RESUMEN
In recent years, the zebrafish has become a well-established laboratory model. We describe here the ZeBraInspector (ZBI) platform for high-content 3D imaging (HCI) of 5 days post-fertilization zebrafish eleuthero-embryos (EEs). This platform includes a mounting method based on 3D-printed stamps to create a grid of wells in an agarose cast, facilitating batch acquisitions with a fast-confocal laser scanning microscope. We describe reference labeling in cleared fish with a fluorescent lipophilic dye. Based on this labeling, the ZBI software registers. EE 3D images, making it possible to visualize numerous identically oriented EEs on a single screen, and to compare their morphologies and any fluorescent patterns at a glance. High-resolution 2D snapshots can be extracted. ZBI software is therefore useful for diverse high-content analyses (HCAs). Following automated segmentation of the lipophilic dye signal, the ZBI software performs volumetric analyses on whole EEs and their nervous system white matter. Through two examples, we illustrate the power of these analyses for obtaining statistically significant results from a small number of samples: the characterization of a phenotype associated with a neurodevelopmental mutation, and of the defects caused by treatments with a toxic anti-cancer compound.
Asunto(s)
Imagenología Tridimensional , Pez Cebra , Animales , Encéfalo/diagnóstico por imagen , Fertilización , Microscopía Confocal/métodos , Pez Cebra/genéticaRESUMEN
We studied cell recruitment following optic tectum (OT) injury in zebrafish (Danio rerio), which has a remarkable ability to regenerate many of its organs, including the brain. The OT is the largest dorsal layered structure in the zebrafish brain. In juveniles, it is an ideal structure for imaging and dissection. We investigated the recruited cells within the juvenile OT during regeneration in a Pdgfrß-Gal4:UAS-EGFP line in which pericytes, vascular, circulating, and meningeal cells are labeled, together with neurons and progenitors. We first performed high-resolution confocal microscopy and single-cell RNA-sequencing (scRNAseq) on EGFP-positive cells. We then tested three types of injury with very different outcomes (needle (mean depth in the OT of 200 µm); deep-laser (depth: 100 to 200 µm depth); surface-laser (depth: 0 to 100 µm)). Laser had the additional advantage of better mimicking of ischemic cerebral accidents. No massive recruitment of EGFP-positive cells was observed following laser injury deep in the OT. This type of injury does not perturb the meninx/brain-blood barrier (BBB). We also performed laser injuries at the surface of the OT, which in contrast create a breach in the meninges. Surprisingly, one day after such injury, we observed the migration to the injury site of various EGFP-positive cell types at the surface of the OT. The migrating cells included midline roof cells, which activated the PI3K-AKT pathway; fibroblast-like cells expressing numerous collagen genes and most prominently in 3D imaging; and a large number of arachnoid cells that probably migrate to the injury site through the activation of cilia motility genes, most likely being direct targets of the FOXJ1a gene. This study, combining high-content imaging and scRNAseq in physiological and pathological conditions, sheds light on meninges repair mechanisms in zebrafish that probably also operate in mammalian meninges.
Asunto(s)
Colículos Superiores , Pez Cebra , Animales , Rayos Láser , Mamíferos , Meninges , Fosfatidilinositol 3-Quinasas , Pez Cebra/genéticaRESUMEN
Neural stem and progenitor cells (NSPCs) are the primary source of new neurons in the brain and serve critical roles in tissue homeostasis and plasticity throughout life. Within the vertebrate brain, NSPCs are located within distinct neurogenic niches differing in their location, cellular composition, and proliferative behaviour. Heterogeneity in the NSPC population is hypothesized to reflect varying capacities for neurogenesis, plasticity and repair between different neurogenic zones. Since the discovery of adult neurogenesis, studies have predominantly focused on the behaviour and biological significance of adult NSPCs (aNSPCs) in rodents. However, compared to rodents, who show lifelong neurogenesis in only two restricted neurogenic niches, zebrafish exhibit constitutive neurogenesis across multiple stem cell niches that provide new neurons to every major brain division. Accordingly, zebrafish are a powerful model to probe the unique cellular and molecular profiles of NSPCs and investigate how these profiles govern tissue homeostasis and regenerative plasticity within distinct stem cell populations over time. Amongst the NSPC populations residing in the zebrafish central nervous system (CNS), proliferating radial-glia, quiescent radial-glia and neuro-epithelial-like cells comprise the majority. Here, we provide insight into the extent to which these distinct NSPC populations function and mature during development, respond to experience, and contribute to successful CNS regeneration in teleost fish. Together, our review brings to light the dynamic biological roles of these individual NSPC populations and showcases their diverse regenerative modes to achieve vertebrate brain repair later in life.
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Células Ependimogliales/fisiología , Células Epiteliales/fisiología , Regeneración Nerviosa/fisiología , Células-Madre Neurales/fisiología , Plasticidad Neuronal/fisiología , Neuronas/fisiología , Animales , Sistema Nervioso Central/crecimiento & desarrollo , Sistema Nervioso Central/fisiología , Pez CebraRESUMEN
Fibrillarin (Fbl) is a highly conserved protein that plays an essential role in ribosome biogenesis and more particularly in the methylation of ribosomal RNAs and rDNA histones. In cellular models, FBL was shown to play an important role in tumorigenesis and stem cell differentiation. We used the zebrafish as an in vivo model to study Fbl function during embryonic development. We show here that the optic tectum and the eye are severely affected by Fbl depletion whereas ventral regions of the brain are less impacted. The morphogenesis defects are associated with impaired neural differentiation and massive apoptosis. Polysome gradient experiments show that fbl mutant larvae display defects in ribosome biogenesis and activity. Strikingly, flow cytometry analyses revealed different S-phase profiles between wild-type and mutant cells, suggesting a defect in S-phase progression.
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Diferenciación Celular/genética , Proteínas Cromosómicas no Histona/metabolismo , Mesencéfalo/embriología , Retina/embriología , Fase S/genética , Animales , Apoptosis , Larva/metabolismo , Mesencéfalo/metabolismo , Morfogénesis/genética , Neurogénesis/genética , ARN Ribosómico/metabolismo , Retina/metabolismo , Pez Cebra/embriologíaRESUMEN
We should start as we mean to go on and try to avoid the confusion most of us experience when bombarded with acronyms with overstated significations. You will be familiar with the situation, you are in a seminar or a meeting and someone who has been using a set of acronyms for years, includes them in sentence after sentence that has you lost because you don't know what some or most of them stand for. Even worse when scientists start making verbs out of them, CRISPR seems to have fallen into this category; how many of us have heard someone asking if a mutation can be CRISPRed! Does it matter though? We are all familiar with informal language in scientific talks and discussions which is replaced by more formal dialect when research is published or presented to the general public. However, when an ill-defined acronym slips outside of laboratory chatter and is widely recognised by the general public, we need to proceed with caution to avoid misinterpretation and misunderstandings.
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Abreviaturas como Asunto , Edición Génica , Genoma , Animales , Edición Génica/métodos , Edición Génica/normas , Ingeniería Genética/métodos , Ingeniería Genética/normas , Humanos , Organismos Modificados Genéticamente , Opinión PúblicaRESUMEN
Alphaviruses, such as chikungunya virus (CHIKV) and Sindbis virus (SINV), are vector-borne pathogens that cause acute illnesses in humans and are sometimes associated with neuropathies, especially in infants and elderly patients. Little is known about their mechanism of entry into the central nervous system (CNS), even for SINV, which has been used extensively as a model for viral encephalopathies. We previously established a CHIKV infection model in the optically transparent zebrafish larva; here we describe a new SINV infection model in this host. We imaged in vivo the onset and progression of the infection caused by intravenous SINV inoculation. Similar to that described for CHIKV, infection in the periphery was detected early and was transient, whereas CNS infection started at later time points and was persistent or progressive. We then tested the possible mechanisms of neuroinvasion by CHIKV and SINV. Neither virus relied on macrophage-mediated transport to access the CNS. CHIKV, but not SINV, always infects endothelial cells of the brain vasculature. By contrast, axonal transport was much more efficient with SINV than CHIKV, both from the periphery to the CNS and between neural tissues. Thus, the preferred mechanisms of neuroinvasion by these two related viruses are distinct, providing a powerful imaging-friendly system to compare mechanisms and prevention methods of encephalopathies.
Asunto(s)
Virus Chikungunya/fisiología , Imagenología Tridimensional , Sistema Nervioso/virología , Virus Sindbis/fisiología , Internalización del Virus , Infecciones por Alphavirus/patología , Infecciones por Alphavirus/virología , Animales , Transporte Axonal , Barrera Hematoencefálica/patología , Barrera Hematoencefálica/virología , Fiebre Chikungunya/patología , Fiebre Chikungunya/virología , Células Endoteliales/patología , Células Endoteliales/virología , Larva/virología , Macrófagos/metabolismo , Microvasos/patología , Sistema Nervioso/patología , Tropismo/fisiología , Replicación Viral/fisiología , Pez CebraRESUMEN
Zebrafish testis has become a powerful model for reproductive biology of teleostean fishes and other vertebrates and encompasses multiple applications in applied and basic research. Many studies have focused on 2D images, which is time consuming and implies extrapolation of results. Three-dimensional imaging of whole organs recently became an important challenge to better understand their architecture and allow cell enumeration. Several protocols have thus been developed to enhance sample transparency, a limiting step for imaging large biological samples. However, none of these methods has been applied to the zebrafish testis. We tested five clearing protocols to determine if some of them could be applied with only small modifications to the testis. We compared clearing efficiency at both macroscopic and microscopic levels. CUBIC and PACT were suitable for an efficient transparency, an optimal optical penetration, the GFP fluorescence preservation and avoiding meaningful tissue deformation. Finally, we succeeded in whole testis 3D capture at a cellular resolution with both CUBIC and PACT, which will be valuable in a standard workflow to investigate the 3D architecture of the testis and its cellular content. This paves the way for further development of high content phenotyping studies in several fields including development, genetic or toxicology.
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Imagenología Tridimensional , Testículo/diagnóstico por imagen , Animales , Animales Modificados Genéticamente/metabolismo , Masculino , Microscopía de Fluorescencia por Excitación Multifotónica , Imagen Óptica , Pez CebraRESUMEN
In mammals, neuroepithelial cells play an essential role in embryonic neurogenesis, whereas glial stem cells are the principal source of neurons at postembryonic stages. By contrast, neuroepithelial-like stem/progenitor (NE) cells have been shown to be present throughout life in teleosts. We used three-dimensional (3D) reconstructions of cleared transgenic wdr12:GFP medaka brains to demonstrate that this cell type is widespread in juvenile and to identify new regions containing NE cells. We established the gene expression profile of optic tectum (OT) NE cells by cell sorting followed by RNA-seq. Our results demonstrate that most OT NE cells are indeed active stem cells and that some of them exhibit long G2 phases. We identified several novel pathways (e.g., DNA repair pathways) potentially involved in NE cell homeostasis. In situ hybridization studies showed that all NE populations in the postembryonic medaka brain have a similar molecular signature. Our findings highlight the importance of NE progenitors in medaka and improve our understanding of NE-cell biology. These cells are potentially useful not only for neural stem cell studies but also for improving the characterization of neurodevelopmental diseases, such as microcephaly. Stem Cells 2017;35:1505-1518.
Asunto(s)
Encéfalo/citología , Encéfalo/crecimiento & desarrollo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Células Neuroepiteliales/metabolismo , Oryzias/crecimiento & desarrollo , Animales , Animales Modificados Genéticamente , Biomarcadores/metabolismo , Proliferación Celular/genética , Reparación del ADN/genética , Fase G2 , Proteínas Fluorescentes Verdes/metabolismo , Oryzias/genética , Análisis de Secuencia de ARN , Colículos Superiores/citología , Regulación hacia ArribaRESUMEN
In zebrafish brains, populations of continuously proliferating cells are present during an entire life span. Under normal conditions, stem cells give rise to rapidly proliferating progenitors that quickly exit the cell cycle and differentiate. Hence fish are favorable models to study what regulates postembryonic neurogenesis. The aim of this study was to determine if optic tectum (OT) cell proliferation is halted during nutritional deprivation (ND) and whether or not it can be restored with refeeding. We examined the effect of ND on the proliferation of Neuroepithelial/Ependymal Progenitor cell (NeEPC) and transitory-amplifying progenitors (TAPs). Following ND, no PCNA immunostaining was found in OT of starved fish, while positive cell populations of PCNA positive progenitors are found at its periphery in control fish. This indicated that active proliferation stopped. To label retaining progenitor cells, BrdU was applied and a chase-period was accompanied by ND. Positive NeEPCs were detected in the external tectum marginal zone of starved fish suggesting that these progenitors are relatively immune to ND. Moreover in the internal tectum marginal zone labeled retaining cells were observed leaving the possibility that some arrested TAPs were present to readily restart proliferation when nutrition was returned. Our results suggest that neurogenesis was maintained during ND and that a normal proliferative situation was recovered after refeeding. We point to the mTOR pathway as a necessary pathway in progenitors to regulate their mitosis activity. Thus, this study highlights mechanisms involved in neural stem and progenitor cell homeostatic maintenance in an adverse situation. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 26-38, 2017.
Asunto(s)
Fenómenos Fisiológicos Nutricionales de los Animales/fisiología , Proliferación Celular/fisiología , Neurogénesis/fisiología , Inanición , Células Madre/fisiología , Colículos Superiores/fisiología , Animales , Epéndimo/citología , Epéndimo/fisiología , Modelos Animales , Células-Madre Neurales/citología , Células-Madre Neurales/fisiología , Células Neuroepiteliales/citología , Células Neuroepiteliales/fisiología , Colículos Superiores/citología , Pez CebraRESUMEN
Somatic mutations activating MAPK and PI3K signalling play a pivotal role in both tumours and brain developmental disorders. We developed a zebrafish model of brain tumours based on somatic expression of oncogenes that activate MAPK and PI3K signalling in neural progenitor cells and found that HRASV12 was the most effective in inducing both heterotopia and invasive tumours. Tumours, but not heterotopias, require persistent activation of phospho (p)-ERK and express a gene signature similar to the mesenchymal glioblastoma subtype, with a strong YAP component. Application of an eight-gene signature to human brain tumours establishes that YAP activation distinguishes between mesenchymal glioblastoma and low grade glioma in a wide The Cancer Genome Atlas (TCGA) sample set including gliomas and glioblastomas (GBMs). This suggests that the activation of YAP might be an important event in brain tumour development, promoting malignant versus benign brain lesions. Indeed, co-expression of dominant-active YAP (YAPS5A) and HRASV12 abolishes the development of heterotopias and leads to the sole development of aggressive tumours. Thus, we have developed a model proving that neurodevelopmental disorders and brain tumours might originate from the same activation of oncogenes through somatic mutations, and established that YAP activation is a hallmark of malignant brain tumours.
Asunto(s)
Neoplasias Encefálicas/enzimología , Neoplasias Encefálicas/patología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transactivadores/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Aminoacil-ARNt Sintetasas/genética , Animales , Neoplasias Encefálicas/genética , Carcinogénesis/genética , Carcinogénesis/patología , Proliferación Celular , Supervivencia Celular , Células Clonales , Modelos Animales de Enfermedad , Elementos de Facilitación Genéticos/genética , Activación Enzimática , Regulación Neoplásica de la Expresión Génica , Genes ras , Glioblastoma/genética , Glioblastoma/patología , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunohistoquímica , Mesodermo/patología , Células-Madre Neurales/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Telencéfalo/patología , Proteínas Señalizadoras YAP , Proteínas de Pez Cebra/genéticaRESUMEN
In studies of brain function, it is essential to understand the underlying neuro-architecture. Very young zebrafish larvae are widely used for neuroarchitecture studies, due to their size and natural transparency. However, this model system has several limitations, due to the immaturity, high rates of development and limited behavioral repertoire of the animals used. We describe here a modified version of the passive clearing technique (PACT) ( Chung et al., 2013 ; Tomer et al., 2014 ; Yang et al., 2014 ; Treweek et al., 2015) , which facilitates neuroanatomical studies on large specimens of aquatic species. This method was initially developed for zebrafish (Danio rerio) ( Frétaud et al., 2017 ; Mayrhofer et al., 2017 ; Xavier et al., 2017 ), but has also been successfully tested on other fish, such as medaka (Oryzias latipes) ( Dambroise et al., 2017 ), Mexican cave fish (Astyanax mexicaus) and African zebra mbuna (Metriaclima zebra), and on other aquatic species, such as Xenopus spp. (Xenopus laevis, Xenopus tropicalis) ( Fini et al., 2017 ) . This protocol, based on the CLARITY method developed and modified by Deisseroth's laboratory and others ( Chung et al., 2013 ; Tomer et al., 2014 ; Yang et al., 2014 ), was adapted for use in aquatic species, including zebrafish in particular (zPACT). This protocol is designed to render zebrafish specimens optically transparent while preserving the overall architecture of the tissue, through crosslinking in a polyacrylamide/formaldehyde mesh. Most of the lipids present in the specimen are then removed by SDS treatment, to homogenize the refractive index of the specimen by eliminating light scattering at the water/lipid interface, which causes opacity. The final clearing step, consists of the incubation of the specimen in a fructose-based mounting medium (derived from SeeDB) ( Ke et al., 2013 ) , with a refractive index matching that of the objective lens of the microscope. The combination of this technique with the use of genetically modified zebrafish in which green fluorescent protein (GFP) is expressed in specific cell populations provides opportunities to describe anatomical details not visible with other techniques.
RESUMEN
The visual systems of vertebrates and many other bilaterian clades consist of complex neural structures guiding a wide spectrum of behaviors. Homologies at the level of cell types and even discrete neural circuits have been proposed, but many questions of how the architecture of visual neuropils evolved among different phyla remain open. In this review we argue that the profound conservation of genetic and developmental steps generating the eye and its target neuropils in fish and fruit flies supports a homology between some core elements of bilaterian visual circuitries. Fish retina and tectum, and fly optic lobe, develop from a partitioned, unidirectionally proliferating neurectodermal domain that combines slowly dividing neuroepithelial stem cells and rapidly amplifying progenitors with shared genetic signatures to generate large numbers and different types of neurons in a temporally ordered way. This peculiar 'conveyor belt neurogenesis' could play an essential role in generating the topographically ordered circuitry of the visual system.
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Evolución Biológica , Drosophila/fisiología , Peces/fisiología , Neurogénesis , Animales , Drosophila/crecimiento & desarrollo , Peces/crecimiento & desarrollo , Neurópilo/fisiología , Lóbulo Óptico de Animales no Mamíferos/crecimiento & desarrollo , Lóbulo Óptico de Animales no Mamíferos/fisiología , Retina/crecimiento & desarrollo , Retina/fisiología , Colículos Superiores/crecimiento & desarrollo , Colículos Superiores/fisiologíaRESUMEN
BACKGROUND: The success of the CRISPR/Cas9 genome editing technique depends on the choice of the guide RNA sequence, which is facilitated by various websites. Despite the importance and popularity of these algorithms, it is unclear to which extent their predictions are in agreement with actual measurements. RESULTS: We conduct the first independent evaluation of CRISPR/Cas9 predictions. To this end, we collect data from eight SpCas9 off-target studies and compare them with the sites predicted by popular algorithms. We identify problems in one implementation but found that sequence-based off-target predictions are very reliable, identifying most off-targets with mutation rates superior to 0.1 %, while the number of false positives can be largely reduced with a cutoff on the off-target score. We also evaluate on-target efficiency prediction algorithms against available datasets. The correlation between the predictions and the guide activity varied considerably, especially for zebrafish. Together with novel data from our labs, we find that the optimal on-target efficiency prediction model strongly depends on whether the guide RNA is expressed from a U6 promoter or transcribed in vitro. We further demonstrate that the best predictions can significantly reduce the time spent on guide screening. CONCLUSIONS: To make these guidelines easily accessible to anyone planning a CRISPR genome editing experiment, we built a new website ( http://crispor.org ) that predicts off-targets and helps select and clone efficient guide sequences for more than 120 genomes using different Cas9 proteins and the eight efficiency scoring systems evaluated here.
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Sistemas CRISPR-Cas/genética , Edición Génica , ARN Guía de Kinetoplastida/genética , Programas Informáticos , Algoritmos , Genoma , Internet , Regiones Promotoras Genéticas , ARN Nuclear Pequeño/genéticaRESUMEN
The 9th European Zebrafish Meeting took place recently in Oslo (June 28-July 2, 2015). A total of 650 participants came to hear the latest research news focused on the zebrafish, Danio rerio, and to its distant evolutionary relative medaka, Oryzias latipes. The packed program included keynote and plenary talks, short oral presentations and poster sessions, workshops, and strategic discussions. The meeting was a great success and revealed dramatically how important the zebrafish in particular has become as a model system for topics, such as developmental biology, functional genomics, biomedicine, toxicology, and drug development. A new emphasis was given to its potential as a model for aquaculture, a topic of great economic interest to the host country Norway and for the future global food supply in general. Zebrafish husbandry as well as its use in teaching were also covered in separate workshops. As has become a tradition in these meetings, there was a well-attended Wellcome Trust Sanger Institute and ZFIN workshop focused on Zebrafish Genome Resources on the first day. The full EZM 2015 program with abstracts can be read and downloaded from the EZM 2015 Web site zebrafish2015.org .
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Acuicultura , Oryzias/genética , Pez Cebra/genética , Animales , Modelos Animales , NoruegaRESUMEN
Although considered a 'house-keeping' function, ribosome biogenesis is regulated differently between cells and can be modulated in a cell-type-specific manner. These differences are required to generate specialized ribosomes that contribute to the translational control of gene expression by selecting mRNA subsets to be translated. Thus, differences in ribosome biogenesis between stem and differentiated cells indirectly contribute to determine cell identity. The concept of the existence of stem cell-specific mechanisms of ribosome biogenesis has progressed from an attractive theory to a useful working model with important implications for basic and medical research.
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Diferenciación Celular/genética , ARN Ribosómico/genética , Ribosomas/genética , Células Madre/metabolismo , Animales , Homeostasis/genética , Humanos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ribosomas/metabolismo , Células Madre/citología , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Presently, human collagen VI-related diseases such as Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) remain incurable, emphasizing the need to unravel their etiology and improve their treatments. In UCMD, symptom onset occurs early, and both diseases aggravate with ageing. In zebrafish fry, morpholinos reproduced early UCMD and BM symptoms but did not allow to study the late phenotype. Here, we produced the first zebrafish line with the human mutation frequently found in collagen VI-related disorders such as UCMD and BM. We used a transcription activator-like effector nuclease (TALEN) to design the col6a1ama605003-line with a mutation within an essential splice donor site, in intron 14 of the col6a1 gene, which provoke an in-frame skipping of exon 14 in the processed mRNA. This mutation at a splice donor site is the first example of a template-independent modification of splicing induced in zebrafish using a targetable nuclease. This technique is readily expandable to other organisms and can be instrumental in other disease studies. Histological and ultrastructural analyzes of homozygous and heterozygous mutant fry and 3 months post-fertilization (mpf) fish revealed co-dominantly inherited abnormal myofibers with disorganized myofibrils, enlarged sarcoplasmic reticulum, altered mitochondria and misaligned sarcomeres. Locomotion analyzes showed hypoxia-response behavior in 9 mpf col6a1 mutant unseen in 3 mpf fish. These symptoms worsened with ageing as described in patients with collagen VI deficiency. Thus, the col6a1ama605003-line is the first adult zebrafish model of collagen VI-related diseases; it will be instrumental both for basic research and drug discovery assays focusing on this type of disorders.
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Colágeno Tipo VI , Exones , Mutación , Sitios de Empalme de ARN , Pez Cebra/genética , Pez Cebra/metabolismo , Animales , Colágeno Tipo VI/biosíntesis , Colágeno Tipo VI/genética , Contractura , Modelos Animales de Enfermedad , Heterocigoto , Homocigoto , Humanos , Distrofias Musculares/congénitoRESUMEN
We aimed to identify cis-regulatory elements that control gene expression in progenitors of the cerebral cortex. A list of 975 putative enhancers were retrieved from a ChIP-Seq experiment performed in NS5 mouse stem cells with antibodies to Sox2, Brn2/Pou3f2, or Brn1/Pou3f3. Through a selection pipeline including gene ontology and expression pattern, we reduced the number of candidate enhancer sequences to 20. Ex vivo electroporation of green fluorescent pProtein (GFP) reporter constructs in the telencephalon of mouse embryos showed that 35% of the 20 selected candidate sequences displayed enhancer activity in the developing cortex at E13.5. In silico transcription factor binding site (TFBS) searches and mutagenesis experiments showed that enhancer activity is related to the presence of Sox/Pou TFBS pairs in the sequence. Comparative genomic analyses showed that enhancer activity is not related to the evolutionary conservation of the sequence. Finally, the combination of in utero electroporation of GFP reporter constructs with immunostaining for Tbr2 (basal progenitor marker) and phospho-histoneH3 (mitotic activity marker) demonstrated that each enhancer is specifically active in precise subpopulations of progenitors in the cortical germinal zone, highlighting the heterogeneity of these progenitors in terms of cis-regulation.
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Corteza Cerebral/citología , Corteza Cerebral/embriología , Regulación del Desarrollo de la Expresión Génica/fisiología , Factores del Dominio POU/metabolismo , Factores de Transcripción SOXB1/metabolismo , Células Madre/fisiología , Animales , Sitios de Unión/genética , Evolución Biológica , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular , Embrión de Mamíferos , Femenino , Histonas/genética , Histonas/metabolismo , Técnicas In Vitro , Ratones , Ratones Transgénicos , Mutagénesis/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Técnicas de Cultivo de Órganos , Factores del Dominio POU/genética , Embarazo , Factores de Transcripción SOXB1/genética , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismoRESUMEN
Investigating neural stem cell (NSC) behaviour in vivo, which is a major area of research, requires NSC models to be developed. We carried out a multilevel characterisation of the zebrafish embryo peripheral midbrain layer (PML) and identified a unique vertebrate progenitor population. Located dorsally in the transparent embryo midbrain, these large slow-amplifying progenitors (SAPs) are accessible for long-term in vivo imaging. They form a neuroepithelial layer adjacent to the optic tectum, which has transitory fast-amplifying progenitors (FAPs) at its margin. The presence of these SAPs and FAPs in separate domains provided the opportunity to data mine the ZFIN expression pattern database for SAP markers, which are co-expressed in the retina. Most of them are involved in nucleotide synthesis, or encode nucleolar and ribosomal proteins. A mutant for the cad gene, which is strongly expressed in the PML, reveals severe midbrain defects with massive apoptosis and sustained proliferation. We discuss how fish midbrain and retina progenitors might derive from ancient sister cell types and have specific features that are not shared with other SAPs.
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Mesencéfalo/embriología , Mesencéfalo/metabolismo , Células-Madre Neurales/metabolismo , Retina/metabolismo , Pez Cebra/embriología , Animales , Ciclo Celular , Diferenciación Celular/genética , Proliferación Celular , Células Cultivadas , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Mitosis , MorfogénesisRESUMEN
Peptide hormones and their receptors are widespread in metazoans, but the knowledge we have of their evolutionary relationships remains unclear. Recently, accumulating genome sequences from many different species have offered the opportunity to reassess the relationships between protostomian and deuterostomian peptidergic systems (PSs). Here we used sequences of all human rhodopsin and secretin-type G protein-coupled receptors as bait to retrieve potential homologs in the genomes of 15 bilaterian species, including nonchordate deuterostomian and lophotrochozoan species. Our phylogenetic analysis of these receptors revealed 29 well-supported subtrees containing mixed sets of protostomian and deuterostomian sequences. This indicated that many vertebrate and arthropod PSs that were previously thought to be phyla specific are in fact of bilaterian origin. By screening sequence databases for potential peptides, we then reconstructed entire bilaterian peptide families and showed that protostomian and deuterostomian peptides that are ligands of orthologous receptors displayed some similarity at the level of their primary sequence, suggesting an ancient coevolution between peptide and receptor genes. In addition to shedding light on the function of human G protein-coupled receptor PSs, this work presents orthology markers to study ancestral neuron types that were probably present in the last common bilaterian ancestor.