RESUMO
DNA polymerase epsilon (Pol ε), a component of the core replisome, is involved in DNA replication. Although genetic defects of Pol ε have been reported to cause immunodeficiency syndromes, its role in haematopoiesis remains unknown. Here, we identified compound heterozygous variants (p.[Asp1131fs];[Thr1891del]) in POLE, encoding Pol ε catalytic subunit A (POLE1), in siblings with a syndromic form of severe congenital transfusion-dependent anaemia. In contrast to Diamond-Blackfan anaemia, marked reticulocytopenia or marked erythroid hypoplasia was not found. Their bone marrow aspirates during infancy revealed erythroid dysplasia with strongly positive TP53 in immunostaining. Repetitive examinations demonstrated trilineage myelodysplasia within 2 years from birth. They had short stature and facial dysmorphism. HEK293 cell-based expression experiments and analyses of patient-derived induced pluripotent stem cells (iPSCs) disclosed a reduced mRNA level of Asp1131fs-POLE1 and defective nuclear translocation of Thr1891del-POLE1. Analysis of iPSCs showed compensatory mRNA upregulation of the other replisome components and increase of the TP53 protein, both suggesting dysfunction of the replisome. We created Pole-knockout medaka fish and found that heterozygous fishes were viable, but with decreased RBCs. Our observations expand the phenotypic spectrum of the Pol ε defect in humans, additionally providing unique evidence linking Pol ε to haematopoiesis.
Assuntos
DNA Polimerase II , Replicação do DNA , Animais , Humanos , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Células HEK293 , Replicação do DNA/genética , Proteína Supressora de Tumor p53/genética , RNA MensageiroRESUMO
The freshwater planarian Dugesia japonica maintains an abundant heterogeneous cell population called neoblasts, which include adult pluripotent stem cells. Thus, it is an excellent model organism for stem cell and regeneration research. Recently, many single-cell RNA sequencing (scRNA-seq) databases of several model organisms, including other planarian species, have become publicly available; these are powerful and useful resources to search for gene expression in various tissues and cells. However, the only scRNA-seq dataset for D. japonica has been limited by the number of genes detected. Herein, we collected D. japonica cells, and conducted an scRNA-seq analysis. A novel, automatic, iterative cell clustering strategy produced a dataset of 3,404 cells, which could be classified into 63 cell types based on gene expression profiles. We introduced two examples for utilizing the scRNA-seq dataset in this study using D. japonica. First, the dataset provided results consistent with previous studies as well as novel functionally relevant insights, that is, the expression of DjMTA and DjP2X-A genes in neoblasts that give rise to differentiated cells. Second, we conducted an integrative analysis of the scRNA-seq dataset and time-course bulk RNA-seq of irradiated animals, demonstrating that the dataset can help interpret differentially expressed genes captured via bulk RNA-seq. Using the R package "Seurat" and GSE223927, researchers can easily access and utilize this dataset.
Assuntos
Células-Tronco Adultas , Planárias , Células-Tronco Pluripotentes , Animais , Planárias/genética , Planárias/metabolismo , Transcriptoma/genética , Perfilação da Expressão GênicaRESUMO
Parkinson's disease is a neurological disorder characterized by reduced motility, depression and dementia. Guamanian parkinsonism dementia with amyotrophic sclerosis is a local case of Parkinson's disease reported in the Western Pacific Islands of Guam and Rota as well as in the Kii Peninsula of Japan. A previous genetic study has suggested that Guamanian parkinsonism is attributable to a variant of the TRPM7 gene, which encodes for melastatin-related transient receptor potential (TRP) ion channels. But the link between parkinsonism and the TRPM7 gene remains elusive. Previous studies have addressed that trpm7-deficient zebrafish embryos showed defects in pigmentation and touch-evoked motor response. In this study, we identified a new viable allele of trpm7 mutant causing an I756N amino acid substitution in the first transmembrane domain. Behavioral analyses revealed that trpm7 mutants showed compromised motility with their movement distance shorter than wild-type larvae. The velocity of the movement was significantly reduced in trpm7 mutants than in wild-type larvae. Along with a previous finding of reduced dopaminergic neurons in zebrafish trpm7 mutants, reduced motility of trpm7 mutants can suggest another similarity between trpm7 phenotypes and Parkinson's disease symptoms.
Assuntos
Canais de Cátion TRPM , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Mutação , Proteínas Serina-Treonina QuinasesRESUMO
Enucleated erythrocytes are characteristic of adult mammals. In contrast, fish, amphibians, reptiles, birds, and fetal mammals possess nucleated erythrocytes in their circulation. Erythroid maturation is regulated by erythropoietin (EPO) and its receptor (EPOR), which are conserved among vertebrates. In mammals, EPOR on the erythroid progenitor membrane disappears after terminal differentiation. However, in western clawed frog, Xenopus tropicalis, mature erythrocytes maintain EPOR expression, suggesting that they have non-canonical functions of the EPO-EPOR axis rather than proliferation and differentiation. In this study, we investigated the non-canonical functions of EPOR in Xenopus mature erythrocytes. EPO stimulation of peripheral erythrocytes did not induce proliferation but induced phosphorylation of intracellular proteins, including signal transducer and activator of transcription 5 (STAT5). RNA-Seq analysis of EPO-stimulated peripheral erythrocytes identified 45 differentially expressed genes (DEGs), including cytokine inducible SH2 containing protein gene (cish) and suppressor of cytokine signaling 3 gene (socs3), negative regulators of the EPOR-Janus kinase (JAK)-STAT pathway. These phosphorylation studies and pathway analysis demonstrated the activation of the JAK-STAT pathway through EPO-EPOR signaling in erythrocytes. Through comparison with EPO-responsive genes in mouse erythroid progenitors obtained from a public database, we identified 31 novel EPO-responsive genes indicating non-canonical functions. Among these, we focused on ornithine decarboxylase 1 gene (odc1), which is the rate-limiting enzyme in polyamine synthesis and affects hematopoietic progenitor differentiation and the endothelial cell response to hypoxic stress. An EPO-supplemented culture of erythrocytes showed increased odc1 expression followed by a decrease in polyamine-rich erythrocytes, suggesting EPO-responsive polyamine excretion. These findings will advance our knowledge of the unknown regulatory systems under the EPO-EPOR axis and functional differences between vertebrates' nucleated and enucleated erythrocytes.
Assuntos
Eritrócitos , Eritropoetina , Receptores da Eritropoetina , Xenopus , Animais , Eritropoetina/metabolismo , Eritropoetina/genética , Receptores da Eritropoetina/metabolismo , Receptores da Eritropoetina/genética , Eritrócitos/metabolismo , Transdução de Sinais , Regulação da Expressão Gênica , Eritroblastos/metabolismoRESUMO
This is a meeting report of "The workshop of research and techniques using next-generation sequencing (NGS) for developmental biology 2022." About 20 researchers attended the workshop. We discuss their NGS studies and techniques.
Assuntos
Biologia do Desenvolvimento , Sequenciamento de Nucleotídeos em Larga Escala , Sequenciamento de Nucleotídeos em Larga Escala/métodosRESUMO
The differences between plants grown in field and in controlled environments have long been recognized. However, few studies have addressed the underlying molecular mechanisms. To evaluate plant responses to fluctuating environments using laboratory equipment, we developed SmartGC, a high-performance growth chamber that reproduces the fluctuating irradiance, temperature and humidity of field environments. We analysed massive transcriptome data of rice plants grown under field and SmartGC conditions to clarify the differences in plant responses to field and controlled environments. Rice transcriptome dynamics in SmartGC mimicked those in the field, particularly during the morning and evening but those in conventional growth chamber conditions did not. Further analysis revealed that fluctuation of irradiance affects transcriptome dynamics in the morning and evening, while fluctuation of temperature affects transcriptome dynamics only in the morning. We found upregulation of genes related to biotic and abiotic stress, and their expression was affected by environmental factors that cannot be mimicked by SmartGC. Our results reveal fillable and unfillable gaps in the transcriptomes of rice grown in field and controlled environments and can accelerate the understanding of plant responses to field environments for both basic biology and agricultural applications.
Assuntos
Oryza , Transcriptoma , Regulação da Expressão Gênica de Plantas , Oryza/metabolismo , Plantas/genética , Estresse Fisiológico/genética , Temperatura , Transcriptoma/genéticaRESUMO
How genetic variations affect gene expression dynamics of field-grown plants remains unclear. Expression quantitative trait loci (eQTL) analysis is frequently used to find genomic regions underlying gene expression polymorphisms. This approach requires transcriptome data for the complete set of the QTL mapping population under the given conditions. Therefore, only a limited range of environmental conditions is covered by a conventional eQTL analysis. We sampled sparse time series of field-grown rice from chromosome segment substitution lines (CSSLs) and conducted RNA sequencing (RNA-Seq). Then, by using statistical analysis integrating meteorological data and the RNA-Seq data, we identified 1,675 eQTLs leading to polymorphisms in expression dynamics under field conditions. A genomic region on chromosome 11 influences the expression of several defense-related genes in a time-of-day- and scaled-age-dependent manner. This includes the eQTLs that possibly influence the time-of-day- and scaled-age-dependent differences in the innate immunity between Koshihikari and Takanari. Based on the eQTL and meteorological data, we successfully predicted gene expression under environments different from training environments and in rice cultivars with more complex genotypes than the CSSLs. Our novel approach of eQTL identification facilitated the understanding of the genetic architecture of expression dynamics under field conditions, which is difficult to assess by conventional eQTL studies. The prediction of expression based on eQTLs and environmental information could contribute to the understanding of plant traits under diverse field conditions.
Assuntos
Genoma de Planta , Oryza/genética , Transcriptoma , Genômica , Oryza/metabolismoRESUMO
Planarians have a remarkable regenerative ability owing to their adult pluripotent stem cells (aPSCs), which are called "neoblasts." Planarians maintain a considerable number of neoblasts throughout their adulthood to supply differentiated cells for the maintenance of tissue homeostasis and asexual reproduction (fission followed by regeneration). Thus, planarians serve as a good model to study the regulatory mechanisms of in vivo aPSCs. In asexually reproducing invertebrates, such as sponge, Hydra, and planaria, piwi family genes are the markers most commonly expressed in aPSCs. While piwi family genes are known as guardians against transposable elements in the germline cells of animals that only sexually propagate, their functions in the aPSC system have remained elusive. In this review, we introduce recent knowledge on the PIWI family proteins in the aPSC system in planarians and other organisms and discuss how PIWI family proteins contribute to the regulation of the aPSC system.
Assuntos
Proteínas Argonautas/metabolismo , Planárias/metabolismo , Células-Tronco Pluripotentes/metabolismo , Reprodução Assexuada , Animais , Proteínas Argonautas/genética , Planárias/genética , Células-Tronco Pluripotentes/citologia , Reprodução Assexuada/genéticaRESUMO
Planarians belong to the phylum Platyhelminthes and can regenerate their missing body parts after injury via activation of somatic pluripotent stem cells called neoblasts. Previous studies suggested that fibroblast growth factor (FGF) signaling plays a crucial role in the regulation of head tissue differentiation during planarian regeneration. To date, however, no FGF homologues in the Platyhelminthes have been reported. Here, we used a planarian Dugesia japonica model and identified an fgf gene termed Djfgf, which encodes a putative secreted protein with a core FGF domain characteristic of the FGF8/17/18 subfamily in bilaterians. Using Xenopus embryos, we found that DjFGF has FGF activity as assayed by Xbra induction. We next examined Djfgf expression in non-regenerating intact and regenerating planarians. In intact planarians, Djfgf was expressed in the auricles in the head and the pharynx. In the early process of regeneration, Djfgf was transiently expressed in a subset of differentiated cells around wounds. Notably, Djfgf expression was highly induced in the process of head regeneration when compared to that in the tail regeneration. Furthermore, assays of head regeneration from tail fragments revealed that combinatorial actions of the anterior extracellular signal-regulated kinase (ERK) and posterior Wnt/ß-catenin signaling restricted Djfgf expression to a certain anterior body part. This is the region where neoblasts undergo active proliferation to give rise to their differentiating progeny in response to wounding. The data suggest the possibility that DjFGF may act as an anterior counterpart of posteriorly localized Wnt molecules and trigger neoblast responses involved in planarian head regeneration.
Assuntos
Fatores de Crescimento de Fibroblastos/genética , Animais , Fatores de Crescimento de Fibroblastos/metabolismo , Filogenia , Planárias/genéticaRESUMO
Leaves within crop canopies experience variable light over the course of a day, which greatly affects photosynthesis and crop productivity. Little is known about the mechanisms of the photosynthetic response to fluctuating light and their genetic control. Here, we examined gas exchange, metabolite levels, and chlorophyll fluorescence during the photosynthetic induction response in an Oryza sativa indica cultivar with high yield (Takanari) and a japonica cultivar with lower yield (Koshihikari). Takanari had a faster induction response to sudden increases in light intensity than Koshihikari, as demonstrated by faster increases in net CO2 assimilation rate, stomatal conductance, and electron transport rate. In a simulated light regime that mimicked a typical summer day, the faster induction response in Takanari increased daily CO2 assimilation by 10%. The faster response of Takanari was explained in part by its maintenance of a larger pool of Calvin-Benson cycle metabolites. Together, the rapid responses of electron transport rate, metabolic flux, and stomatal conductance in Takanari contributed to the greater daily carbon gain under fluctuating light typical of natural environments.
Assuntos
Luz , Oryza/metabolismo , Fotossíntese , Folhas de Planta/metabolismo , Oryza/crescimento & desenvolvimento , Oryza/efeitos da radiação , Folhas de Planta/efeitos da radiaçãoRESUMO
Nuclear PIWIs together with their guide RNAs (piRNAs) epigenetically silence various genes including transposons in many organisms. In planarians, the nuclear piwi family gene, DjpiwiB is specifically transcribed in adult pluripotent stem cells (adult PSC, neoblast), but not in differentiated cells. However, the protein accumulates in the nuclei of both neoblasts and their descendant differentiated cells. Interestingly, PIWI(DjPiwiB)-piRNA complexes are indispensable for the repression of transposable genes at the onset of differentiation from neoblasts. Here, we conducted a comparative transcriptome analysis between control and DjpiwiB(RNAi) animals to identify non-transposable target genes of the DjPiwiB-piRNA complexes. Using bioinformatic analyses and RNAi we demonstrate that DjPiwiB-piRNA complexes are required for the proper expression of Djmcm2 and Djhistone h4 in neoblasts and that DjPiwiB-piRNA complexes regulate the transient expression of Djcalu during neoblast differentiation. Thus, DjPiwiB-piRNA complexes regulate the correct expression patterns during neoblast self-renewal and differentiation.
Assuntos
Planárias/citologia , Planárias/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Biologia Computacional , Regulação da Expressão Gênica no Desenvolvimento , Interferência de RNA/fisiologia , RNA de Cadeia Dupla/genética , RNA Mensageiro/genéticaRESUMO
The robust regenerative ability of planarians is known to be dependent on adult pluripotent stem cells called neoblasts. One of the morphological features of neoblasts is cytoplasmic ribonucleoprotein granules (chromatoid bodies: CBs), which resemble germ granules present in germline cells in other animals. Previously, we showed by immuno-electron microscopic analysis that DjCBC-1, a planarian Me31B/Dhh1/DDX6 homologue, which is a component of ribonucleoprotein granules, was localized in CBs in the planarian Dugesia japonica. Also, recently it was reported using another planarian species that Y12 antibody recognizing symmetrical dimethylarginine (sDMA) specifically binds to CBs in which histone mRNA is co-localized. Here, we showed by double immunostaining and RNA interference (RNAi) that DjCBC-1-containing CBs and Y12-immunoreactive CBs are distinct structures, suggesting that CBs are composed of heterogeneous populations. We also found that the Y12-immunoreactive CBs specifically contained a cytoplasmic type of planarian PIWI protein (DjPiwiC). We revealed by RNAi experiments that Y12-immunoreactive CBs may have anti-transposable element activity involving the DjPiwiC protein in the neoblasts.
Assuntos
Células-Tronco Adultas/metabolismo , Proteínas de Helminto/metabolismo , Planárias/metabolismo , Células-Tronco Pluripotentes/metabolismo , Regeneração/fisiologia , Células-Tronco Adultas/citologia , Animais , Planárias/citologia , Células-Tronco Pluripotentes/citologiaRESUMO
Planarian's strong regenerative ability is dependent on stem cells (called neoblasts) that are X-ray-sensitive and proliferative stem cells. In addition to neoblasts, another type of X-ray-sensitive cells was newly identified by recent research. Thus, planarian's X-ray-sensitive cells can be divided into at least two populations, Type 1 and Type 2, the latter corresponding to planarian's classically defined "neoblasts". Here, we show that Type 1 cells were distributed in the outer region (OR) immediately underneath the muscle layer at all axial levels from head to tail, while the Type 2 cells were distributed in a more internal region (IR) of the mesenchymal space at the axial levels from neck to tail. To elucidate the biological significance of these two regions, we searched for genes expressed in differentiated cells that were locate close to these X-ray-sensitive cell populations in the mesenchymal space, and identified six genes mainly expressed in the OR or IR, named OR1, OR2, OR3, IR1, IR2 and IR3. The predicted amino acid sequences of these genes suggested that differentiated cells expressing OR1, OR3, IR1, or IR2 provide Type 1 and Type 2 cells with specific extracellular matrix (ECM) environments.
Assuntos
Biomarcadores/análise , Diferenciação Celular , Mesoderma , Planárias/embriologia , Planárias/efeitos da radiação , Tolerância a Radiação , Animais , Animais Geneticamente Modificados , Biomarcadores/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/efeitos da radiação , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos da radiação , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Mesoderma/efeitos da radiação , Planárias/genética , Planárias/metabolismo , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/farmacologia , Raios XRESUMO
Planarian feeding behavior involves three steps: moving toward food, extending the pharynx from their planarian's ventral side after arriving at the food, and ingesting the food through the pharynx. Although pharynx extension is a remarkable behavior, it remains unknown what neuronal cell types are involved in its regulation. To identify neurons involved in regulating pharynx extension, we quantitatively analyzed pharynx extension and sought to identify these neurons by RNA interference (RNAi) and in situ hybridization. This assay, when performed using planarians with amputation of various body parts, clearly showed that the head portion is indispensable for inducing pharynx extension. We thus tested the effects of knockdown of brain neurons such as serotonergic, GABAergic, and dopaminergic neurons by RNAi, but did not observe any effects on pharynx extension behavior. However, animals with RNAi of the Prohormone Convertase 2 (PC2, a neuropeptide processing enzyme) gene did not perform the pharynx extension behavior, suggesting the possible involvement of neuropeptide(s in the regulation of pharynx extension. We screened 24 neuropeptide-coding genes, analyzed their functions by RNAi using the pharynx extension assay system, and identified at least five neuropeptide genes involved in pharynx extension. These was expressed in different cells or neurons, and some of them were expressed in the brain, suggesting complex regulation of planarian feeding behavior by the nervous system.
Assuntos
Comportamento Alimentar/fisiologia , Neuropeptídeos/genética , Planárias/fisiologia , Sequência de Aminoácidos , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hibridização In Situ , Neurônios/fisiologia , Neuropeptídeos/química , Faringe/metabolismo , Planárias/genética , Pró-Proteína Convertase 2/genética , Interferência de RNARESUMO
Electromagnetic fields (EMFs) have received widespread attention as effective, noninvasive, and safe therapies across a range of clinical applications for bone disorders. However, due to the various frequencies of devices, their effects on tissues/cells are vary, which has been a bottleneck in understanding the effects of EMFs on bone tissue. Here, we developed an in vivo model system using zebrafish scales to investigate the effects of extremely low-frequency EMFs (ELF-EMFs) on fracture healing. Exposure to 10 millitesla (mT) of ELF-EMFs at 60 Hz increased the number of both osteoblasts and osteoclasts in the fractured scale, whereas 3 or 30 mT did not. Gene expression analysis revealed that exposure to 10 mT ELF-EMFs upregulated wnt10b and Wnt target genes in the fractured scale. Moreover, ß-catenin expression was enhanced by ELF-EMFs predominantly at the fracture site of the zebrafish scale. Inhibition of Wnt/ß-catenin signaling by IWR-1-endo treatment reduced both osteoblasts and osteoclasts in the fractured scale exposed to ELF-EMFs. These results suggest that ELF-EMFs promote both osteoblast and osteoclast activity through activation of Wnt/ß-catenin signaling in fracture healing. Our data provide in vivo evidence that ELF-EMFs generated with a widely used commercial AC power supply have a facilitative effect on fracture healing.
RESUMO
Skeletal muscle development is a highly ordered process orchestrated transcriptionally by the myogenic regulatory factors. However, the downstream molecular mechanisms of myogenic regulatory factor functions in myogenesis are not fully understood. Here, we identified the RNA-binding protein Musashi2 (Msi2) as a myogenin target gene and a post-transcriptional regulator of myoblast differentiation. Msi2 knockdown in murine myoblasts blocked differentiation without affecting the expression of MyoD or myogenin. Msi2 overexpression was also sufficient to promote myoblast differentiation and myocyte fusion. Msi2 loss attenuated autophagosome formation via down-regulation of the autophagic protein MAPL1LC3/ATG8 (LC3) at the early phase of myoblast differentiation. Moreover, forced activation of autophagy effectively suppressed the differentiation defects incurred by Msi2 loss. Consistent with its functions in myoblasts in vitro, mice deficient for Msi2 exhibited smaller limb skeletal muscles, poorer exercise performance, and muscle fiber-type switching in vivo. Collectively, our study demonstrates that Msi2 is a novel regulator of mammalian myogenesis and establishes a new functional link between muscular development and autophagy regulation.
Assuntos
Desenvolvimento Muscular , Músculo Esquelético , Animais , Camundongos , Miogenina/genética , Miogenina/metabolismo , Músculo Esquelético/metabolismo , Desenvolvimento Muscular/genética , Autofagia/genética , Proteínas de Ligação a RNA/genética , Mamíferos/metabolismoRESUMO
Inbred strains of organisms are genetically highly uniform and thus useful for life science research. We have previously reported the ongoing generation of the zebrafish IM strain from the India (IND) strain through full sib-pair mating for 16 generations. However, the IM fish laid a small number of offspring and had a short lifespan, implying the need for discreet care in breeding. Here, we report the subsequent establishment of IM strain as well as the generation of a new inbred zebrafish strain, Mishima-AB (M-AB). M-AB was derived from the *AB strain by full sib-pair mating for over 20 generations, which fulfills the general criterion for the establishment of an inbred strain. In contrast to the IM case, maintenance of the M-AB strain by sib-pair mating required almost no special handling. Genome sequencing of IM individuals from the 47th generation and M-AB individuals from the 27th generation revealed that SNP-based genomic heterogeneity across whole-genome nucleotides was 0.008% and 0.011%, respectively. These percentages were much lower than those of the parental IND (0.197%) and *AB (0.086%) strains. These results indicate that the genomes of these inbred strains were highly homogenous. We also demonstrated the successful microinjection of antisense morpholinos, CRISPR/Cas9, and foreign genes into M-AB embryos at the 1-cell stage. Overall, we report the establishment of a zebrafish inbred strain, M-AB, which is capable of regular breeding and genetic manipulation. This strain will be useful for the analysis of genetically susceptible phenotypes such as behaviors, microbiome features and drug susceptibility.
Assuntos
Endogamia , Peixe-Zebra , Animais , Peixe-Zebra/genética , Genoma , Mapeamento Cromossômico , FenótipoRESUMO
The unfolded protein response is triggered in vertebrates by ubiquitously expressed IRE1α/ß (although IRE1ß is gut-specific in mice), PERK, and ATF6α/ß, transmembrane-type sensor proteins in the ER, to cope with ER stress, the accumulation of unfolded and misfolded proteins in the ER. Here, we burdened medaka fish, a vertebrate model organism, with ER stress persistently from fertilization by knocking out the AXER gene encoding an ATP/ADP exchanger in the ER membrane, leading to decreased ATP concentration-mediated impairment of the activity of Hsp70- and Hsp90-type molecular chaperones in the ER lumen. ER stress and apoptosis were evoked from 4 and 6 dpf, respectively, leading to the death of all AXER-KO medaka by 12 dpf because of heart failure (medaka hatch at 7 dpf). Importantly, constitutive activation of IRE1α signaling-but not ATF6α signaling-rescued this heart failure and allowed AXER-KO medaka to survive 3 d longer, likely because of XBP1-mediated transcriptional induction of ER-associated degradation components. Thus, activation of a specific pathway of the unfolded protein response can cure defects in a particular organ.
Assuntos
Insuficiência Cardíaca , Oryzias , Proteína 1 de Ligação a X-Box , Animais , Trifosfato de Adenosina , Endorribonucleases/genética , Proteínas de Membrana , Proteínas Serina-Treonina Quinases/genética , Proteína 1 de Ligação a X-Box/genética , Fator 6 Ativador da TranscriçãoRESUMO
The freshwater planarian Dugesia japonica is a good in vivo model for studying the function of piwi genes in adult pluripotent stem cell (aPSC) due to their abundant aPSCs. Generally, PIWI family proteins encoded by piwi genes bind to small noncoding RNAs called piRNAs (PIWI-interacting piRNAs). The analysis of PIWI-piRNA complexes in the planarian is useful for revealing the functions of piwi genes in the aPSC system. In this chapter, we present an immunoprecipitation protocol for PIWI-piRNA complexes from whole planarians.
Assuntos
Células-Tronco Adultas , Planárias , Células-Tronco Pluripotentes , Células-Tronco Adultas/metabolismo , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Imunoprecipitação , Planárias/genética , Planárias/metabolismo , Células-Tronco Pluripotentes/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
BACKGROUND: Quantification of gene expression such as RNA-Seq is a popular approach to study various biological phenomena. Despite the development of RNA-Seq library preparation methods and sequencing platforms in the last decade, RNA extraction remains the most laborious and costly step in RNA-Seq of tissue samples of various organisms. Thus, it is still difficult to examine gene expression in thousands of samples. RESULTS: Here, we developed Direct-RT buffer in which homogenization of tissue samples and direct-lysate reverse transcription can be conducted without RNA purification. The DTT concentration in Direct-RT buffer prevented RNA degradation but not RT in the lysates of several plant tissues, yeast, and zebrafish larvae. Direct reverse transcription on these lysates in Direct-RT buffer produced comparable amounts of cDNA to those synthesized from purified RNA. To maximize the advantage of the Direct-RT buffer, we integrated Direct-RT and targeted RNA-Seq to develop a cost-effective, high-throughput quantification method for the expressions of hundreds of genes: DeLTa-Seq (Direct-Lysate reverse transcription and Targeted RNA-Seq). The DeLTa-Seq method could drastically improve the efficiency and accuracy of gene expression analysis. DeLTa-Seq analysis of 1056 samples revealed the temperature-dependent effects of jasmonic acid and salicylic acid in Arabidopsis thaliana. CONCLUSIONS: The DeLTa-Seq method can realize large-scale studies using thousands of animal, plant, and microorganism samples, such as chemical screening, field experiments, and studies focusing on individual variability. In addition, Direct-RT is also beneficial for gene expression analysis in small tissues from which it is difficult to purify enough RNA for the experiments.