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How the timing of development is linked to organismal size is a longstanding question. Although numerous studies have reported a correlation of temporal and spatial traits, the developmental or selective constraints underlying this link remain largely unexplored. We address this question by studying the periodic process of embryonic axis segmentation in-vivo in Oryzias fish. Interspecies comparisons reveal that the timing of segmentation correlates to segment, tissue and organismal size. Segment size in turn scales according to tissue and organism size. To probe for underlying causes, we genetically hybridised two closely related species. Quantitative analysis in ~600 phenotypically diverse F2 embryos reveals a decoupling of timing from size control, while spatial scaling is preserved. Using developmental quantitative trait loci (devQTL) mapping we identify distinct genetic loci linked to either the control of segmentation timing or tissue size. This study demonstrates that a developmental constraint mechanism underlies spatial scaling of axis segmentation, while its spatial and temporal control are dissociable modules.
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Motivation: Crosses among inbred lines are a fundamental tool for the discovery of genetic loci associated with phenotypes of interest. In organisms for which large reference panels or SNP chips are not available, imputation from low-pass whole-genome sequencing is an effective method for obtaining genotype data from a large number of individuals. To date, a structured analysis of the conditions required for optimal genotype imputation has not been performed. Results: We report a systematic exploration of the effect of several design variables on imputation performance in F2 crosses of inbred medaka lines using the imputation software STITCH. We determined that, depending on the number of samples, imputation performance reaches a plateau when increasing the per-sample sequencing coverage. We also systematically explored the trade-offs between cost, imputation accuracy, and sample numbers. We developed a computational pipeline to streamline the process, enabling other researchers to perform a similar cost-benefit analysis on their population of interest. Availability and implementation: The source code for the pipeline is available at https://github.com/birneylab/stitchimpute. While our pipeline has been developed and tested for an F2 population, the software can also be used to analyse populations with a different structure.
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Medaka fish (Oryzias latipes) is a powerful model to study genetics underlying the developmental and functional traits of the vertebrate visual system. We established a simple and high-throughput optomotor response (OMR) assay utilizing medaka larvae to study visual functions including visual acuity and contrast sensitivity. Our assay presents multiple adjustable stripes in motion to individual fish in a linear arena. For that the OMR assay employs a tablet display and the Fish Stripes software to adjust speed, width, color, and contrast of the stripes. Our results demonstrated that optomotor responses were robustly induced by black and white stripes presented from below in the linear-pool-arena. We detected robust strain specific differences in the OMR when comparing long established medaka inbred strains. We observed an interesting training effect upon the initial exposure of larvae to thick stripes, which allowed them to better respond to narrower stripes. The OMR setup and protocol presented here provide an efficient tool for quantitative phenotype mapping, addressing visual acuity, trainability of cortical neurons, color sensitivity, locomotor response, retinal regeneration and others. Our open-source setup presented here provides a crucial prerequisite for ultimately addressing the genetic basis of those processes.
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Larva , Oryzias , Animales , Oryzias/fisiología , Larva/fisiología , Agudeza Visual/fisiología , Estimulación Luminosa , Sensibilidad de Contraste/fisiología , Visión Ocular/fisiología , Ensayos Analíticos de Alto Rendimiento/métodosRESUMEN
Multi-photon 3D laser printing has gathered much attention in recent years as a means of manufacturing biocompatible scaffolds that can modify and guide cellular behavior in vitro. However, in vivo tissue engineering efforts have been limited so far to the implantation of beforehand 3D printed biocompatible scaffolds and in vivo bioprinting of tissue constructs from bioinks containing cells, biomolecules, and printable hydrogel formulations. Thus, a comprehensive 3D laser printing platform for in vivo and in situ manufacturing of microimplants raised from synthetic polymer-based inks is currently missing. Here, a platform for minimal-invasive manufacturing of microimplants directly in the organism is presented by one-photon photopolymerization and multi-photon 3D laser printing. Employing a commercially available elastomeric ink giving rise to biocompatible synthetic polymer-based microimplants, first applicational examples of biological responses to in situ printed microimplants are demonstrated in the teleost fish Oryzias latipes and in embryos of the fruit fly Drosophila melanogaster. This provides a framework for future studies addressing the suitability of inks for in vivo 3D manufacturing. The platform bears great potential for the direct engineering of the intricate microarchitectures in a variety of tissues in model organisms and beyond.
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Drosophila melanogaster , Rayos Láser , Impresión Tridimensional , Ingeniería de Tejidos , Impresión Tridimensional/instrumentación , Animales , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Oryzias , Materiales Biocompatibles , Bioimpresión/métodos , TintaRESUMEN
The development of an organism is orchestrated by the spatial and temporal expression of genes. Accurate visualisation of gene expression patterns in the context of the surrounding tissues offers a glimpse into the mechanisms that drive morphogenesis. We developed correlative light-sheet fluorescence microscopy and X-ray computed tomography approach to map gene expression patterns to the whole organism`s 3D anatomy. We show that this multimodal approach is applicable to gene expression visualized by protein-specific antibodies and fluorescence RNA in situ hybridisation offering a detailed understanding of individual phenotypic variations in model organisms. Furthermore, the approach offers a unique possibility to identify tissues together with their 3D cellular and molecular composition in anatomically less-defined in vitro models, such as organoids. We anticipate that the visual and quantitative insights into the 3D distribution of gene expression within tissue architecture, by multimodal approach developed here, will be equally valuable for reference atlases of model organisms development, as well as for comprehensive screens, and morphogenesis studies of in vitro models.
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Anticuerpos , Tomografía Computarizada por Rayos X , Hibridación Fluorescente in Situ , Microscopía Fluorescente , Expresión GénicaRESUMEN
The polygenic contribution to heart development and function along the health-disease continuum remains unresolved. To gain insight into the genetic basis of quantitative cardiac phenotypes, we utilize highly inbred Japanese rice fish models, Oryzias latipes, and Oryzias sakaizumii. Employing automated quantification of embryonic heart rates as core metric, we profiled phenotype variability across five inbred strains. We observed maximal phenotypic contrast between individuals of the HO5 and the HdrR strain. HO5 showed elevated heart rates associated with embryonic ventricular hypoplasia and impaired adult cardiac function. This contrast served as the basis for genome-wide mapping. In a segregation population of 1192 HO5 x HdrR F2 embryos, we mapped 59 loci (173 genes) associated with heart rate. Experimental validation of the top 12 candidate genes in loss-of-function models revealed their causal and distinct impact on heart rate, development, ventricle size, and arrhythmia. Our study uncovers new diagnostic and therapeutic targets for developmental and electrophysiological cardiac diseases and provides a novel scalable approach to investigate the intricate genetic architecture of the vertebrate heart.
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High-throughput DNA sequencing studies increasingly associate DNA variants with congenital heart disease (CHD). However, functional modeling is a crucial prerequisite for translating genomic data into clinical care. We used CRISPR-Cas9-mediated targeting of 12 candidate genes in the vertebrate model medaka (Oryzias latipes), five of which displayed a novel cardiovascular phenotype spectrum in F0 (crispants): mapre2, smg7, cdc42bpab, ankrd11 and myrf, encoding a transcription factor recently linked to cardiac-urogenital syndrome. Our myrf mutant line showed particularly prominent embryonic cardiac defects recapitulating phenotypes of pediatric patients, including hypoplastic ventricle. Mimicking human mutations, we edited three sites to generate specific myrf single-nucleotide variants via cytosine and adenine base editors. The Glu749Lys missense mutation in the conserved intramolecular chaperon autocleavage domain fully recapitulated the characteristic myrf mutant phenotype with high penetrance, underlining the crucial function of this protein domain. The efficiency and scalability of base editing to model specific point mutations accelerate gene validation studies and the generation of human-relevant disease models.
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Edición Génica , Cardiopatías Congénitas , Humanos , Niño , Mutación/genética , Mutación Puntual , Factores de Transcripción/metabolismo , Cardiopatías Congénitas/genética , Sistemas CRISPR-Cas/genéticaRESUMEN
The discovery and study of adult stem cells have revolutionized regenerative medicine by offering new opportunities for treating various medical conditions. Anamniote stem cells, which retain their full proliferative capacity and full differentiation range throughout their lifetime, harbour a greater potential compared to mammalian adult stem cells, which only exhibit limited stem cell potential. Therefore, understanding the mechanisms underlying these differences is of significant interest. In this review, we examine the similarities and differences of adult retinal stem cells in anamniotes and mammals, from their embryonic stages in the optic vesicle to their residence in the postembryonic retinal stem cell niche, the ciliary marginal zone located in the retinal periphery. In anamniotes, developing precursors of retinal stem cells are exposed to various environmental cues during their migration in the complex morphogenetic remodelling of the optic vesicle to the optic cup. In contrast, their mammalian counterparts in the retinal periphery are primarily instructed by neighbouring tissues once they are in place. We explore the distinct modes of optic cup morphogenesis in mammals and teleost fish and highlight molecular mechanisms governing morphogenesis and stem cells instruction. The review concludes with the molecular mechanisms of ciliary marginal zone formation and offers a perspective on the impact of comparative single cell transcriptomic studies to reveal the evolutionary similarities and differences.
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Células Madre Adultas , Disco Óptico , Animales , Retina , Peces , Desarrollo Embrionario , MamíferosRESUMEN
Reception of Wnt signals by cells is predominantly mediated by Frizzled receptors in conjunction with a co-receptor, the latter being LRP6 or LRP5 for the Wnt/ß-catenin signalling pathway. It is important that cells maintain precise control of receptor activation events in order to properly regulate Wnt/ß-catenin signalling as aberrant signalling can result in disease in humans. Phosphorylation of the intracellular domain (ICD) of LRP6 is well known to regulate Wntß-catenin signalling; however, less is known for regulatory post-translational modification events within the extracellular domain (ECD). Using a cell culture-based expression screen for functional regulators of LRP6, we identified a glycosyltransferase, B3GnT2-like, from a teleost fish (medaka) cDNA library, that modifies LRP6 and regulates Wnt/ß-catenin signalling. We provide both gain-of-function and loss-of-function evidence that the single human homolog, B3GnT2, promotes extension of polylactosamine chains at multiple N-glycans on LRP6, thereby enhancing trafficking of LRP6 to the plasma membrane and promoting Wnt/ß-catenin signalling. Our findings further highlight the importance of LRP6 as a regulatory hub in Wnt signalling and provide one of the few examples of how a specific glycosyltransferase appears to selectively target a signalling pathway component to alter cellular signalling events.
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Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad , beta Catenina , Animales , Humanos , beta Catenina/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Glicosilación , Vía de Señalización Wnt , Glicosiltransferasas/metabolismo , N-Acetilglucosaminiltransferasas/metabolismoRESUMEN
Base editing by CRISPR crucially depends on the presence of a protospacer adjacent motif (PAM) at the correct distance from the editing site. Here, we present and validate an efficient one-shot approach termed 'inception' that expands the editing range. This is achieved by sequential, combinatorial base editing: de novo generated synonymous, non-synonymous or intronic PAM sites facilitate subsequent base editing at nucleotide positions that were initially inaccessible, further opening the targeting range of highly precise editing approaches. We demonstrate the applicability of the inception concept in medaka (Oryzias latipes) in three settings: loss of function, by introducing a pre-termination STOP codon in the open reading frame of oca2; locally confined multi-codon changes to generate allelic variants with different phenotypic severity in kcnh6a; and the removal of a splice acceptor site by targeting intronic sequences of rx3. Using sequentially acting base editors in the described combinatorial approach expands the number of accessible target sites by 65% on average. This allows the use of well-established tools with NGG PAM recognition for the establishment of thus far unreachable disease models, for hypomorphic allele studies and for efficient targeted mechanistic investigations in a precise and predictable manner.
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Sistemas CRISPR-Cas , Edición Génica , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Oryzias/genéticaRESUMEN
CRISPR/Cas-based approaches have largely replaced conventional gene targeting strategies. However, homology-directed repair (HDR) in the mouse genome is not very efficient, and precisely inserting longer sequences using HDR remains challenging given that donor constructs preferentially integrate as concatemers. Here, we showed that injecting 5' biotinylated donor DNA into mouse embryos at the two-cell stage led to efficient single-copy HDR (scHDR) allele generation. Our dedicated genotyping strategy showed that these alleles occurred with frequencies of 19%, 20%, and 26% at three independent gene loci, indicating that scHDR was dramatically increased by 5' biotinylation. Thus, we suggest that the combination of a 5' biotinylated donor and diligent analysis of concatemer integration are prerequisites for efficiently and reliably generating conditional alleles or other large fragment knock-ins in the mouse genome.
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Embrión de Mamíferos , Edición Génica , Animales , Ratones , Sistemas CRISPR-Cas , ADN , Reparación del ADN por Unión de Extremidades , Edición Génica/métodos , Marcación de Gen , Reparación del ADN por RecombinaciónRESUMEN
The dorsal axial muscles, or epaxial muscles, are a fundamental structure covering the spinal cord and vertebrae, as well as mobilizing the vertebrate trunk. To date, mechanisms underlying the morphogenetic process shaping the epaxial myotome are largely unknown. To address this, we used the medaka zic1/zic4-enhancer mutant Double anal fin (Da), which exhibits ventralized dorsal trunk structures resulting in impaired epaxial myotome morphology and incomplete coverage over the neural tube. In wild type, dorsal dermomyotome (DM) cells reduce their proliferative activity after somitogenesis. Subsequently, a subset of DM cells, which does not differentiate into the myotome population, begins to form unique large protrusions extending dorsally to guide the epaxial myotome dorsally. In Da, by contrast, DM cells maintain the high proliferative activity and mainly form small protrusions. By combining RNA- and ChIP-sequencing analyses, we revealed direct targets of Zic1, which are specifically expressed in dorsal somites and involved in various aspects of development, such as cell migration, extracellular matrix organization, and cell-cell communication. Among these, we identified wnt11 as a crucial factor regulating both cell proliferation and protrusive activity of DM cells. We propose that dorsal extension of the epaxial myotome is guided by a non-myogenic subpopulation of DM cells and that wnt11 empowers the DM cells to drive the coverage of the neural tube by the epaxial myotome.
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Oryzias , Somitos , Proteínas Wnt/metabolismo , Animales , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , MorfogénesisRESUMEN
Single nucleotide variants (SNVs) are prevalent genetic factors shaping individual trait profiles and disease susceptibility. The recent development and optimizations of base editors, rubber and pencil genome editing tools now promise to enable direct functional assessment of SNVs in model organisms. However, the lack of bioinformatic tools aiding target prediction limits the application of base editing in vivo. Here, we provide a framework for adenine and cytosine base editing in medaka (Oryzias latipes) and zebrafish (Danio rerio), ideal for scalable validation studies. We developed an online base editing tool ACEofBASEs (a careful evaluation of base-edits), to facilitate decision-making by streamlining sgRNA design and performing off-target evaluation. We used state-of-the-art adenine (ABE) and cytosine base editors (CBE) in medaka and zebrafish to edit eye pigmentation genes and transgenic GFP function with high efficiencies. Base editing in the genes encoding troponin T and the potassium channel ERG faithfully recreated known cardiac phenotypes. Deep-sequencing of alleles revealed the abundance of intended edits in comparison to low levels of insertion or deletion (indel) events for ABE8e and evoBE4max. We finally validated missense mutations in novel candidate genes of congenital heart disease (CHD) dapk3, ube2b, usp44, and ptpn11 in F0 and F1 for a subset of these target genes with genotype-phenotype correlation. This base editing framework applies to a wide range of SNV-susceptible traits accessible in fish, facilitating straight-forward candidate validation and prioritization for detailed mechanistic downstream studies.
DNA contains sequences of four different molecules known as bases that represent our genetic code. In a mutation called a single nucleotide variant (or SNV for short), a single base in the sequence is swapped for another base. This can lead the individual carrying this SNV to produce a slightly different version of a protein to that found in other people. This slightly different protein may not work properly, or may perform a different task. In recent years, researchers have identified thousands of SNVs in humans linked with congenital heart diseases, but the roles of many of these SNVs remain unclear. Tools known as base editors allow researchers to efficiently modify single bases in DNA. Base editors use molecules known as short guide RNAs (or sgRNAs for short) to direct enzymes to specific positions in the DNA to swap, delete or insert a base. The sgRNAs need to be carefully designed to target the correct bases, however, which is a time consuming process. Furthermore, base editors were developed in cells grown in laboratories and so far only a few studies have demonstrated how they could be used in living animals. To overcome these limitations, Cornean, Gierten, Welz et al. developed a framework for base editing in two species of fish that are often used as models in research, namely medaka and zebrafish. The framework uses existing base editors that swap individual target bases and a new online tool referred to as ACEofBASEs to help design the required sgRNAs. The team were able to use the framework to characterize the medaka equivalents of four SNVs that have been previously associated with congenital heart disease in humans. The new framework developed here will help researchers to investigate the roles of SNVs in fish and other animals and validate human disease candidates. This approach could also be used to study the various ways that cells modify proteins by changing the specific bases involved in such modifications.
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Edición Génica , Pez Cebra , Adenina , Animales , Sistemas CRISPR-Cas , Citosina , ADN , Mutación , Pez Cebra/genéticaRESUMEN
Precise, targeted genome editing by CRISPR/Cas9 is key for basic research and translational approaches in model and non-model systems. While active in all species tested so far, editing efficiencies still leave room for improvement. The bacterial Cas9 needs to be efficiently shuttled into the nucleus as attempted by fusion with nuclear localization signals (NLSs). Additional peptide tags such as FLAG- or myc-tags are usually added for immediate detection or straightforward purification. Immediate activity is usually granted by administration of preassembled protein/RNA complexes. We present the 'hei-tag (high efficiency-tag)' which boosts the activity of CRISPR/Cas genome editing tools already when supplied as mRNA. The addition of the hei-tag, a myc-tag coupled to an optimized NLS via a flexible linker, to Cas9 or a C-to-T (cytosine-to-thymine) base editor dramatically enhances the respective targeting efficiency. This results in an increase in bi-allelic editing, yet reduction of allele variance, indicating an immediate activity even at early developmental stages. The hei-tag boost is active in model systems ranging from fish to mammals, including tissue culture applications. The simple addition of the hei-tag allows to instantly upgrade existing and potentially highly adapted systems as well as to establish novel highly efficient tools immediately applicable at the mRNA level.
The genetic code stored within DNA provides cells with the instructions they need to carry out their role in the body. Any changes to these genes, or the DNA sequence around them, has the potential to completely alter how a cell behaves. Scientists have developed various tools that allow them to experimentally modify the genome of cells or even entire living organisms. This includes the popular Cas9 enzyme which cuts DNA at specific sites, and base editors which can precisely change bits of genetic code without cutting DNA. While there are lots of Cas9 enzymes and base editors currently available, these often differ greatly in their activity depending on which cell type or organism they are applied to. Finding a tool that can effectively modify the genome of an organism at the right time during development also poses a challenge. All the cells in an organism arise from a single fertilized cell. If this cell is genetically edited, all its subsequent daughter cells (which make up the entire organism) will contain the genetic modification. However, most genome editing tools only work efficiently later in development, resulting in an undesirable mosaic organism composed of both edited and non-edited cells. Here, Thumberger et al. have developed a new 'high efficiency-tag' (also known as hei-tag for short) that can enhance the activity of gene editing tools and overcome this barrier. The tag improves the efficiency of gene editing by immediately shuttling a Cas9 enzyme to the nucleus, the cellular compartment that stores DNA. In all cases, gene editing tools with hei-tag worked better than those without in fish embryos and mouse cells grown in the laboratory. When Cas9 enzymes connected to a hei-tag were injected into the first fertilized cell of a fish embryo, this resulted in an even distribution of edited genes spread throughout the whole organism. To understand how a gene affects an organism, researchers need to be able to edit it as early in development as possible. Attaching the 'hei-tag' to already available tools could help boost their activity and make them more efficient. It could also allow advances in medical research aimed at replacing faulty genes with fully functioning ones.
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Sistemas CRISPR-Cas , Edición Génica , Animales , Sistemas CRISPR-Cas/genética , Citosina , Edición Génica/métodos , Mamíferos , Señales de Localización Nuclear , ARN Mensajero/genéticaRESUMEN
Organized intestinal mucosal immune response appears to be restricted to tetrapods. In teleost fish, there is no evidence for the existence of a particular intestinal region that facilitates the interaction of antigen-presenting cells (APCs) and T cells, such as secondary lymphoid organs. Indeed, despite their importance in the defense against pathogens, the location and manner of APC-T cell interaction within the fish gut is unknown. Here, using non-invasive live imaging of newly developed transgenic reporter lines, we addressed the spatial organization and behavior of APCs and T cells in the intestine of medaka fish both during homeostasis and inflammation. We report that Ccr9a+ T cells are recruited to a band in the lamina propria next to the muscularis mucosa in which Ccl25-expressing cells are present. Ccr9a+ T cells contact APCs for several minutes, in a process mediated by connexin 43. This type of interaction was observed in homeostasis and inflammation, with the interaction being longer and more frequent during inflammation. Thus, our results demonstrate that the mucosal immune response in the intestine of medaka is organized and endowed with a specific region with specialized microenvironment and function.
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Células Presentadoras de Antígenos/inmunología , Mucosa Intestinal/inmunología , Linfocitos T/inmunología , Animales , Quimiocinas CC/metabolismo , Oryzias/inmunología , Receptores CCR/metabolismoRESUMEN
BACKGROUND: Unraveling the relationship between genetic variation and phenotypic traits remains a fundamental challenge in biology. Mapping variants underlying complex traits while controlling for confounding environmental factors is often problematic. To address this, we establish a vertebrate genetic resource specifically to allow for robust genotype-to-phenotype investigations. The teleost medaka (Oryzias latipes) is an established genetic model system with a long history of genetic research and a high tolerance to inbreeding from the wild. RESULTS: Here we present the Medaka Inbred Kiyosu-Karlsruhe (MIKK) panel: the first near-isogenic panel of 80 inbred lines in a vertebrate model derived from a wild founder population. Inbred lines provide fixed genomes that are a prerequisite for the replication of studies, studies which vary both the genetics and environment in a controlled manner, and functional testing. The MIKK panel will therefore enable phenotype-to-genotype association studies of complex genetic traits while allowing for careful control of interacting factors, with numerous applications in genetic research, human health, drug development, and fundamental biology. CONCLUSIONS: Here we present a detailed characterization of the genetic variation across the MIKK panel, which provides a rich and unique genetic resource to the community by enabling large-scale experiments for mapping complex traits.
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Oryzias , Animales , Genoma , Endogamia , Oryzias/genética , FenotipoRESUMEN
BACKGROUND: The teleost medaka (Oryzias latipes) is a well-established vertebrate model system, with a long history of genetic research, and multiple high-quality reference genomes available for several inbred strains. Medaka has a high tolerance to inbreeding from the wild, thus allowing one to establish inbred lines from wild founder individuals. RESULTS: We exploit this feature to create an inbred panel resource: the Medaka Inbred Kiyosu-Karlsruhe (MIKK) panel. This panel of 80 near-isogenic inbred lines contains a large amount of genetic variation inherited from the original wild population. We use Oxford Nanopore Technologies (ONT) long read data to further investigate the genomic and epigenomic landscapes of a subset of the MIKK panel. Nanopore sequencing allows us to identify a large variety of high-quality structural variants, and we present results and methods using a pan-genome graph representation of 12 individual medaka lines. This graph-based reference MIKK panel genome reveals novel differences between the MIKK panel lines and standard linear reference genomes. We find additional MIKK panel-specific genomic content that would be missing from linear reference alignment approaches. We are also able to identify and quantify the presence of repeat elements in each of the lines. Finally, we investigate line-specific CpG methylation and performed differential DNA methylation analysis across these 12 lines. CONCLUSIONS: We present a detailed analysis of the MIKK panel genomes using long and short read sequence technologies, creating a MIKK panel-specific pan genome reference dataset allowing for investigation of novel variation types that would be elusive using standard approaches.
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Oryzias , Animales , Epigenómica , Genoma , Genómica/métodos , Humanos , Oryzias/genéticaRESUMEN
The differentiation of T cells from lymphoid progenitors in the thymus follows sequential developmental stages that constantly require interaction with thymic epithelial cells. Several distinct aspects of early T cell development depend on the activation of Notch receptors on thymocytes, while the selection of thymocytes at later stages are believed to be Notch independent. Using reverse genetic approaches and whole-thymus live imaging in an in vivo teleost model, the medaka, we report that Notch1 signals is required for proliferation and specification of developing T cells as well as involved in their selection in the thymus. We reveal that Notch1 controls the migratory behavior of thymocytes through controlling the chemokine receptor Ccr9b and thereby influence the T cell receptor (TCR) activation. Hence, we propose that, in lower vertebrates, the function of Notch signaling extends to all stages of T cell development, except when thymocytes undergo TCRß rearrangement.
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Movimiento Celular , Proteínas de Peces/inmunología , Oryzias , Receptor Notch1/deficiencia , Transducción de Señal , Linfocitos T/inmunología , Timo/inmunología , Animales , Movimiento Celular/genética , Movimiento Celular/inmunología , Proteínas de Peces/deficiencia , Oryzias/genética , Oryzias/inmunología , Receptor Notch1/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
Genetics crucially contributes to cardiovascular diseases (CVDs), the global leading cause of death. Since the majority of CVDs can be prevented by early intervention there is a high demand for the identification of predictive causative genes. While genome wide association studies (GWAS) correlate genes and CVDs after diagnosis and provide a valuable resource for such causative candidate genes, often preferentially those with previously known or suspected function are addressed further. To tackle the unaddressed blind spot of understudied genes, we particularly focused on the validation of human heart phenotype-associated GWAS candidates with little or no apparent connection to cardiac function. Building on the conservation of basic heart function and underlying genetics from fish to human we combined CRISPR/Cas9 genome editing of the orthologs of human GWAS candidates in isogenic medaka with automated high-throughput heart rate analysis. Our functional analyses of understudied human candidates uncovered a prominent fraction of heart rate associated genes from adult human patients impacting on the heart rate in embryonic medaka already in the injected generation. Following this pipeline, we identified 16 GWAS candidates with potential diagnostic and predictive power for human CVDs.
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Enfermedades Cardiovasculares/genética , Frecuencia Cardíaca/genética , Cadenas Ligeras de Miosina/genética , Oryzias/genética , Animales , Animales Modificados Genéticamente , Sistemas CRISPR-Cas/genética , Enfermedades Cardiovasculares/diagnóstico , Enfermedades Cardiovasculares/patología , Edición Génica , Estudio de Asociación del Genoma Completo , Humanos , Regiones Promotoras Genéticas/genéticaRESUMEN
αß and γδ T cells are two distinct sublineages that develop in the vertebrate thymus. Thus far, their differentiation from a common progenitor is mostly understood to be regulated by intrinsic mechanisms. However, the proportion of αß/γδ T cells varies in different vertebrate taxa. How this process is regulated in species that tend to produce a high frequency of γδ T cells is unstudied. Using an in vivo teleost model, the medaka, we report that progenitors first enter a thymic niche where their development into γδ T cells is favored. Translocation from this niche, mediated by chemokine receptor Ccr9b, is a prerequisite for their differentiation into αß T cells. On the other hand, the thymic niche also generates opposing gradients of the cytokine interleukin-7 and chemokine Ccl25a, and, together, they influence the lineage outcome. We propose a previously unknown mechanism that determines the proportion of αß/γδ lineages within species.