressão GênicaRESUMO
The gradual reprogramming of haematopoietic precursors into the T-cell fate is characterized by at least two sequential developmental stages. Following Notch1-dependent T-cell lineage specification during which the first T-cell lineage genes are expressed and myeloid and dendritic cell potential is lost, T-cell specific transcription factors subsequently induce T-cell commitment by repressing residual natural killer (NK)-cell potential. How these processes are regulated in human is poorly understood, especially since efficient T-cell lineage commitment requires a reduction in Notch signalling activity following T-cell specification. Here, we show that GATA3, in contrast to TCF1, controls human T-cell lineage commitment through direct regulation of three distinct processes: repression of NK-cell fate, upregulation of T-cell lineage genes to promote further differentiation and restraint of Notch activity. Repression of the Notch1 target gene DTX1 hereby is essential to prevent NK-cell differentiation. Thus, GATA3-mediated positive and negative feedback mechanisms control human T-cell lineage commitment.
Assuntos
Linhagem da Célula/genética , Retroalimentação Fisiológica , Fator de Transcrição GATA3/genética , Células-Tronco Hematopoéticas/imunologia , Timócitos/imunologia , Diferenciação Celular , Linhagem da Célula/imunologia , Reprogramação Celular , Criança , Fator de Transcrição GATA3/imunologia , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/citologia , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/imunologia , Humanos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Cultura Primária de Células , Receptor Notch1/genética , Receptor Notch1/imunologia , Transdução de Sinais , Timócitos/citologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologiaRESUMO
Stem cells or undifferentiated cells can cope more easily with external stresses. To evaluate the impact of toxic compounds on stem cell dynamics in vivo, in relation to other biological responses, we use the carcinogenic element cadmium and the regenerating model organism Macrostomum lignano. Through both BrdU and anti-histone H3 immunostainings, cadmium-induced effects were investigated at different stages of the stem cell cycle. A 24-h exposure to 100 and 250 µM CdCl2 significantly decreased the number of stem cells (neoblasts) in mitosis, whereas the number of cells in the S phase remained unchanged. After this short-term exposure, the ultrastructure of the neoblasts was minimally affected in contrast to the epidermal tissues. These results were supported by gene expression data: transcripts of cdc2 and pig3 were significantly upregulated during all treatments. Both genes are involved in the cell cycle progression and are transcribed in the gonadal region, where stem cells are highly represented. Based on a substantial increase in gene expression of heat shock proteins (HSP) and their high activity in the gonadal region, we hypothesize that these proteins are key players in the protection of stem cells against external stresses. Apart from the strong HSP induction, other protective processes including cell division, apoptosis and anti-oxidative defence, were also activated. We, therefore, conclude that the protection of stem cells against external stressors may be based on the interplay between stem cell maintenance, i.e. repair and recovery through division, on one hand and apoptosis on the other hand. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1217-1228, 2016.
Assuntos
Cloreto de Cádmio/toxicidade , Transcriptoma/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Proteína Quinase CDC2/metabolismo , Epiderme/efeitos dos fármacos , Epiderme/ultraestrutura , Gônadas/efeitos dos fármacos , Gônadas/metabolismo , Proteínas de Choque Térmico/metabolismo , Histonas/metabolismo , Hibridização In Situ , Microscopia Eletrônica , Mitose/efeitos dos fármacos , Platelmintos/citologia , Proteínas Proto-Oncogênicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fase S , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Although hematopoietic precursor activity can be generated in vitro from human embryonic stem cells, there is no solid evidence for the appearance of multipotent, self-renewing and transplantable hematopoietic stem cells. This could be due to short half-life of hematopoietic stem cells in culture or, alternatively, human embryonic stem cell-initiated hematopoiesis may be hematopoietic stem cell-independent, similar to yolk sac hematopoiesis, generating multipotent progenitors with limited expansion capacity. Since a MYB was reported to be an excellent marker for hematopoietic stem cell-dependent hematopoiesis, we generated a MYB-eGFP reporter human embryonic stem cell line to study formation of hematopoietic progenitor cells in vitro. We found CD34(+) hemogenic endothelial cells rounding up and developing into CD43(+) hematopoietic cells without expression of MYB-eGFP. MYB-eGFP(+) cells appeared relatively late in embryoid body cultures as CD34(+)CD43(+)CD45(-/lo) cells. These MYB-eGFP(+) cells were CD33 positive, proliferated in IL-3 containing media and hematopoietic differentiation was restricted to the granulocytic lineage. In agreement with data obtained on murine Myb(-/-) embryonic stem cells, bright eGFP expression was observed in a subpopulation of cells, during directed myeloid differentiation, which again belonged to the granulocytic lineage. In contrast, CD14(+) macrophage cells were consistently eGFP(-) and were derived from eGFP-precursors only. In summary, no evidence was obtained for in vitro generation of MYB(+) hematopoietic stem cells during embryoid body cultures. The observed MYB expression appeared late in culture and was confined to the granulocytic lineage.
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Diferenciação Celular , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Proteínas Proto-Oncogênicas c-myb/metabolismo , Saco Vitelino/citologia , Células Cultivadas , Corpos Embrioides , Células-Tronco Embrionárias/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Citometria de Fluxo , Granulócitos/citologia , Granulócitos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Técnicas In Vitro , Macrófagos/citologia , Macrófagos/metabolismo , Proteínas Proto-Oncogênicas c-myb/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transgenes/fisiologia , Saco Vitelino/metabolismoRESUMO
Sex allocation theory is considered as a touchstone of evolutionary biology, providing some of the best supported examples for Darwinian adaptation. In particular, Hamilton's local mate competition theory has been shown to generate precise predictions for extraordinary sex ratios observed in many separate-sexed organisms. In analogy to local mate competition, Charnov's mating group size model predicts how sex allocation in simultaneous hermaphrodites is affected by the mating group size (i.e., the number of mating partners plus one). Until now, studies have not directly explored the relationship between mating group size and sex allocation, which we here achieve in the simultaneously hermaphroditic flatworm Macrostomum lignano. Using transgenic focal worms with ubiquitous expression of green-fluorescent protein (GFP), we assessed the number of wild-type mating partners carrying GFP+ sperm from these focal worms when raised in different social group sizes. This allowed us to test directly how mating group size was related to the sex allocation of focal worms. We find that the proportion of male investment initially increases with increasing mating group size, but then saturates as predicted by theory. To our knowledge, this is the first direct test of the mating group size model in a simultaneously hermaphroditic animal.
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Organismos Hermafroditas/fisiologia , Comportamento Sexual Animal , Turbelários/fisiologia , Animais , Densidade Demográfica , Reprodução , Razão de MasculinidadeRESUMO
Sperm production is a key male reproductive trait and an important parameter in sperm competition theory. Under sperm competition, paternity success is predicted to depend strongly on male allocation to sperm production. Furthermore, because sperm production is inherently costly, individuals should economize in sperm expenditure, and conditional adjustment of the copulation frequency according to their sperm availability may be expected. However, experimental studies showing effects of sperm production on mating behaviour and paternity success have so far been scarce, mainly because sperm production is difficult to manipulate directly in animals. Here, we used phenotypic engineering to manipulate sperm-production rate, by employing dose-dependent RNA interference (RNAi) of a spermatogenesis-specific gene, macbol1, in the free-living flatworm Macrostomum lignano. We demonstrate (i) that our novel dose-dependent RNAi approach allows us to induce high variability in sperm-production rate; (ii) that a reduced sperm-production rate is associated with a decreased copulation frequency, suggesting conditional adjustment of mating behaviour; and (iii) that both sperm production and copulation frequency are important determinants of paternity success in a competitive situation, as predicted by sperm competition theory. Our study clearly documents the potential of phenotypic engineering via dose-dependent RNAi to test quantitative predictions of evolutionary theory.
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Evolução Biológica , Proteínas de Helminto/genética , Modelos Biológicos , Platelmintos/fisiologia , Comportamento Sexual Animal , Espermatogênese/genética , Animais , Masculino , Fenótipo , Platelmintos/genética , Interferência de RNARESUMO
Macrostomum lignano (Platyhelminthes) possesses pluripotent stem cells, also called neoblasts, which power its extraordinary regeneration capacity. We have examined the cellular dynamics of neoblasts during regeneration of the rostrum in M. lignano. First, using live squeeze observations, the growth curve of the rostrum was determined. Second, neoblasts were labelled with 5-bromo-2'-deoxyuridine (BrdU) and an anti-phospho-histone H3 mitosis marker (anti-phos-H3) to analyze their proliferative response to amputation. During the regeneration process, both S- and M-phase cells were present anterior to the eyes, a region that is devoid of proliferating cells during homeostasis. Furthermore, BrdU pulse experiments revealed a biphasic S-phase pattern, different from the pattern known to occur during regeneration of the tail plate in M. lignano. During a first systemic phase, S-phase numbers significantly increased, both in the region adjacent to the wound (the anterior segment) and the region far from the wound (the posterior segment). During the second, spatially restricted phase, S-phase numbers in the anterior segment rose to a peak at 3 to 5 days post-amputation (p-a), while in the posterior segment, S-phase activity approached control values again. A blastema, characterized as a build-up of S- and M-phase cells, was formed 1 day p-a. Altogether, our data present new insights into the cellular response of the neoblast system upon amputation, clearly demonstrating important differences from the situation known to occur during regeneration of the tail plate. Furthermore, the presence of proliferating cells in the region anterior to the eyes shows a clear alteration in stem cell regulation during regeneration.
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Proliferação de Células , Células-Tronco/citologia , Turbelários/fisiologia , Animais , Bromodesoxiuridina/farmacologia , Diferenciação Celular , Olho/crescimento & desenvolvimento , Regeneração , Fase S , Células-Tronco/metabolismo , Cauda/crescimento & desenvolvimentoRESUMO
Adult stem cells are proposed to have acquired special features to prevent an accumulation of DNA-replication errors. Two such mechanisms, frequently suggested to serve this goal are cellular quiescence, and non-random segregation of DNA strands during stem cell division, a theory designated as the immortal strand hypothesis. To date, it has been difficult to test the in vivo relevance of both mechanisms in stem cell systems. It has been shown that in the flatworm Macrostomum lignano pluripotent stem cells (neoblasts) are present in adult animals. We sought to address by which means M. lignano neoblasts protect themselves against the accumulation of genomic errors, by studying the exact mode of DNA-segregation during their division. In this study, we demonstrated four lines of in vivo evidence in favor of cellular quiescence. Firstly, performing BrdU pulse-chase experiments, we localized 'Label-Retaining Cells' (LRCs). Secondly, EDU pulse-chase combined with Vasa labeling demonstrated the presence of neoblasts among the LRCs, while the majority of LRCs were differentiated cells. We showed that stem cells lose their label at a slow rate, indicating cellular quiescence. Thirdly, CldU/IdU- double labeling studies confirmed that label-retaining stem cells showed low proliferative activity. Finally, the use of the actin inhibitor, cytochalasin D, unequivocally demonstrated random segregation of DNA-strands in LRCs. Altogether, our data unambiguously demonstrated that the majority of neoblasts in M. lignano distribute their DNA randomly during cell division, and that label-retention is a direct result of cellular quiescence, rather than a sign of co-segregation of labeled strands.
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DNA/genética , DNA/metabolismo , Platelmintos/genética , Células-Tronco Pluripotentes/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Divisão Celular/fisiologia , Segregação de Cromossomos/efeitos dos fármacos , Segregação de Cromossomos/genética , Citocalasina D/farmacologia , Imuno-Histoquímica , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacosRESUMO
Members of the DAZ (Deleted in AZoospermia) gene family are important players in the process of gametogenesis and their dysregulation accounts for 10% of human male infertility. Boule, the ancestor of the family, is mainly involved in male meiosis in most organisms. With the exception of Drosophila and C. elegans, nothing is known on the function of boule in non-vertebrate animals. In the present study, we report on three boule orthologues in the flatworm Macrostomum lignano. We demonstrate that macbol1 and macbol2 are expressed in testes whilst macbol3 is expressed in ovaries and developing eggs. Macbol1 RNAi blocked spermatocyte differentiation whereas macbol2 showed no effect upon RNAi treatment. Macbol3 RNAi resulted in aberrant egg maturation and led to female sterility. We further demonstrated the evolutionary functional conservation of macbol1 by introducing this gene into Drosophila bol(1) mutants. Macbol1 was able to rescue the progression of fly meiotic divisions. In summary, our findings provide evidence for an involvement of boule genes in male and female gamete development in one organism. Furthermore, boule gene function is shown here for the first time in a lophotrochozoan. Our results point to a more diverse functional assignment of boule genes. Therefore, a better understanding of boule function in flatworms can help to elucidate the molecular mechanisms of and concomitant infertility in higher organisms including humans.
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Proteínas de Helminto/fisiologia , Oogênese , Espermatogênese , Turbelários/genética , Sequência de Aminoácidos , Animais , Azoospermia/genética , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Feminino , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Masculino , Meiose , Dados de Sequência Molecular , Oogênese/genética , Filogenia , Espermatogênese/genética , Turbelários/embriologiaRESUMO
Molecular phylogenies place the acoel flatworms as the sister-group to the remaining Bilateria, a position that should prove very valuable when trying to understand the evolutionary origins of the bilaterian body plan. A major feature characterizing Bilateria is the presence of two, orthogonal, body axis. In this article we aim at tackling the problem of how the bilaterian anterior-posterior (AP) axis is organized, and how this axis have been established over evolutionary time. To this purpose we have studied the role of some key regulatory genes involved in the control of the AP axis, the Hox family of transcription factors. All acoels studied to date contain a minimal complement of three Hox genes that are all expressed in nested domains along this major axis, providing the oldest evidence for a "Hox vectorial system" working in Bilateria. However, this proposition is not based in the analysis of Hox functions. Here we document the specific roles of one posterior Hox gene, IpHoxPost, in the postembryonic development of the acoel Isodiametra pulchra. The analysis has been done using RNA interference technologies, for the first time in acoels, and we demonstrate that the functions of this gene are restricted to the posterior region of the animal, within the muscular and neural tissues. We conclude, therefore, that the posterior Hox genes were used to specify and maintain defined anatomical regions within the AP axis of animals since the beginning of bilaterian evolution.
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Genes Homeobox , Sistema Nervoso/anatomia & histologia , Platelmintos/genética , Animais , Sequência de Bases , Primers do DNA , Regulação da Expressão Gênica , Homeostase , Hibridização In Situ , Platelmintos/anatomia & histologia , Platelmintos/fisiologia , Reação em Cadeia da Polimerase , Interferência de RNA , ReproduçãoRESUMO
Comparison of microRNA expression identified tissues present in the last common ancestor of Bilaterians and put evolution of microRNAs in the context of tissue evolution.
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Evolução Biológica , Invertebrados/embriologia , Invertebrados/genética , MicroRNAs/genética , Fenótipo , Filogenia , Animais , MicroRNAs/metabolismoRESUMO
Stem cells are the only proliferating cells in flatworms and can be eliminated by irradiation with no damage to differentiated cells. We investigated the effect of fractionated irradiation schemes on Macrostomum lignano, namely, on survival, gene expression, morphology and regeneration. Proliferating cells were almost undetectable during the first week post-treatment. Cell proliferation and gene expression were restored within 1 month in a dose-dependent manner following exposure to up to 150 Gy irradiation. During recovery, stem cells did not cross the midline but were restricted within lateral compartments. An accumulated dose of 210 Gy resulted in a lethal phenotype. Our findings demonstrate that M. lignano represents a suitable model system for elucidating the effect of irradiation on the stem cell system in flatworms and for improving our understanding of the recovery potential of severely damaged stem-cell systems.
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Raios gama , Regeneração/efeitos da radiação , Turbelários/fisiologia , Turbelários/efeitos da radiação , Envelhecimento/genética , Envelhecimento/efeitos da radiação , Animais , Bromodesoxiuridina/metabolismo , Compartimento Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Relação Dose-Resposta à Radiação , Regulação da Expressão Gênica no Desenvolvimento/efeitos da radiação , Células-Tronco/citologia , Células-Tronco/metabolismo , Células-Tronco/efeitos da radiação , Análise de Sobrevida , Turbelários/genética , Turbelários/crescimento & desenvolvimentoRESUMO
BACKGROUND: Tissue plasticity and a substantial regeneration capacity based on stem cells are the hallmark of several invertebrate groups such as sponges, cnidarians and Platyhelminthes. Traditionally, Acoela were seen as an early branching clade within the Platyhelminthes, but became recently positioned at the base of the Bilateria. However, little is known on how the stem cell system in this new phylum is organized. In this study, we wanted to examine if Acoela possess a neoblast-like stem cell system that is responsible for development, growth, homeostasis and regeneration. RESULTS: We established enduring laboratory cultures of the acoel Isodiametra pulchra (Acoela, Acoelomorpha) and implemented in situ hybridization and RNA interference (RNAi) for this species. We used BrdU labelling, morphology, ultrastructure and molecular tools to illuminate the morphology, distribution and plasticity of acoel stem cells under different developmental conditions. We demonstrate that neoblasts are the only proliferating cells which are solely mesodermally located within the organism. By means of in situ hybridisation and protein localisation we could demonstrate that the piwi-like gene ipiwi1 is expressed in testes, ovaries as well as in a subpopulation of somatic stem cells. In addition, we show that germ cell progenitors are present in freshly hatched worms, suggesting an embryonic formation of the germline. We identified a potent stem cell system that is responsible for development, homeostasis, regeneration and regrowth upon starvation. CONCLUSIONS: We introduce the acoel Isodiametra pulchra as potential new model organism, suitable to address developmental questions in this understudied phylum. We show that neoblasts in I. pulchra are crucial for tissue homeostasis, development and regeneration. Notably, epidermal cells were found to be renewed exclusively from parenchymally located stem cells, a situation known only from rhabditophoran flatworms so far. For further comparison, it will be important to analyse the stem cell systems of other key-positioned understudied taxa.
Assuntos
Células-Tronco/citologia , Turbelários/citologia , Animais , Evolução Biológica , Diferenciação Celular , Proteínas de Helminto/genética , Proteínas de Helminto/fisiologia , Hibridização In Situ , Filogenia , Turbelários/fisiologiaRESUMO
BACKGROUND: Failure of sperm differentiation is one of the major causes of male sterility. During spermiogenesis, spermatids undergo a complex metamorphosis, including chromatin condensation and cell elongation. Although the resulting sperm morphology and property can vary depending on the species, these processes are fundamental in many organisms. Studying genes involved in such processes can thus provide important information for a better understanding of spermatogenesis, which might be universally applied to many other organisms. RESULTS: In a screen for genes that have gonad-specific expression we isolated an elav-like gene, melav2, from Macrostomum lignano, containing the three RNA recognition motifs characteristic of elav-like genes. We found that melav2 mRNA was expressed exclusively in the testis, as opposed to the known elav genes, which are expressed in the nervous system. The RNAi phenotype of melav2 was characterized by an aberrant spermatid morphology, where sperm elongation often failed, and an empty seminal vesicle. Melav2 RNAi treated worms were thus male-sterile. Further analysis revealed that in melav2 RNAi treated worms precocious chromatin condensation occurred during spermatid differentiation, resulting in an abnormally tightly condensed chromatin and large vacuoles in round spermatids. In addition, immunostaining using an early-spermatid specific antibody revealed that melav2 RNAi treated worms had a larger amount of signal positive cells, suggesting that many cells failed the transition from early spermatid stage. CONCLUSION: We characterize a new function for elav-like genes, showing that melav2 plays a crucial role during spermatid differentiation, especially in the regulation of chromatin condensation and/or cell elongation.
Assuntos
Antígenos de Superfície/metabolismo , Platelmintos/embriologia , Platelmintos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Superfície/genética , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Masculino , Dados de Sequência Molecular , Platelmintos/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Alinhamento de Sequência , Espermatogênese , Testículo/citologia , Testículo/metabolismoRESUMO
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.
Assuntos
Platelmintos/crescimento & desenvolvimento , Regeneração/fisiologia , Células-Tronco/citologia , Turbelários/crescimento & desenvolvimento , Animais , Diferenciação Celular/fisiologia , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Homeostase , Imuno-Histoquímica , Microscopia Eletrônica , Filogenia , Platelmintos/fisiologia , Interferência de RNA , Células-Tronco/metabolismo , Cauda/fisiologiaRESUMO
Since first described, acoels were considered members of the flatworms (Platyhelminthes). However, no clear synapomorphies among the three large flatworm taxa -- the Catenulida, the Acoelomorpha and the Rhabditophora -- have been characterized to date. Molecular phylogenies, on the other hand, commonly positioned acoels separate from other flatworms. Accordingly, our own multi-locus phylogenetic analysis using 43 genes and 23 animal species places the acoel flatworm Isodiametra pulchra at the base of all Bilateria, distant from other flatworms. By contrast, novel data on the distribution and proliferation of stem cells and the specific mode of epidermal replacement constitute a strong synapomorphy for the Acoela plus the major group of flatworms, the Rhabditophora. The expression of a piwi-like gene not only in gonadal, but also in adult somatic stem cells is another unique feature among bilaterians. These two independent stem-cell-related characters put the Acoela into the Platyhelminthes-Lophotrochozoa clade and account for the most parsimonious evolutionary explanation of epidermal cell renewal in the Bilateria. Most available multigene analyses produce conflicting results regarding the position of the acoels in the tree of life. Given these phylogenomic conflicts and the contradiction of developmental and morphological data with phylogenomic results, the monophyly of the phylum Platyhelminthes and the position of the Acoela remain unresolved. By these data, both the inclusion of Acoela within Platyhelminthes, and their separation from flatworms as basal bilaterians are well-supported alternatives.