Bioelectrical signals generated by ion channels play crucial roles in many cellular processes in both excitable and nonexcitable cells. Some ion channels are directly implemented in chemical ...signaling pathways, the others are involved in regulation of cytoplasmic or vesicular ion concentrations, pH, cell volume, and membrane potentials. Together with ion transporters and gap junction complexes, ion channels form steady-state voltage gradients across the cell membranes in nonexcitable cells. These membrane potentials are involved in regulation of such processes as migration guidance, cell proliferation, and body axis patterning during development and regeneration. While the importance of membrane potential in stem cell maintenance, proliferation, and differentiation is evident, the mechanisms of this bioelectric control of stem cell activity are still not well understood, and the role of specific ion channels in these processes remains unclear. Here we introduce the flatworm Macrostomum lignano as a versatile model organism for addressing these topics. We discuss biological and experimental properties of M. lignano, provide an overview of the recently developed experimental tools for this animal model, and demonstrate how manipulation of membrane potential influences regeneration in M. lignano.
MicroRNAs (miRNAs) are small noncoding RNAs that function in literally all cellular processes. miRNAs interact with Argonaute (Ago) proteins and guide them to specific target sites located in the ...3'-untranslated region (3'-UTR) of target mRNAs leading to translational repression and deadenylation-induced mRNA degradation. Most miRNAs are processed from hairpin-structured precursors by the consecutive action of the RNase III enzymes Drosha and Dicer. However, processing of miR-451 is Dicer independent and cleavage is mediated by the endonuclease Ago2. Here we have characterized miR-451 sequence and structure requirements for processing as well as sorting of miRNAs into different Ago proteins. Pre-miR-451 appears to be optimized for Ago2 cleavage and changes result in reduced processing. In addition, we show that the mature miR-451 only associates with Ago2 suggesting that mature miRNAs are not exchanged between different members of the Ago protein family. Based on cloning and deep sequencing of endogenous miRNAs associated with Ago1-3, we do not find evidence for miRNA sorting in human cells. However, Ago identity appears to influence the length of some miRNAs, while others remain unaffected.
The Hippo pathway orchestrates activity of stem cells during development and tissue regeneration and is crucial for controlling organ size. However, roles of the Hippo pathway in highly regenerative ...organisms, such as flatworms, are unknown. Here we show that knockdown of the Hippo pathway core genes in the flatworm Macrostomum lignano affects tissue homeostasis and causes formation of outgrowths through hyperproliferation of stem cells (neoblasts), and leads to disruption of allometric scaling during regeneration and increased size of regenerated parts. We further show that Yap, the downstream effector of the Hippo pathway, is a potential neoblast marker gene, as it is expressed in dividing cells in M. lignano and is essential for neoblast self-renewal. The phenotypes we observe in M. lignano upon knockdown of the Hippo pathway core genes and Yap are consistent with the known functions of the pathway in other model organisms and demonstrate that the Hippo pathway is functionally conserved between flatworms and mammals. This work establishes M. lignano as a productive model for investigation of the Hippo pathway.
Diverse stresses and aging alter expression levels of microRNAs, suggesting a role for these posttranscriptional regulators of gene expression in stress modulation and longevity. Earlier studies ...demonstrated a central role for the miR-34 family in promoting cell cycle arrest and cell death following stress in human cells. However, the biological significance of this response was unclear. Here we show that in C. elegans mir-34 upregulation is necessary for developmental arrest, correct morphogenesis, and adaptation to a lower metabolic state to protect animals against stress-related damage. Either deletion or overexpression of mir-34 lead to an impaired stress response, which can largely be explained by perturbations in DAF-16/FOXO target gene expression. We demonstrate that mir-34 expression is regulated by the insulin signaling pathway via a negative feedback loop between miR-34 and DAF-16/FOXO. We propose that mir-34 provides robustness to stress response programs by controlling noise in the DAF-16/FOXO-regulated gene network.
To identify the type 2 diabetes gene located at chromosome 18p11.
We investigated the region in a young genetically isolated population by genotyping 34 single nucleotide polymorphisms (SNPs) in 78 ...case subjects and 101 control subjects. Two SNPs were selected and followed up in two cohorts. The first cohort came from a general Dutch population. In this cohort, association with type 2 diabetes was investigated using 616 type 2 diabetic case subjects and 2,890 control subjects; association with oral glucose tolerance test data was performed in 361 normoglycemic people. Association with fat distribution was studied in the second replication cohort, consisting of 836 people from the genetically isolated population.
At the initial step, we found that the common C allele of SNP rs3745012 was associated with type 2 diabetes (odds ratio 2.01, P = 0.03). This SNP is located at the 3' untranslated region of the LPIN2 gene, which is a plausible candidate for type 2 diabetes and obesity. In the cohort from the general Dutch population, we demonstrated that rs3745012 interacts with BMI in determination of type 2 diabetes: whereas in subjects with high BMI, the common C allele is associated with type 2 diabetes, the same allele exhibits a neutral or protective effect in lean subjects (P = 0.05 overall effect, P = 0.02 interaction). Most remarkably, rs3745012 strongly affected composite insulin sensitivity index (P = 0.006 for overall effect, P = 0.004 for interaction). In the second replication cohort, we found that the allele C of rs3745012 increases trunk-to-legs fat mass ratio (P = 0.001) and may affect other fat-related measurements.
rs3745012 SNP of the LPIN2 gene is associated with type 2 diabetes and fat distribution.
Regeneration-capable flatworms are highly informative research models to study the mechanisms of stem cell regulation, regeneration, and tissue patterning. Transgenesis is a powerful research tool ...for investigating gene function, but until recently, a transgenesis method was missing in flatworms, hampering their wider adoption in biomedical research. Here we describe a detailed protocol to create stable transgenic lines of the flatworm M. lignano using random integration of DNA constructs through microinjection into single-cell stage embryos.
Mirtrons are alternative precursors for microRNA biogenesis that were recently described in invertebrates. These short hairpin introns use splicing to bypass Drosha cleavage, which is otherwise ...essential for the generation of canonical animal microRNAs. Using computational and experimental strategies, we now establish that mammals have mirtrons as well. We identified 3 mirtrons that are well conserved and expressed in diverse mammals, 16 primate-specific mirtrons, and 46 candidates supported by limited cloning evidence in primates. As with some fly and worm mirtrons, the existence of well-conserved mammalian mirtrons indicates their relatively ancient incorporation into endogenous regulatory pathways. However, as worms, flies, and mammals each have different sets of mirtrons, we hypothesize that different animals may have independently evolved the capacity for this hybrid small RNA pathway. This notion is supported by our observation of several clade-specific features of mammalian and invertebrate mirtrons.
Fine-tuning the brain: MicroRNAs Vreugdenhil, Erno; Berezikov, Eugene
Frontiers in neuroendocrinology,
04/2010, Volume:
31, Issue:
2
Journal Article
Peer reviewed
Abstract The brain is of bewildering complexity and numerous genes and signaling molecules have been described that affect the architecture and functioning of specific neuronal circuits. Recent ...evidence from genome analysis revealed the existence of a large group of novel RNA molecules with unexpected properties. One such group is called microRNAs, which are small 21–23 nucleotides RNA molecules that are transcribed by the genome. However, they are not translated into proteins but rather control translation of coding mRNA. Particularly in the brain, numerous different microRNAs are expressed in a cell type specific fashion both during development and in adulthood. Aberrant microRNA expression has been implicated in several human diseases including CNS diseases. The aim of this review is to emphasize their role in the development of the brain and their function. In addition, we highlight recent findings on the evolution of mammalian microRNAs and their effect on steroid signaling in the brain.
Approaches to microRNA discovery Berezikov, Eugene; Cuppen, Edwin; Plasterk, Ronald H A
Nature genetics,
06/2006, Volume:
38 Suppl, Issue:
6s
Journal Article
Peer reviewed
MicroRNAs (miRNAs) are noncoding RNAs that can regulate gene expression. Several hundred genes encoding miRNAs have been experimentally identified in animals, and many more are predicted by ...computational methods. How can new miRNAs be discovered and distinguished from other types of small RNA? Here we summarize current methods for identifying and validating miRNAs and discuss criteria used to define an miRNA.
Abstract
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.
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