Proof of principle for piggyBac-mediated transgenesis in the flatworm Macrostomum lignano

Regeneration-capable flatworms are informative research models to study the mechanisms of stem cell regulation, regeneration, and tissue patterning. The free-living flatworm Macrostomum lignano is currently the only flatworm where stable transgenesis is available, and as such it offers a powerful experimental platform to address questions that were previously difficult to answer. The published transgenesis approach relies on random integration of DNA constructs into the genome. Despite its efficiency, there is room and need for further improvement and diversification of transgenesis methods in M. lignano. Transposon-mediated transgenesis is an alternative approach, enabling easy mapping of the integration sites and the possibility of insertional mutagenesis studies. Here, we report for the first time that transposon-mediated transgenesis using piggyBac can be performed in M. lignano to create stable transgenic lines with single-copy transgene insertions.     

Boule-like genes regulate male and female gametogenesis in the flatworm Macrostomum lignano

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 bol¹ 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.     

Resilience to aging in the regeneration‐capable flatworm Macrostomum lignano

Animals show a large variability of lifespan, ranging from short‐lived as Caenorhabditis elegans to immortal as Hydra. A fascinating case is flatworms, in which reversal of aging by regeneration is proposed, yet conclusive evidence for this rejuvenation‐by‐regeneration hypothesis is lacking. We tested this hypothesis by inducing regeneration in the sexual free‐living flatworm Macrostomum lignano. We studied survival, fertility, morphology, and gene expression as a function of age. Here, we report that after regeneration, genes expressed in the germline are upregulated at all ages, but no signs of rejuvenation are observed. Instead, the animal appears to be substantially longer lived than previously appreciated, and genes expressed in stem cells are upregulated with age, while germline genes are downregulated. Remarkably, several genes with known beneficial effects on lifespan when overexpressed in mice and C. elegans are naturally upregulated with age in M. lignano, suggesting that molecular mechanism for offsetting negative consequences of aging has evolved in this animal. We therefore propose that M. lignano represents a novel powerful model for molecular studies of aging attenuation, and the identified aging gene expression patterns provide a valuable resource for further exploration of anti‐aging strategies.     

Stem cells propagate their DNA by random segregation in the flatworm Macrostomum lignano

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.     

Ontogeny of the complex sperm in the macrostomid flatworm Macrostomum lignano (Macrostomorpha,Rhabditophora)

Spermiogenesis in Macrostomum lignano (Macrostomorpha, Rhabditophora) is described using light‐ and electron microscopy of the successive stages in sperm development. Ovoid spermatids develop to highly complex, elongated sperm possessing an undulating distal (anterior) process (or “feeler”), bristles, and a proximal (posterior) brush. In particular, we present a detailed account of the morphology and ontogeny of the bristles, describing for the first time the formation of a highly specialized bristle complex consisting of several parts. This complex is ultimately reduced when sperm are mature. The implications of the development of this bristle complex on both sperm maturation and the evolution and function of the bristles are discussed. The assumed homology between bristles and flagellae questioned. J. Morphol., 2009. © 2008 Wiley‐Liss, Inc.     

Dual functions of Macpiwi1 in transposon silencing and stem cell maintenance in the flatworm Macrostomum lignano

PIWI proteins and piRNA pathways are essential for transposon silencing and some aspects of gene regulation during animal germline development. In contrast to most animal species, some flatworms also express PIWIs and piRNAs in somatic stem cells, where they are required for tissue renewal and regeneration. Here, we have identified and characterized piRNAs and PIWI proteins in the emerging model flatworm Macrostomum lignano. We found that M. lignano encodes at least three PIWI proteins. One of these, Macpiwi1, acts as a key component of the canonical piRNA pathway in the germline and in somatic stem cells. Knockdown of Macpiwi1 dramatically reduces piRNA levels, derepresses transposons, and severely impacts stem cell maintenance. Knockdown of the piRNA biogenesis factor Macvasa caused an even greater reduction in piRNA levels with a corresponding increase in transposons. Yet, in Macvasa knockdown animals, we detected no major impact on stem cell self-renewal. These results may suggest stem cell maintenance functions of PIWI proteins in flatworms that are distinguishable from their impact on transposons and that might function independently of what are considered canonical piRNA populations.     

The caudal regeneration blastema is an accumulation of rapidly proliferating stem cells in the flatworm Macrostomum lignano

Background  

Macrostomum lignano is a small free-living flatworm capable of regenerating all body parts posterior of the pharynx and anterior to the brain. We quantified the cellular composition of the caudal-most body region, the tail plate, and investigated regeneration of the tail plate in vivo and in semithin sections labeled with bromodeoxyuridine, a marker for stem cells (neoblasts) in S-phase.     

Effects of two seminal fluid transcripts on post‐mating behaviour in the simultaneously hermaphroditic flatworm Macrostomum lignano

The seminal fluid proteins (SFPs) transferred to mating partners along with sperm often play crucial roles in mediating post‐mating sexual selection. One way in which sperm donors can maximize their own reproductive success is by modifying the partner's (sperm recipient's) post‐copulatory behaviour to prevent or delay re‐mating, thereby decreasing the likelihood or intensity of sperm competition. Here, we adopted a quantitative genetic approach combining gene expression and behavioural data to identify candidates that could mediate such a response in the simultaneously hermaphroditic flatworm Macrostomum lignano. We identified two putative SFPs—Mlig‐pro46 and Mlig‐pro63—linked to both mating frequency and ‘suck’ frequency, a distinctive behaviour, in which, upon ejaculate receipt, the worm places its pharynx over its female genital opening and apparently attempts to remove the received ejaculate. We, therefore, performed a manipulative experiment using RNA interference‐induced knockdown to ask how the loss of Mlig‐pro46 and Mlig‐pro63 expression, singly and in combination, affects mating frequency, partner suck propensity and sperm competitive ability. None of the knockdown treatments impacted strongly on the mating frequency or sperm competitive ability, but knockdown of Mlig‐pro63 resulted in a significantly decreased suck propensity of mating partners. This suggests that Mlig‐pro63 may normally act as a cue in the ejaculate to trigger recipient suck behaviour and—given that other proteins in the ejaculate have the opposite effect—could be one component of an ongoing arms race between donors and recipients over the control of ejaculate fate. However, the adaptive significance of Mlig‐pro46 and Mlig‐pro63 from a donor perspective remains enigmatic.     

Genome-wide screening of functional long noncoding RNAs in the epicardial adipose tissues of atrial fibrillation

Atrial fibrillation (AF) is the most common arrhythmias, and patients with AF are facing increased risk of heart failure and ischemic stroke. However, the AF pathogenesis, especially the long noncoding RNAs (lncRNA)-related mechanism, has not been fully understood. In this study, we collected RNA sequencing data of the epicardial adipose tissues (EAT) from 6 AF and 6 sinus rhythm (SR) to identify the differentially expressed protein-coding genes (PCGs) and lncRNAs. Functionally, the differentially expressed PCGs were significantly enriched in bone development disease, chronic kidney failure, and kidney disease. Particularly, we found that homeobox (HOX) genes, especially the antisense RNAs, HOTAIRM1, HOXA-AS2 and HOXB-AS2, were significantly downregulated in EAT of AF. The biological function predictions for the dysregulated lncRNAs revealed that TNF signaling pathway was the most frequent pathway that the lncRNAs might participate in. In addition, SNHG16 and RP11-471B22.2 might participate in TGF-beta signaling and ECM-receptor interaction by interacting with the proteins involved in the pathways, respectively. Collectively, we provided some potentially pathogenic lncRNAs in AF, which might be useful for the related researchers to study their functionality and develop new therapeutics.     

Molecular mechanisms of long noncoding RNAs

Long noncoding RNAs (lncRNAs) are an important class of pervasive genes involved in a variety of biological functions. Here we discuss the emerging archetypes of molecular functions that lncRNAs execute-as signals, decoys, guides, and scaffolds. For each archetype, examples from several disparate biological contexts illustrate the commonality of the molecular mechanisms, and these mechanistic views provide useful explanations and predictions of biological outcomes. These archetypes of lncRNA function may be a useful framework to consider how lncRNAs acquire properties as biological signal transducers and hint at their possible origins in evolution. As new lncRNAs are being discovered at a rapid pace, the molecular mechanisms of lncRNAs are likely to be enriched and diversified.     

长非编码RNA

人类基因组序列的约5%～10%被稳定转录,蛋白质编码基因仅约占1%,其余4%～9%的序列虽能转录,但转录物功能尚不明确。尽管如此,已确证在非蛋白质编码转录物中,含有具备调节功能的非编码RNA(noncoding RNA,ncRNA)。与具有调节功能的短链非编码RNA[如微RNA(microRNA)、小干扰RNA(siRNA),、Piwi-RNA]相比,长非编码RNA(long noncoding RNA,lncRNA)在数量上占大多数。lncRNA通过多种方式产生,以多种途径调节靶基因表达,参与调控生物体生长、发育、衰老、死亡等过程;lncRNA功能异常往往导致疾病发生。本文综述了lncRNA的起源、分类、作用分子机制及lncRNA异常与疾病的相关性等内容,旨在充分了解这一重要新型调控分子。