RESUMEN
Vertebrate calcitonin-producing cells (C-cells) are neuroendocrine cells that secrete the small peptide hormone calcitonin in response to elevated blood calcium levels. Whereas mouse C-cells reside within the thyroid gland and derive from pharyngeal endoderm, avian C-cells are located within ultimobranchial glands and have been reported to derive from the neural crest. We use a comparative cell lineage tracing approach in a range of vertebrate model systems to resolve the ancestral embryonic origin of vertebrate C-cells. We find, contrary to previous studies, that chick C-cells derive from pharyngeal endoderm, with neural crest-derived cells instead contributing to connective tissue intimately associated with C-cells in the ultimobranchial gland. This endodermal origin of C-cells is conserved in a ray-finned bony fish (zebrafish) and a cartilaginous fish (the little skate, Leucoraja erinacea). Furthermore, we discover putative C-cell homologs within the endodermally-derived pharyngeal epithelium of the ascidian Ciona intestinalis and the amphioxus Branchiostoma lanceolatum, two invertebrate chordates that lack neural crest cells. Our findings point to a conserved endodermal origin of C-cells across vertebrates and to a pre-vertebrate origin of this cell type along the chordate stem.
Asunto(s)
Calcitonina , Linaje de la Célula , Ciona intestinalis , Endodermo , Cresta Neural , Células Neuroendocrinas , Animales , Endodermo/metabolismo , Endodermo/citología , Calcitonina/metabolismo , Células Neuroendocrinas/metabolismo , Células Neuroendocrinas/citología , Ciona intestinalis/metabolismo , Ciona intestinalis/embriología , Cresta Neural/metabolismo , Cresta Neural/citología , Embrión de Pollo , Ratones , Vertebrados/embriología , Vertebrados/metabolismo , Pez Cebra/embriología , Anfioxos/embriología , Anfioxos/metabolismo , Anfioxos/genética , Cuerpo Ultimobranquial/metabolismoRESUMEN
Transcriptome-wide association study (TWAS) methodologies aim to identify genetic effects on phenotypes through the mediation of gene transcription. In TWAS, in silico models of gene expression are trained as functions of genetic variants and then applied to genome-wide association study (GWAS) data. This post-GWAS analysis identifies gene-trait associations with high interpretability, enabling follow-up functional genomics studies and the development of genetics-anchored resources. We provide an overview of commonly used TWAS approaches, their advantages and limitations, and some widely used applications. © 2024 Wiley Periodicals LLC.
Asunto(s)
Estudio de Asociación del Genoma Completo , Transcriptoma , Transcriptoma/genética , Estudio de Asociación del Genoma Completo/métodos , Sitios de Carácter Cuantitativo , Simulación por Computador , FenotipoRESUMEN
Rgp1 was previously identified as a component of a guanine nucleotide exchange factor (GEF) complex to activate Rab6a-mediated trafficking events in and around the Golgi. While the role of Rgp1 in protein trafficking has been examined in vitro and in yeast, the role of Rgp1 during vertebrate embryogenesis and protein trafficking in vivo is unknown. Using genetic, CRISPR-induced zebrafish mutants for Rgp1 loss-of-function, we found that Rgp1 is required for craniofacial cartilage development. Within live rgp1-/- craniofacial chondrocytes, we observed altered movements of Rab6a+ vesicular compartments, consistent with a conserved mechanism described in vitro. Using transmission electron microscopy (TEM) and immunofluorescence analyses, we show that Rgp1 plays a role in the secretion of collagen II, the most abundant protein in cartilage. Our overexpression experiments revealed that Rab8a is a part of the post-Golgi collagen II trafficking pathway. Following loss of Rgp1, chondrocytes activate an Arf4b-mediated stress response and subsequently respond with nuclear DNA fragmentation and cell death. We propose that an Rgp1-regulated Rab6a-Rab8a pathway directs secretion of ECM cargoes such as collagen II, a pathway that may also be utilized in other tissues where coordinated trafficking and secretion of collagens and other large cargoes is required for normal development and tissue function.
Asunto(s)
Cartílago , Pez Cebra , Animales , Pez Cebra/genética , Cartílago/metabolismo , Condrocitos/metabolismo , Colágeno/metabolismo , Aparato de Golgi/metabolismo , Factores de Intercambio de Guanina Nucleótido/genéticaRESUMEN
BACKGROUND: Movement of the lower jaw, a common behavior observed among vertebrates, is required for eating and processing food. This movement is controlled by signals sent from the trigeminal motor nerve through neuromuscular junctions (NMJs) to the masticatory muscles. Dysfunctional jaw movements contribute to craniomandibular disorders, yet the pathophysiology of these disorders is not well understood, as limited studies have been conducted on the molecular mechanisms of jaw movement. RESULTS: Using erc1b/kimm533 genetic loss of function mutant, we evaluated lower jaw muscle organization and innervation by the cranial motor nerves in developing zebrafish. Using time-lapse confocal imaging of the erc1b mutant in a transgenic fluorescent reporter line, we found delayed trigeminal nerve growth and disrupted nerve branching architecture during muscle innervation. By automated 3D image analysis of NMJ distribution, we identified an increased number of small, disorganized NMJ clusters in erc1b mutant larvae compared to WT siblings. Using genetic replacement experiments, we determined the Rab GTPase binding domain of Erc1b is required for cranial motor nerve branching, but not NMJ organization or muscle attachment. CONCLUSIONS: We identified Erc1b/ERC1 as a novel component of a genetic pathway contributing to muscle organization, trigeminal nerve outgrowth, and NMJ spatial distribution during development that is required for jaw movement.
Asunto(s)
Neuronas Motoras , Pez Cebra , Animales , Neuronas Motoras/fisiología , Unión Neuromuscular/metabolismo , Músculos , MaxilaresRESUMEN
Coessentiality mapping has been useful to systematically cluster genes into biological pathways and identify gene functions1-3. Here, using the debiased sparse partial correlation (DSPC) method3, we construct a functional coessentiality map for cellular metabolic processes across human cancer cell lines. This analysis reveals 35 modules associated with known metabolic pathways and further assigns metabolic functions to unknown genes. In particular, we identify C12orf49 as an essential regulator of cholesterol and fatty acid metabolism in mammalian cells. Mechanistically, C12orf49 localizes to the Golgi, binds membrane-bound transcription factor peptidase, site 1 (MBTPS1, site 1 protease) and is necessary for the cleavage of its substrates, including sterol regulatory element binding protein (SREBP) transcription factors. This function depends on the evolutionarily conserved uncharacterized domain (DUF2054) and promotes cell proliferation under cholesterol depletion. Notably, c12orf49 depletion in zebrafish blocks dietary lipid clearance in vivo, mimicking the phenotype of mbtps1 mutants. Finally, in an electronic health record (EHR)-linked DNA biobank, C12orf49 is associated with hyperlipidaemia through phenome analysis. Altogether, our findings reveal a conserved role for C12orf49 in cholesterol and lipid homeostasis and provide a platform to identify unknown components of other metabolic pathways.
Asunto(s)
Colesterol/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo , Animales , Línea Celular , Proliferación Celular , Regulación de la Expresión Génica , Aparato de Golgi/metabolismo , Humanos , Hiperlipidemias/genética , Metabolismo de los Lípidos/genética , Proproteína Convertasas/metabolismo , Serina Endopeptidasas/metabolismo , Pez CebraRESUMEN
Discovery of genotype-phenotype relationships remains a major challenge in clinical medicine. Here, we combined three sources of phenotypic data to uncover a new mechanism for rare and common diseases resulting from collagen secretion deficits. Using a zebrafish genetic screen, we identified the ric1 gene as being essential for skeletal biology. Using a gene-based phenome-wide association study (PheWAS) in the EHR-linked BioVU biobank, we show that reduced genetically determined expression of RIC1 is associated with musculoskeletal and dental conditions. Whole-exome sequencing identified individuals homozygous-by-descent for a rare variant in RIC1 and, through a guided clinical re-evaluation, it was discovered that they share signs with the BioVU-associated phenome. We named this new Mendelian syndrome CATIFA (cleft lip, cataract, tooth abnormality, intellectual disability, facial dysmorphism, attention-deficit hyperactivity disorder) and revealed further disease mechanisms. This gene-based, PheWAS-guided approach can accelerate the discovery of clinically relevant disease phenome and associated biological mechanisms.
Asunto(s)
Anomalías Múltiples/patología , Bancos de Muestras Biológicas , Factores de Intercambio de Guanina Nucleótido/genética , Fenómica , Proteínas de Pez Cebra/genética , Animales , Conducta Animal , Condrocitos/patología , Condrocitos/ultraestructura , Modelos Animales de Enfermedad , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Fibroblastos/ultraestructura , Humanos , Modelos Biológicos , Sistema Musculoesquelético/patología , Osteogénesis , Fenotipo , Procolágeno/metabolismo , Transporte de Proteínas , Vías Secretoras , Síndrome , Pez CebraRESUMEN
Although the use of model systems for studying the mechanism of mutations that have a large effect is common, we highlight here the ways that zebrafish-model-system studies of a gene, GRIK5, that contributes to the polygenic liability to develop eye diseases have helped to illuminate a mechanism that implicates vascular biology in eye disease. A gene-expression prediction derived from a reference transcriptome panel applied to BioVU, a large electronic health record (EHR)-linked biobank at Vanderbilt University Medical Center, implicated reduced GRIK5 expression in diverse eye diseases. We tested the function of GRIK5 by depletion of its ortholog in zebrafish, and we observed reduced blood vessel numbers and integrity in the eye and increased vascular permeability. Analyses of EHRs in >2.6 million Vanderbilt subjects revealed significant comorbidity of eye and vascular diseases (relative risks 2-15); this comorbidity was confirmed in 150 million individuals from a large insurance claims dataset. Subsequent studies in >60,000 genotyped BioVU participants confirmed the association of reduced genetically predicted expression of GRIK5 with comorbid vascular and eye diseases. Our studies pioneer an approach that allows a rapid iteration of the discovery of gene-phenotype relationships to the primary genetic mechanism contributing to the pathophysiology of human disease. Our findings also add dimension to the understanding of the biology driven by glutamate receptors such as GRIK5 (also referred to as GLUK5 in protein form) and to mechanisms contributing to human eye diseases.
Asunto(s)
Bancos de Muestras Biológicas , Registros Electrónicos de Salud , Embrión no Mamífero/patología , Oftalmopatías/patología , Regulación de la Expresión Génica , Receptores de Ácido Kaínico/genética , Enfermedades Vasculares/patología , Animales , Embrión no Mamífero/metabolismo , Oftalmopatías/genética , Oftalmopatías/metabolismo , Genotipo , Humanos , Fenómica , Fenotipo , Receptores de Ácido Kaínico/metabolismo , Enfermedades Vasculares/genética , Enfermedades Vasculares/metabolismo , Pez CebraRESUMEN
The COPII coat complex, which mediates secretory cargo trafficking from the endoplasmic reticulum, is a key control point for subcellular protein targeting. Because misdirected proteins cannot function, protein sorting by COPII is critical for establishing and maintaining normal cell and tissue homeostasis. Indeed, mutations in COPII genes cause a range of human pathologies, including cranio-lenticulo-sutural dysplasia (CLSD), which is characterized by collagen trafficking defects, craniofacial abnormalities, and skeletal dysmorphology. Detailed knowledge of the COPII pathway is required to understand its role in normal cell physiology and to devise new treatments for disorders in which it is disrupted. However, little is known about how vertebrates dynamically regulate COPII activity in response to developmental, metabolic, or pathological cues. Several COPII proteins are modified by O-linked ß-N-acetylglucosamine (O-GlcNAc), a dynamic form of intracellular protein glycosylation, but the biochemical and functional effects of these modifications remain unclear. Here, we use a combination of chemical, biochemical, cellular, and genetic approaches to demonstrate that site-specific O-GlcNAcylation of COPII proteins mediates their protein-protein interactions and modulates cargo secretion. In particular, we show that individual O-GlcNAcylation sites of SEC23A, an essential COPII component, are required for its function in human cells and vertebrate development, because mutation of these sites impairs SEC23A-dependent in vivo collagen trafficking and skeletogenesis in a zebrafish model of CLSD. Our results indicate that O-GlcNAc is a conserved and critical regulatory modification in the vertebrate COPII-dependent trafficking pathway.
Asunto(s)
Acetilglucosamina/metabolismo , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Acilación , Animales , Línea Celular , Colágeno/metabolismo , Anomalías Craneofaciales/metabolismo , Modelos Animales de Enfermedad , Glicosilación , Humanos , Orgánulos/metabolismo , Conformación Proteica , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Vertebrados , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Pez CebraRESUMEN
The evolutionary origins of the hypoxia-sensitive cells that trigger amniote respiratory reflexes - carotid body glomus cells, and 'pulmonary neuroendocrine cells' (PNECs) - are obscure. Homology has been proposed between glomus cells, which are neural crest-derived, and the hypoxia-sensitive 'neuroepithelial cells' (NECs) of fish gills, whose embryonic origin is unknown. NECs have also been likened to PNECs, which differentiate in situ within lung airway epithelia. Using genetic lineage-tracing and neural crest-deficient mutants in zebrafish, and physical fate-mapping in frog and lamprey, we find that NECs are not neural crest-derived, but endoderm-derived, like PNECs, whose endodermal origin we confirm. We discover neural crest-derived catecholaminergic cells associated with zebrafish pharyngeal arch blood vessels, and propose a new model for amniote hypoxia-sensitive cell evolution: endoderm-derived NECs were retained as PNECs, while the carotid body evolved via the aggregation of neural crest-derived catecholaminergic (chromaffin) cells already associated with blood vessels in anamniote pharyngeal arches.
Asunto(s)
Hipoxia de la Célula , Linaje de la Célula , Células Neuroendocrinas , Células Neuroepiteliales , Animales , Anuros , Evolución Biológica , Lampreas , Pez CebraRESUMEN
The zebrafish skeleton shares many similarities with human and other vertebrate skeletons. Over the past years, work in zebrafish has provided an extensive understanding of the basic developmental mechanisms and cellular pathways directing skeletal development and homeostasis. This review will focus on the cell biology of cartilage and bone and how the basic cellular processes within chondrocytes and osteocytes function to assemble the structural frame of a vertebrate body. We will discuss fundamental functions of skeletal cells in production and secretion of extracellular matrix and cellular activities leading to differentiation of progenitors to mature cells that make up the skeleton. We highlight important examples where findings in zebrafish provided direction for the search for genes causing human skeletal defects and also how zebrafish research has proven important for validating candidate human disease genes. The work we cover here illustrates utility of zebrafish in unraveling molecular mechanisms of cellular functions necessary to form and maintain a healthy skeleton.
Asunto(s)
Enfermedades Óseas/patología , Modelos Animales de Enfermedad , Pez Cebra/embriología , Animales , Cartílago/embriología , Matriz Extracelular/metabolismo , Humanos , Sustancias Macromoleculares/metabolismoRESUMEN
Anderson disease (ANDD) or chylomicron retention disease (CMRD) is a rare, hereditary lipid malabsorption syndrome associated with mutations in the SAR1B gene that is characterized by failure to thrive and hypocholesterolemia. Although the SAR1B structure has been resolved and its role in formation of coat protein II (COPII)-coated carriers is well established, little is known about the requirement for SAR1B during embryogenesis. To address this question, we have developed a zebrafish model of Sar1b deficiency based on antisense oligonucleotide knockdown. We show that zebrafish sar1b is highly conserved among vertebrates; broadly expressed during development; and enriched in the digestive tract organs, brain, and craniofacial skeleton. Consistent with ANDD symptoms of chylomicron retention, we found that dietary lipids in Sar1b-deficient embryos accumulate in enterocytes. Transgenic expression analysis revealed that Sar1b is required for growth of exocrine pancreas and liver. Furthermore, we found abnormal differentiation and maturation of craniofacial cartilage associated with defects in procollagen II secretion and absence of select, neuroD-positive neurons of the midbrain and hindbrain. The model presented here will help to systematically dissect developmental roles of Sar1b and to discover molecular and cellular mechanisms leading to organ-specific ANDD pathology. Key messages: Sar1b depletion phenotype in zebrafish resembles Anderson disease deficits. Sar1b deficiency results in multi-organ developmental deficits. Sar1b is required for dietary cholesterol uptake into enterocytes.
Asunto(s)
Hipobetalipoproteinemias/genética , Hipobetalipoproteinemias/metabolismo , Metabolismo de los Lípidos/genética , Síndromes de Malabsorción/genética , Síndromes de Malabsorción/metabolismo , Proteínas de Unión al GTP Monoméricas/deficiencia , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo/genética , Huesos/embriología , Huesos/metabolismo , Encéfalo/embriología , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Tracto Gastrointestinal/embriología , Tracto Gastrointestinal/metabolismo , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Organogénesis/genética , Fenotipo , Pez CebraRESUMEN
Sec13 is a dual function protein, being a core component of both the COPII coat, which mediates protein trafficking from the endoplasmic reticulum to the Golgi apparatus, and the nuclear pore complex (NPC), which facilitates nucleo-cytoplasmic traffic. Here, we present a genetic model to differentiate the roles of these two functions of Sec13 in vivo. We report that sec13(sq198) mutant embryos develop small eyes that exhibit disrupted retinal lamination and that the mutant retina contains an excessive number of apoptotic cells. Surprisingly, we found that loss of COPII function by oligonucleotide-mediated gene knockdown of sec31a and sec31b or brefeldin A treatment did not disrupt retinal lamination, although it did result in digestive organ defects similar to those seen in sec13(sq198), suggesting that the digestive organ defects observed in sec13(sq198) are due to loss of COPII function, whereas the retinal lamination defects are due to loss of the NPC function. We showed that the retinal cells of sec13(sq198) failed to form proper nuclear pores, leading to a nuclear accumulation of total mRNA and abnormal activation of the p53-dependent apoptosis pathway, causing the retinal defect in sec13(sq198). Furthermore, we found that a mutant lacking Nup107, a key NPC-specific component, phenocopied the retinal lamination phenotype as observed in sec13(sq198). Our results demonstrate a requirement for the nuclear pore function of Sec13 in development of the retina and provide the first genetic evidence to differentiate the contributions of the NPC and the COPII functions of Sec13 during organogenesis.
Asunto(s)
Poro Nuclear/fisiología , Retina/embriología , Proteínas de Pez Cebra/fisiología , Animales , Secuencia de Bases , Cartilla de ADN , Hibridación in Situ , Microscopía Electrónica de Transmisión , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
The bone morphogenetic protein antagonist Gremlin 2 (Grem2) is required for atrial differentiation and establishment of cardiac rhythm during embryonic development. A human Grem2 variant has been associated with familial atrial fibrillation, suggesting that abnormal Grem2 activity causes arrhythmias. However, it is not known how Grem2 integrates into signaling pathways to direct atrial cardiomyocyte differentiation. Here, we demonstrate that Grem2 expression is induced concurrently with the emergence of cardiovascular progenitor cells during differentiation of mouse embryonic stem cells (ESCs). Grem2 exposure enhances the cardiogenic potential of ESCs by 20-120-fold, preferentially inducing genes expressed in atrial myocytes such as Myl7, Nppa, and Sarcolipin. We show that Grem2 acts upstream to upregulate proatrial transcription factors CoupTFII and Hey1 and downregulate atrial fate repressors Irx4 and Hey2. The molecular phenotype of Grem2-induced atrial cardiomyocytes was further supported by induction of ion channels encoded by Kcnj3, Kcnj5, and Cacna1d genes and establishment of atrial-like action potentials shown by electrophysiological recordings. We show that promotion of atrial-like cardiomyocytes is specific to the Gremlin subfamily of BMP antagonists. Grem2 proatrial differentiation activity is conveyed by noncanonical BMP signaling through phosphorylation of JNK and can be reversed by specific JNK inhibitors, but not by dorsomorphin, an inhibitor of canonical BMP signaling. Taken together, our data provide novel mechanistic insights into atrial cardiomyocyte differentiation from pluripotent stem cells and will assist the development of future approaches to study and treat arrhythmias.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/fisiología , Sistema de Señalización de MAP Quinasas , Proteínas/fisiología , Animales , Células Cultivadas , Citocinas , Atrios Cardíacos/citología , Ratones , Miocitos Cardíacos/fisiologíaRESUMEN
Over the past decades, studies using zebrafish have significantly advanced our understanding of the cellular basis for development and human diseases. Zebrafish have rapidly developing transparent embryos that allow comprehensive imaging of embryogenesis combined with powerful genetic approaches. However, forward genetic screens in zebrafish have generated unanticipated findings that are mirrored by human genetic studies: disruption of genes implicated in basic cellular processes, such as protein secretion or cytoskeletal dynamics, causes discrete developmental or disease phenotypes. This is surprising because many processes that were assumed to be fundamental to the function and survival of all cell types appear instead to be regulated by cell-specific mechanisms. Such discoveries are facilitated by experiments in whole animals, where zebrafish provides an ideal model for visualization and manipulation of organelles and cellular processes in a live vertebrate. Here, we review well-characterized mutants and newly developed tools that underscore this notion. We focus on the secretory pathway and microtubule-based trafficking as illustrative examples of how studying cell biology in vivo using zebrafish has broadened our understanding of the role fundamental cellular processes play in embryogenesis and disease.
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Biología Celular , Desarrollo Embrionario/genética , Proteínas de Transporte Vesicular/genética , Pez Cebra/embriología , Animales , Movimiento Celular/genética , Humanos , Microtúbulos/genética , Microtúbulos/metabolismo , Fenotipo , Vertebrados/genética , Proteínas de Transporte Vesicular/metabolismo , Pez Cebra/genéticaRESUMEN
Cellular life depends on protein transport and membrane traffic. In multicellular organisms, membrane traffic is required for extracellular matrix deposition, cell adhesion, growth factor release, and receptor signaling, which are collectively required to integrate the development and physiology of tissues and organs. Understanding the regulatory mechanisms that govern cargo and membrane flow presents a prime challenge in cell biology. Extracellular matrix (ECM) secretion remains poorly understood, although given its essential roles in the regulation of cell migration, differentiation, and survival, ECM secretion mechanisms are likely to be tightly controlled. Recent studies in vertebrate model systems, from fishes to mammals and in human patients, have revealed complex and diverse loss-of-function phenotypes associated with mutations in components of the secretory machinery. A broad spectrum of diseases from skeletal and cardiovascular to neurological deficits have been linked to ECM trafficking. These discoveries have directly challenged the prevailing view of secretion as an essential but monolithic process. Here, we will discuss the latest findings on mechanisms of ECM trafficking in vertebrates.
Asunto(s)
Matriz Extracelular/metabolismo , Animales , Adhesión Celular/fisiología , Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Modelos Animales de Enfermedad , HumanosRESUMEN
ENOX1 is a highly conserved NADH oxidase that helps to regulate intracellular nicotinamide adenine dinucleotide levels in many cell types, including endothelial cells. Pharmacologic and RNA interference (RNAi)-mediated suppression of ENOX1 impairs surrogate markers of tumor angiogenesis/vasculogenesis, providing support for the concept that ENOX1 represents an antiangiogenic druggable target. However, direct genetic evidence that demonstrates a role for ENOX1 in vascular development is lacking. In this study, we exploited a zebrafish embryonic model of development to address this question. Whole-mount in situ hybridization coupled with immunofluorescence performed on zebrafish embryos demonstrate that enox1 message and translated protein are expressed in most tissues, and its expression is enriched in blood vessels and heart. Morpholino-mediated suppression of Enox1 in Tg(fli1-eGFP) and Tg(flk1-eGFP) zebrafish embryos significantly impairs the development of vasculature and blood circulation. Using in vivo multiphoton microscopy, we show that morpholino-mediated knockdown of enox1 increases NADH levels, consistent with loss of enzyme. VJ115 is a small-molecule inhibitor of Enox1's oxidase activity shown to increase intracellular NADH in endothelial cells; we used VJ115 to determine if the oxidase activity was crucial for vascular development. We found that VJ115 suppressed vasculogenesis in Tg(fli1-eGFP) embryos and impaired circulation. Previously, it was shown that suppression of ENOX1 radiosensitizes proliferating tumor vasculature, a consequence of enhanced endothelial cell apoptosis. Thus, our current findings, coupled with previous research, support the hypothesis that ENOX1 represents a potential cancer therapy target, one that combines molecular targeting with cytotoxic sensitization.
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Endotelio Vascular/embriología , Endotelio Vascular/crecimiento & desarrollo , Complejos Multienzimáticos/fisiología , NADH NADPH Oxidorreductasas/fisiología , Animales , Animales Modificados Genéticamente , Endotelio Vascular/enzimología , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/metabolismo , Neovascularización Fisiológica/fisiología , Pez CebraRESUMEN
The neural crest is a multipotent stem cell population that arises from the dorsal aspect of the neural tube and generates both non-ectomesenchymal (melanocytes, peripheral neurons and glia) and ectomesenchymal (skeletogenic, odontogenic, cartilaginous and connective tissue) derivatives. In amniotes, only cranial neural crest generates both classes, with trunk neural crest restricted to non-ectomesenchyme. By contrast, it has been suggested that anamniotes might generate derivatives of both classes at all axial levels, with trunk neural crest generating fin osteoblasts, scale mineral-forming cells and connective tissue cells; however, this has not been fully tested. The cause and evolutionary significance of this cranial/trunk dichotomy, and its absence in anamniotes, are debated. Recent experiments have disputed the contribution of fish trunk neural crest to fin osteoblasts and scale mineral-forming cells. This prompted us to test the contribution of anamniote trunk neural crest to fin connective tissue cells. Using genetics-based lineage tracing in zebrafish, we find that these fin mesenchyme cells derive entirely from the mesoderm and that neural crest makes no contribution. Furthermore, contrary to previous suggestions, larval fin mesenchyme cells do not generate the skeletogenic cells of the adult fin, but persist to form fibroblasts associated with adult fin rays. Our data demonstrate that zebrafish trunk neural crest does not generate ectomesenchymal derivatives and challenge long-held ideas about trunk neural crest fate. These findings have important implications for the ontogeny and evolution of the neural crest.
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Aletas de Animales/embriología , Cresta Neural/fisiología , Pez Cebra/embriología , Aletas de Animales/citología , Animales , Evolución Biológica , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Femenino , Fibroblastos/citología , Masculino , Mesodermo/citología , Pez Cebra/genéticaRESUMEN
Atrial fibrillation (AF) is the most common cardiac arrhythmia and carries a significant risk of stroke and heart failure. The molecular etiologies of AF are poorly understood, leaving patients with limited therapeutic options. AF has been recognized as an inherited disease in almost 30% of patient cases. However, few genetic loci have been identified and the mechanisms linking genetic variants to AF susceptibility remain unclear. By sequencing 193 probands with lone AF, we identified a Q76E variant within the coding sequence of the bone morphogenetic protein (BMP) antagonist gremlin-2 (GREM2) that increases its inhibitory activity. Functional modeling in zebrafish revealed that, through regulation of BMP signaling, GREM2 is required for cardiac laterality and atrial differentiation during embryonic development. GREM2 overactivity results in slower cardiac contraction rates in zebrafish, and induction of previously identified AF candidate genes encoding connexin-40, sarcolipin and atrial natriuretic peptide in differentiated mouse embryonic stem cells. By live heart imaging in zebrafish overexpressing wild-type or variant GREM2, we found abnormal contraction velocity specifically in atrial cardiomyocytes. These results implicate, for the first time, regulators of BMP signaling in human AF, providing mechanistic insights into the pathogenesis of the disease and identifying potential new therapeutic targets.
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Fibrilación Atrial/genética , Diferenciación Celular/genética , Modelos Animales de Enfermedad , Atrios Cardíacos/fisiopatología , Péptidos y Proteínas de Señalización Intercelular/genética , Miocitos Cardíacos/patología , Proteínas de Pez Cebra/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Arritmias Cardíacas/complicaciones , Arritmias Cardíacas/genética , Arritmias Cardíacas/patología , Arritmias Cardíacas/fisiopatología , Fibrilación Atrial/complicaciones , Fibrilación Atrial/fisiopatología , Proteínas Morfogenéticas Óseas/metabolismo , Citocinas , Femenino , Regulación del Desarrollo de la Expresión Génica , Atrios Cardíacos/embriología , Atrios Cardíacos/patología , Frecuencia Cardíaca/fisiología , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Datos de Secuencia Molecular , Miocitos Cardíacos/metabolismo , Organogénesis/genética , Linaje , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/metabolismoRESUMEN
The neural crest is a stem cell-like population exclusive to vertebrates that gives rise to many different cell types including chondrocytes, neurons and melanocytes. Arising from the neural plate border at the intersection of Wnt and Bmp signaling pathways, the complexity of neural crest gene regulatory networks has made the earliest steps of induction difficult to elucidate. Here, we report that tfap2a and foxd3 participate in neural crest induction and are necessary and sufficient for this process to proceed. Double mutant tfap2a (mont blanc, mob) and foxd3 (mother superior, mos) mob;mos zebrafish embryos completely lack all neural crest-derived tissues. Moreover, tfap2a and foxd3 are expressed during gastrulation prior to neural crest induction in distinct, complementary, domains; tfap2a is expressed in the ventral non-neural ectoderm and foxd3 in the dorsal mesendoderm and ectoderm. We further show that Bmp signaling is expanded in mob;mos embryos while expression of dkk1, a Wnt signaling inhibitor, is increased and canonical Wnt targets are suppressed. These changes in Bmp and Wnt signaling result in specific perturbations of neural crest induction rather than general defects in neural plate border or dorso-ventral patterning. foxd3 overexpression, on the other hand, enhances the ability of tfap2a to ectopically induce neural crest around the neural plate, overriding the normal neural plate border limit of the early neural crest territory. Although loss of either Tfap2a or Foxd3 alters Bmp and Wnt signaling patterns, only their combined inactivation sufficiently alters these signaling gradients to abort neural crest induction. Collectively, our results indicate that tfap2a and foxd3, in addition to their respective roles in the differentiation of neural crest derivatives, also jointly maintain the balance of Bmp and Wnt signaling in order to delineate the neural crest induction domain.
Asunto(s)
Células Madre Embrionarias/metabolismo , Factores de Transcripción Forkhead/metabolismo , Cresta Neural/embriología , Cresta Neural/metabolismo , Factor de Transcripción AP-2/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Animales , Secuencia de Bases , Tipificación del Cuerpo , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Muerte Celular , Cartilla de ADN/genética , Células Madre Embrionarias/citología , Factores de Transcripción Forkhead/genética , Gastrulación , Regulación del Desarrollo de la Expresión Génica , Genes p53 , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Mutación , Cresta Neural/citología , Neurogénesis , Factor de Transcripción AP-2/genética , Vía de Señalización Wnt , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
A surprising diversity of mechanisms controls sex determination of vertebrate organisms, even among closely related species. Both genetic and temperature-dependent systems of sex determination have been described in teleost fish. In the common zebrafish model organism, heteromorphic sex chromosomes are not observed, and the potential role of a genetic component of sex determination remains largely unknown. Here we report a genome-wide linkage study of sex determination in zebrafish using a novel SNP genetic map. We identified loci on zebrafish chromosomes 5 (LOD score 7.9) and 16 (LOD score 9.3) governing sex determination as a complex trait, rather than as an XY or ZW genetic system. Each of these loci contains a prominent candidate gene with a conserved role in sex determination across additional species that suggest potential mechanisms of sex determination in zebrafish. The chromosome 5 locus harbors dmrt1, a key gene in sex determination from fruit flies to humans; mutation of the human DMRT1 ortholog is a cause of complete sex reversal of XY individuals. The chromosome 16 locus harbors cyp21a2; mutation of the human CYP21A2 ortholog is one of the more common causes of pseudohermaphroditism. Mutation detection at each of these candidate genes within the zebrafish cross identified hypomorphic variants on the female-associated allele of each locus. The two loci together accounted for 16% of variance of the trait. Interacting environmental cues are likely to be an additional important component of sex determination in zebrafish.