RESUMO
Human in vitro oogenesis provides a framework for clarifying the mechanism of human oogenesis. To create its benchmark, it is vital to promote in vitro oogenesis using a model physiologically close to humans. Here, we establish a foundation for in vitro oogenesis in cynomolgus (cy) monkeys (Macaca fascicularis): cy female embryonic stem cells harboring one active and one inactive X chromosome (Xa and Xi, respectively) differentiate robustly into primordial germ cell-like cells, which in xenogeneic reconstituted ovaries develop efficiently into oogonia and, remarkably, further into meiotic oocytes at the zygotene stage. This differentiation entails comprehensive epigenetic reprogramming, including Xi reprogramming, yet Xa and Xi remain epigenetically asymmetric with, as partly observed in vivo, incomplete Xi reactivation. In humans and monkeys, the Xi epigenome in pluripotent stem cells functions as an Xi-reprogramming determinant. We further show that developmental pathway over-activations with suboptimal up-regulation of relevant meiotic genes impede in vitro meiotic progression. Cy in vitro oogenesis exhibits critical homology with the human system, including with respect to bottlenecks, providing a salient model for advancing human in vitro oogenesis.
Assuntos
Oócitos , Oogênese , Animais , Feminino , Humanos , Macaca fascicularis , Oogênese/fisiologia , Ovário , Células-Tronco EmbrionáriasRESUMO
In vitro oogenesis is key to elucidating the mechanism of human female germ-cell development and its anomalies. Accordingly, pluripotent stem cells have been induced into primordial germ cell-like cells and into oogonia with epigenetic reprogramming, yet further reconstitutions remain a challenge. Here, we demonstrate ex vivo reconstitution of fetal oocyte development in both humans and cynomolgus monkeys (Macaca fascicularis). With an optimized culture of fetal ovary reaggregates over three months, human and monkey oogonia enter and complete the first meiotic prophase to differentiate into diplotene oocytes that form primordial follicles, the source for oogenesis in adults. The cytological and transcriptomic progressions of fetal oocyte development in vitro closely recapitulate those in vivo. A comparison of single-cell transcriptomes among humans, monkeys, and mice unravels primate-specific and conserved programs driving fetal oocyte development, the former including a distinct transcriptomic transformation upon oogonia-to-oocyte transition and the latter including two active X chromosomes with little X-chromosome upregulation. Our study provides a critical step forward for realizing human in vitro oogenesis and uncovers salient characteristics of fetal oocyte development in primates.
Assuntos
Meiose , Oogênese , Animais , Feminino , Humanos , Macaca fascicularis , Camundongos , Oócitos , Oogênese/fisiologia , OvárioRESUMO
Runt-related transcription factor (RUNX) family members play critical roles in the development of multiple organs. Mammalian RUNX family members, consisting of RUNX1, RUNX2, and RUNX3, have distinct tissue-specific expression and function. In this study, we examined the spatiotemporal expression patterns of RUNX family members in developing kidneys and analyzed the role of RUNX1 during kidney development. In the developing mouse kidney, RUNX1 protein was strongly expressed in the ureteric bud (UB) tip and weakly expressed in the distal segment of the renal vesicle (RV), comma-shaped body (CSB), and S-shaped body (SSB). In contrast, RUNX2 protein was restricted to the stroma, and RUNX3 protein was only expressed in immune cells. We also analyzed the expression of RUNX family members in the cynomolgus monkey kidney. We found that expression patterns of RUNX2 and RUNX3 were conserved between rodents and primates, whereas RUNX1 was only expressed in the UB tip, not in the RV, CSB, or SSB of cynomolgus monkeys, suggesting a species differences. We further evaluated the roles of RUNX1 using two different conditional knockout mice: Runx1f/f:HoxB7-Cre and Runx1f/f:R26-CreERT2 and found no abnormalities in the kidney. Our findings showed that RUNX1, which is mainly expressed in the UB tip, is not essential for kidney development.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core , Rim , Animais , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Rim/metabolismo , Rim/embriologia , Rim/crescimento & desenvolvimento , Camundongos , Macaca fascicularis , Regulação da Expressão Gênica no Desenvolvimento , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Subunidades alfa de Fatores de Ligação ao Core/metabolismo , Subunidades alfa de Fatores de Ligação ao Core/genética , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
In vitro reconstitution of germ-cell development from pluripotent stem cells (PSCs) has created key opportunities to explore the fundamental mechanisms underlying germ-cell development, particularly in mice and humans. Importantly, such investigations have clarified critical species differences in the mechanisms regulating mouse and human germ-cell development, highlighting the necessity of establishing an in vitro germ-cell development system in other mammals, such as non-human primates. Here, we show that multiple lines of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in cynomolgus monkeys (Macaca fascicularis; cy) can be maintained stably in an undifferentiated state under a defined condition with an inhibitor for WNT signaling, and such PSCs are induced efficiently into primordial germ cell-like cells (PGCLCs) bearing a transcriptome similar to early cyPGCs. Interestingly, the induction kinetics of cyPGCLCs from cyPSCs is faster than that of human (h) PGCLCs from hPSCs, and while the transcriptome dynamics during cyPGCLC induction is relatively similar to that during hPGCLC induction, it is substantially divergent from that during mouse (m) PGCLC induction. Our findings delineate common as well as species-specific traits for PGC specification, creating a foundation for parallel investigations into the mechanism for germ-cell development in mice, monkeys, and humans.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Pluripotentes/citologia , Animais , Células-Tronco Pluripotentes Induzidas/citologia , Macaca fascicularis , TranscriptomaRESUMO
Nonhuman primates (NHPs) are considered to be the most valuable models for human transgenic (Tg) research into disease because human pathology is more closely recapitulated in NHPs than rodents. Previous studies have reported the generation of Tg NHPs that ubiquitously overexpress a transgene using various promoters, but it is not yet clear which promoter is most suitable for the generation of NHPs overexpressing a transgene ubiquitously and persistently in various tissues. To clarify this issue, we evaluated four putative ubiquitous promoters, cytomegalovirus (CMV) immediate-early enhancer and chicken beta-actin (CAG), elongation factor 1α (EF1α), ubiquitin C (UbC), and CMV, using an in vitro differentiation system of cynomolgus monkey embryonic stem cells (ESCs). While the EF1α promoter drove Tg expression more strongly than the other promoters in undifferentiated pluripotent ESCs, the CAG promoter was more effective in differentiated cells such as embryoid bodies and ESC-derived neurons. When the CAG and EF1α promoters were used to generate green fluorescent protein (GFP)-expressing Tg monkeys, the CAG promoter drove GFP expression in skin and hematopoietic tissues more strongly than in ΕF1α-GFP Tg monkeys. Notably, the EF1α promoter underwent more silencing in both ESCs and Tg monkeys. Thus, the CAG promoter appears to be the most suitable for ubiquitous and stable expression of transgenes in the differentiated tissues of Tg cynomolgus monkeys and appropriate for the establishment of human disease models.
Assuntos
Animais Geneticamente Modificados , Vetores Genéticos , Macaca fascicularis/genética , Regiões Promotoras Genéticas , Transgenes , Actinas/genética , Animais , Antígenos Virais/genética , Células Cultivadas , Galinhas/genética , Clonagem de Organismos/métodos , Clonagem de Organismos/normas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Elementos Facilitadores Genéticos/genética , Feminino , Técnicas de Transferência de Genes/normas , Vetores Genéticos/genética , Proteínas Imediatamente Precoces/genética , Masculino , Camundongos , Fator 1 de Elongação de Peptídeos/genéticaRESUMO
Transgenic mice are important tools for genetic analysis. A current prominent method for producing transgenic mice involves pronuclear microinjection into 1-cell embryos. However, the total transgenic efficiency obtained using this method is less than 10%. Here, we demonstrate that highly efficient transgenesis in mice can be achieved by cytoplasmic microinjection using a hyperactive piggyBac system. In embryos in which hyPBase mRNA and pPB-CAG-TagRFP DNA were co-injected into the cytoplasm, TagRFP fluorescence was observed after the 2-cell stage; when 30 ng/µl pPB-CAG-TagRFP DNA and 30 ng/µl hyPBase mRNA were co-injected, 94.4% of blastocysts were TagRFP positive. Furthermore, a high concentration of hyPBase mRNA resulted in creation of mosaic embryos in which the TagRFP signals partially disappeared. However, suitable concentrations of injected DNA and hyPBase mRNA produced embryos in which almost all blastomeres were TagRFP positive. Thus, the hyperactive piggyBac transposon system is an easy-to-implement and highly effective method that can contribute to production of transgenic mice.
Assuntos
Blastocisto/metabolismo , Elementos de DNA Transponíveis , Técnicas de Transferência de Genes , Animais , Blastocisto/citologia , Citoplasma/metabolismo , Técnicas de Cultura Embrionária/métodos , Feminino , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Microinjeções , RNA Mensageiro/metabolismo , Transgenes , Proteína Vermelha FluorescenteRESUMO
Nuclear reprogramming of differentiated cells can be induced by oocyte factors. Despite numerous attempts, these factors and mechanisms responsible for successful reprogramming remain elusive. Here, we identify one such factor, necessary for the development of nuclear transfer embryos, using porcine oocyte extracts in which some reprogramming events are recapitulated. After incubating somatic nuclei in oocyte extracts from the metaphase II stage, the oocyte proteins that were specifically and abundantly incorporated into the nuclei were identified by mass spectrometry. Among 25 identified proteins, we especially focused on a multifunctional protein, DJ-1. DJ-1 is present at a high concentration in oocytes from the germinal vesicle stage until embryos at the four-cell stage. Inhibition of DJ-1 function compromises the development of nuclear transfer embryos but not that of fertilized embryos. Microarray analysis of nuclear transfer embryos in which DJ-1 function is inhibited shows perturbed expression of P53 pathway components. In addition, embryonic arrest of nuclear transfer embryos injected with anti-DJ-1 antibody is rescued by P53 inhibition. We conclude that DJ-1 is an oocyte factor that is required for development of nuclear transfer embryos. This study presents a means for identifying natural reprogramming factors in mammalian oocytes and a unique insight into the mechanisms underlying reprogramming by nuclear transfer.
Assuntos
Blastocisto/metabolismo , Desdiferenciação Celular/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Técnicas de Transferência Nuclear , Oócitos/química , Oócitos/metabolismo , Animais , Blastocisto/citologia , Feminino , Metáfase/fisiologia , Oócitos/citologia , Suínos , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismoRESUMO
Exocyst is an octameric protein complex implicated in exocytosis. The exocyst complex is highly conserved among mammalian species, but the physiological function of each subunit in exocyst remains unclear. Previously, we identified exocyst complex component 3-like (Exoc3l) as a gene abundantly expressed in embryonic endothelial cells and implicated in the process of angiogenesis in human umbilical cord endothelial cells. Here, to reveal the physiological roles of Exoc3l during development, we generated Exoc3l knockout (KO) mice by genome editing with CRISPR/Cas9. Exoc3l KO mice were viable and showed no significant phenotype in embryonic angiogenesis or postnatal retinal angiogenesis. Exoc3l KO mice also showed no significant alteration in cholesterol homeostasis or insulin secretion, although several reports suggest an association of Exoc3l with these processes. Despite the implied roles, Exoc3l KO mice exhibited no apparent phenotype in vascular development, cholesterol homeostasis, or insulin secretion.
Assuntos
Mutação com Perda de Função , Proteínas de Transporte Vesicular , Animais , Camundongos , Humanos , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Células Endoteliais/metabolismo , Secreção de Insulina , Colesterol , Mamíferos/metabolismoRESUMO
Familial hypercholesterolemia (FH) is an inherited autosomal dominant disorder that is associated with a high plasma level of low-density lipoprotein (LDL) cholesterol, leading to an increased risk of cardiovascular diseases. To develop basic and translational research on FH, we here generated an FH model in a non-human primate (cynomolgus monkeys) by deleting the LDL receptor (LDLR) gene using the genome editing technique. Six LDLR knockout (KO) monkeys were produced, all of which were confirmed to have mutations in the LDLR gene by sequence analysis. The levels of plasma cholesterol and triglyceride were quite high in the monkeys, and were similar to those in FH patients with homozygous mutations in the LDLR gene. In addition, periocular xanthoma was observed only 1 year after birth. Lipoprotein profile analysis showed that the plasma very low-density lipoprotein and LDL were elevated, while the plasma high density lipoprotein was decreased in LDLR KO monkeys. The LDLR KO monkeys were also strongly resistant to medications for hypercholesterolemia. Taken together, we successfully generated a non-human primate model of hypercholesterolemia in which the phenotype is similar to that of homozygous FH patients.
Assuntos
Traumatismos Craniocerebrais , Hipercolesterolemia , Hiperlipoproteinemia Tipo II , Animais , Humanos , Primatas , Hiperlipoproteinemia Tipo II/genética , Lipoproteínas LDL , Macaca fascicularisRESUMO
PiggyBac (PB) transposition of reprogramming factors (Oct3/4 (O), Sox2 (S), Klf4 (K) and c-Myc) is a safe, nonviral method for generating induced pluripotent stem cells (iPSCs). However, compared with retroviral methods, the reprogramming efficiency of the PB-mediated methods is relatively low. In this study, we describe a simple and efficient system for generating high-quality iPSCs by a single transfection of multiple plasmids that does not require the use of a virus, special instruments or skilled techniques. To improve reprogramming efficiency, we modified the components of the polycistronic 2A vectors used in this study and also investigated the combination of another reprogramming-related factor (L-Myc). By simultaneous transposition of multiple PB vectors containing an EOS (early transposon promoter and Oct3/4 and Sox2 enhancers) reporter and modified polycistronic doxycycline (Dox)-inducible factors, we reprogrammed mouse somatic cells with an efficiency higher than is usually obtained with retroviral methods and we established some iPSC lines that contributed highly to chimeras. By using the Dox-inducible system, we also showed that the appropriate elimination of exogenous-factor expression at appropriate time accelerated the induction of Oct3/4 when a combination of OKS and c-Myc vectors were used.
Assuntos
Elementos de DNA Transponíveis/genética , Doxiciclina/farmacologia , Genes Reporter/genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Animais , Diferenciação Celular , Reprogramação Celular/genética , Vetores Genéticos/genética , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Fator 4 Semelhante a Kruppel , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Regiões Promotoras Genéticas/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismoRESUMO
Angiogenesis is a process to generate new blood vessels from pre-existing vessels and to maintain vessels, and plays critical roles in normal development and disease. However, the molecular mechanisms underlying angiogenesis are not fully understood. This study examined the roles of exocyst complex component (Exoc) 3-like 2 (Exoc3l2) during development in mice. We found that Exoc3l1, Exoc3l2, Exoc3l3 and Exoc3l4 are expressed abundantly in endothelial cells at embryonic day 8.5. The generation of Exoc3l2 knock-out (KO) mice showed that disruption of Exoc3l2 resulted in lethal in utero. Substantial numbers of Exoc3l2 KO embryos exhibited hemorrhaging. Deletion of Exoc3l2 using Tie2-Cre transgenic mice demonstrated that Exoc3l2 in hematopoietic and endothelial lineages was responsible for the phenotype. Taken together, these findings reveal that Exoc3l2 is essential for cardiovascular and brain development in mice.
RESUMO
Genetic mutations in fused in sarcoma (FUS) cause amyotrophic lateral sclerosis (ALS). Although mitochondrial dysfunction and stress granule have been crucially implicated in FUS proteinopathy, the molecular basis remains unclear. Here, we show that DHX30, a component of mitochondrial RNA granules required for mitochondrial ribosome assembly, interacts with FUS, and plays a crucial role in ALS-FUS. WT FUS did not affect mitochondrial localization of DHX30, but the mutant FUS lowered the signal of mitochondrial DHX30 and promoted the colocalization of cytosolic FUS aggregates and stress granule markers. The immunohistochemistry of the spinal cord from an ALS-FUS patient also confirmed the colocalization, and the immunoelectron microscope demonstrated decreased mitochondrial DHX30 signal in the spinal motor neurons. Subcellular fractionation by the detergent-solubility and density-gradient ultracentrifugation revealed that mutant FUS also promoted cytosolic mislocalization of DHX30 and aggregate formation. Interestingly, the mutant FUS disrupted the DHX30 conformation with aberrant disulfide formation, leading to impaired mitochondrial translation. Moreover, blue-native gel electrophoresis revealed an OXPHOS assembly defect caused by the FUS mutant, which was similar to that caused by DHX30 knockdown. Collectively, our study proposes DHX30 as a pivotal molecule in which disulfide-mediated conformational change mediates mitochondrial dysfunction and cytosolic aggregate formation in ALS-FUS.
Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Esclerose Lateral Amiotrófica/genética , Detergentes , Dissulfetos , Humanos , Mitocôndrias/genética , Mutação , RNA , RNA Helicases/genética , Proteína FUS de Ligação a RNA/química , Proteína FUS de Ligação a RNA/genéticaRESUMO
Human development has been studied for over a century, but the molecular mechanisms underlying human embryogenesis remain largely unknown due to technical difficulties and ethical issues. Accordingly, mice have been used as a model for mammalian development and studied extensively to infer human biology based on the conservation of fundamental processes between the two species. As research has progressed, however, species-specific differences in characteristics between rodents and primates have become apparent. Non-human primates (NHPs) have also been used for biomedical research, and are now attracting attention as a model for human development. Here, we summarize primate species from the evolutionary and genomic points of view. Then we review the current issues and progress in gene modification technology for NHPs. Finally, we discuss recent studies on the early embryogenesis of primates and future perspectives.
Assuntos
Desenvolvimento Embrionário , Modelos Biológicos , Primatas/embriologia , Animais , Evolução Biológica , Pesquisa Biomédica , Células Germinativas/citologia , HumanosRESUMO
Cynomolgus macaque (Macaca fascicularis) and common marmoset (Callithrix jacchus) have been widely used in human biomedical research. Long-standing primate genome assemblies used the human genome as a reference for ordering and orienting the assembled fragments into chromosomes. Here we performed de novo genome assembly of these two species without any human genome-based bias observed in the genome assemblies released earlier. We assembled PacBio long reads, and the resultant contigs were scaffolded with Hi-C data, which were further refined based on Hi-C contact maps and alternate de novo assemblies. The assemblies achieved scaffold N50 lengths of 149 Mb and 137 Mb for cynomolgus macaque and common marmoset, respectively. The high fidelity of our assembly is also ascertained by BAC-end concordance in common marmoset. Our assembly of cynomolgus macaque outperformed all the available assemblies of this species in terms of contiguity. The chromosome-scale genome assemblies produced in this study are valuable resources for non-human primate models and provide an important baseline in human biomedical research.
Assuntos
Callithrix/genética , Mapeamento de Sequências Contíguas , Macaca fascicularis/genética , Animais , Cromossomos , Ordem dos GenesRESUMO
The in vitro reconstitution of human germ-cell development provides a robust framework for clarifying key underlying mechanisms. Here, we explored transcription factors (TFs) that engender the germ-cell fate in their pluripotent precursors. Unexpectedly, SOX17, TFAP2C, and BLIMP1, which act under the BMP signaling and are indispensable for human primordial germ-cell-like cell (hPGCLC) specification, failed to induce hPGCLCs. In contrast, GATA3 or GATA2, immediate BMP effectors, combined with SOX17 and TFAP2C, generated hPGCLCs. GATA3/GATA2 knockouts dose-dependently impaired BMP-induced hPGCLC specification, whereas GATA3/GATA2 expression remained unaffected in SOX17, TFAP2C, or BLIMP1 knockouts. In cynomolgus monkeys, a key model for human development, GATA3, SOX17, and TFAP2C were co-expressed exclusively in early PGCs. Crucially, the TF-induced hPGCLCs acquired a hallmark of bona fide hPGCs to undergo epigenetic reprogramming and mature into oogonia/gonocytes in xenogeneic reconstituted ovaries. By uncovering a TF circuitry driving the germ line program, our study provides a paradigm for TF-based human gametogenesis.
Assuntos
Células Germinativas/metabolismo , Fatores de Transcrição SOXF/metabolismo , Fator de Transcrição AP-2/metabolismo , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Feminino , Fatores de Transcrição GATA/genética , Fatores de Transcrição GATA/metabolismo , Células Germinativas/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Macaca fascicularis , Camundongos , Camundongos Endogâmicos ICR , Fatores de Transcrição SOXF/genética , Transdução de Sinais/genética , Fator de Transcrição AP-2/genética , Fatores de Transcrição/metabolismoRESUMO
Alzheimer's disease (AD) is the most common cause of dementia and understanding its pathogenesis should lead to improved therapeutic and diagnostic methods. Although several groups have developed transgenic mouse models overexpressing the human amyloid-ß precursor protein (APP) gene with AD mutations, with and without presenilin mutations, as well as APP gene knock-in mouse models, these animals display amyloid pathology but do not show neurofibrillary tangles or neuronal loss. This presumably is due to differences between the etiology of the aged-related human disease and the mouse models. Here we report the generation of two transgenic cynomolgus monkeys overexpressing the human gene for APP with Swedish, Artic, and Iberian mutations, and demonstrated expression of gene tagged green fluorescent protein marker in the placenta, amnion, hair follicles, and peripheral blood. We believe that these nonhuman primate models will be very useful to study the pathogenesis of dementia and AD. However, generated Tg monkeys still have some limitations. We employed the CAG promoter, which will promote gene expression in a non-tissue specific manner. Moreover, we used transgenic models but not knock-in models. Thus, the inserted transgene destroys endogenous gene(s) and may affect the phenotype(s). Nevertheless, it will be of great interest to determine whether these Tg monkeys will develop tauopathy and neurodegeneration similar to human AD.
Assuntos
Precursor de Proteína beta-Amiloide/genética , Encéfalo/metabolismo , Macaca fascicularis/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Regiões Promotoras GenéticasRESUMO
Cynomolgus monkey ES (Cyn ES) cells can be generated in a similar manner as human ES cells. However, Cyn ES cells are difficult to maintain in an undifferentiated state by untrained researchers. For easier culture, we generated an OCT3/4-P2A tdTomato IRES ZeocinR Cyn ES cell line using CRISPR/Cas9 genome editing technology. The stop codon of the endogenous OCT3/4 locus was replaced with the P2A tdTomato IRES ZeocinR pA cassette by homologous recombination. This cell line enables us to isolate pluripotent stem cells and exclude differentiated cells by addition of zeocin, especially for culture without feeder cells.
Assuntos
Sistemas CRISPR-Cas , Diferenciação Celular , Separação Celular/métodos , Células-Tronco Embrionárias/citologia , Edição de Genes , Células-Tronco Pluripotentes Induzidas/citologia , Fator 3 de Transcrição de Octâmero/genética , Animais , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Células Alimentadoras , Recombinação Homóloga , Células-Tronco Pluripotentes Induzidas/metabolismo , Macaca fascicularis , Fator 3 de Transcrição de Octâmero/antagonistas & inibidoresRESUMO
Vascular endothelial growth factor receptor 3 (Vegfr3) has been widely used as a marker for lymphatic and vascular endothelial cells during mouse embryonic development and in adult mouse, making it valuable for studying angiogenesis and lymphangiogenesis under normal and pathological conditions. Here, we report the generation of a novel transgenic (Tg) mouse that expresses a membrane-localized fluorescent reporter protein, Gap43-Venus, under the control of the Vegfr3 regulatory sequence. Vegfr3-Gap43-Venus BAC Tg recapitulated endogenous Vegfr3 expression in vascular and lymphatic endothelial cells during embryonic development and tumor development. Thus, this Tg mouse line contributes a valuable model to study angiogenesis and lymphangiogenesis in physiological and pathological contexts.
Assuntos
Proteínas de Bactérias/metabolismo , Células Endoteliais/metabolismo , Proteína GAP-43/metabolismo , Proteínas Luminescentes/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Proteínas de Bactérias/genética , Vasos Sanguíneos/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Embrião de Mamíferos/irrigação sanguínea , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Proteína GAP-43/genética , Expressão Gênica , Proteínas Luminescentes/genética , Vasos Linfáticos/citologia , Vasos Linfáticos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Microscopia Confocal , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Autosomal dominant polycystic kidney disease (ADPKD) caused by PKD1 mutations is one of the most common hereditary disorders. However, the key pathological processes underlying cyst development and exacerbation in pre-symptomatic stages remain unknown, because rodent models do not recapitulate critical disease phenotypes, including disease onset in heterozygotes. Here, using CRISPR/Cas9, we generate ADPKD models with PKD1 mutations in cynomolgus monkeys. As in humans and mice, near-complete PKD1 depletion induces severe cyst formation mainly in collecting ducts. Importantly, unlike in mice, PKD1 heterozygote monkeys exhibit cyst formation perinatally in distal tubules, possibly reflecting the initial pathology in humans. Many monkeys in these models survive after cyst formation, and cysts progress with age. Furthermore, we succeed in generating selective heterozygous mutations using allele-specific targeting. We propose that our models elucidate the onset and progression of ADPKD, which will serve as a critical basis for establishing new therapeutic strategies, including drug treatments.
Assuntos
Macaca fascicularis , Rim Policístico Autossômico Dominante/genética , Canais de Cátion TRPP/genética , Alelos , Animais , Modelos Animais de Doenças , Feminino , Heterozigoto , Humanos , Rim/metabolismo , Rim/patologia , Macaca fascicularis/genética , Macaca fascicularis/metabolismo , Masculino , Mutação , Rim Policístico Autossômico Dominante/metabolismo , Rim Policístico Autossômico Dominante/patologia , Canais de Cátion TRPP/metabolismoRESUMO
In mammals, the Y chromosome strictly influences the maintenance of male germ cells. Almost all mammalian species require genetic contributors to generate testes. An endangered species, Tokudaia osimensis, has a unique sex chromosome composition XO/XO, and genetic differences between males and females have not been confirmed. Although a distinctive sex-determining mechanism may exist in T. osimensis, it has been difficult to examine thoroughly in this rare animal species. To elucidate the discriminative sex-determining mechanism in T. osimensis and to find a strategy to prevent its possible extinction, we have established induced pluripotent stem cells (iPSCs) and derived interspecific chimeras using mice as the hosts and recipients. Generated iPSCs are considered to be in the so-called "true naïve" state, and T. osimensis iPSCs may contribute as interspecific chimeras to several different tissues and cells in live animals. Surprisingly, female T. osimensis iPSCs not only contributed to the female germ line in the interspecific mouse ovary but also differentiated into spermatocytes and spermatids that survived in the adult interspecific mouse testes. Thus, T. osimensis cells have high sexual plasticity through which female somatic cells can be converted to male germline cells. These findings suggest flexibility in T. osimensis cells, which can adapt their germ cell sex to the gonadal niche. The probable reduction of the extinction risk of an endangered species through the use of iPSCs is indicated by this study.