RESUMO
Adult frogs that are well-cared-for will give high-quality eggs and embryos for use in every Xenopus protocol. Thoughtful frog husbandry is thus pivotal to successful research using these organisms. Protocols for successfully raising tadpoles, establishing and maintaining water quality, and detecting specific pathogens are key to maintaining healthy frog populations.
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Xenopus laevis , Animais , LarvaRESUMO
Collecting eggs from adult Xenopus laevis and Xenopus tropicalis to raise healthy embryos and tadpoles is relatively simple but requires careful handling of the frog. Eggs can be fertilized through natural matings or by in vitro fertilization and examined visually. Here we review how eggs are obtained and how to recognize healthy eggs that will develop into high-quality embryos.
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Fertilização in vitro , Fertilização , Animais , Xenopus laevis , Xenopus , LarvaRESUMO
The complex 16p11.2 deletion syndrome (16pdel) is accompanied by neurological disorders, including epilepsy, autism spectrum disorder, and intellectual disability. We demonstrated that 16pdel iPSC differentiated neurons from affected people show augmented local field potential activity and altered ceramide-related lipid species relative to unaffected. FAM57B, a poorly characterized gene in the 16p11.2 interval, has emerged as a candidate tied to symptomatology. We found that FAM57B modulates ceramide synthase (CerS) activity, but is not a CerS per se. In FAM57B mutant human neuronal cells and zebrafish brain, composition and levels of sphingolipids and glycerolipids associated with cellular membranes are disrupted. Consistently, we observed aberrant plasma membrane architecture and synaptic protein mislocalization, which were accompanied by depressed brain and behavioral activity. Together, these results suggest that haploinsufficiency of FAM57B contributes to changes in neuronal activity and function in 16pdel syndrome through a crucial role for the gene in lipid metabolism.
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Zebrafish are an important and expanding experimental system for brain research. We describe a noninvasive electrophysiology technique that can be used in living larvae to measure spontaneous activity in the brain and spinal cord simultaneously. This easy-to-use method uses a commercially available multielectrode array to detect local field potential parameters, and allows for relative coordinated (network) measurements of activity. We demonstrate sensitivity of this system by measuring activity in larvae treated with the antiepileptic drug valproic acid. Valproic acid decreased larval movement and startle response, and decreased spontaneous brain activity. Spinal cord activity did not change after treatment, suggesting valproic acid primarily affects brain function. The observed differences in brain activity, but not spinal cord activity, after valproic acid treatment indicates that brain activity differences are not a secondary effect of decreased startle response and movement. We provide a step-by-step protocol for experiments presented that a novice could easily follow. This electrophysiological method will be useful to the zebrafish neuroscience community.
Assuntos
Encéfalo/fisiologia , Eletrodos , Medula Espinal/fisiologia , Peixe-Zebra/fisiologia , Animais , Fenômenos Eletrofisiológicos , Larva/crescimento & desenvolvimento , Larva/fisiologia , Ácido Valproico/farmacologia , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
The brain ventricular system is a series of connected cavities, filled with cerebrospinal fluid (CSF), that forms within the vertebrate central nervous system (CNS). The hollow neural tube is a hallmark of the chordate CNS, and a closed neural tube is essential for normal development. Development and function of the ventricular system is examined, emphasizing three interdigitating components that form a functional system: ventricle walls, CSF fluid properties, and activity of CSF constituent factors. The cellular lining of the ventricle both can produce and is responsive to CSF. Fluid properties and conserved CSF components contribute to normal CNS development. Anomalies of the CSF/ventricular system serve as diagnostics and may cause CNS disorders, further highlighting their importance. This review focuses on the evolution and development of the brain ventricular system, associated function, and connected pathologies. It is geared as an introduction for scholars with little background in the field.
Assuntos
Ventrículos Cerebrais/crescimento & desenvolvimento , Ventrículos Cerebrais/metabolismo , Líquido Cefalorraquidiano/metabolismo , Animais , Evolução Biológica , Encefalopatias/metabolismo , Ventrículos Cerebrais/citologia , Pressão do Líquido Cefalorraquidiano/fisiologia , Proteínas do Líquido Cefalorraquidiano/metabolismo , Cílios/metabolismo , Epitélio/crescimento & desenvolvimento , Epitélio/metabolismo , Humanos , Cinética , Tubo Neural/citologia , Tubo Neural/crescimento & desenvolvimento , Tubo Neural/metabolismo , Transdução de SinaisRESUMO
Basal constriction occurs at the zebrafish midbrain-hindbrain boundary constriction (MHBC) and is likely a widespread morphogenetic mechanism. 3D reconstruction demonstrates that MHBC cells are wedge-shaped, and initially constrict basally, with subsequent apical expansion. wnt5b is expressed in the MHB and is required for basal constriction. Consistent with a requirement for this pathway, expression of dominant negative Gsk3ß overcomes wnt5b knockdown. Immunostaining identifies focal adhesion kinase (Fak) as active in the MHB region, and knockdown demonstrates Fak is a regulator of basal constriction. Tissue specific knockdown further indicates that Fak functions cell autonomously within the MHBC. Fak acts downstream of wnt5b, suggesting that Wnt5b signals locally as an early step in basal constriction and acts together with more widespread Fak activation. This study delineates signaling pathways that regulate basal constriction during brain morphogenesis.
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The RNAi pathway provides both innate immunity and efficient gene-knockdown tools in many eukaryotic species, but curiously not in zebrafish. We discovered that RNAi is less effective in zebrafish at least partly because Argonaute2-catalyzed mRNA slicing is impaired. This defect is due to two mutations that arose in an ancestor of most teleost fish, implying that most fish lack effective RNAi. Despite lacking efficient slicing activity, these fish have retained the ability to produce miR-451, a microRNA generated by a cleavage reaction analogous to slicing. This ability is due to a G-G mismatch within the fish miR-451 precursor, which substantially enhances its cleavage. An analogous G-G mismatch (or sometimes also a G-A mismatch) enhances target slicing, despite disrupting seed pairing important for target binding. These results provide a strategy for restoring RNAi to zebrafish and reveal unanticipated opposing effects of a seed mismatch with implications for mechanism and guide-RNA design.
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Proteínas Argonautas/metabolismo , Pareamento Incorreto de Bases , MicroRNAs/metabolismo , Interferência de RNA , RNA Guia de Cinetoplastídeos/genética , RNA Mensageiro/genética , Peixe-Zebra/genética , Animais , Proteínas Argonautas/genética , Técnicas de Silenciamento de Genes , Humanos , MicroRNAs/genética , RNA Mensageiro/metabolismo , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/metabolismo , Peixe-Zebra/fisiologiaRESUMO
Deletion of the 16p11.2 CNV affects 25 core genes and is associated with multiple symptoms affecting brain and body, including seizures, hyperactivity, macrocephaly, and obesity. Available data suggest that most symptoms are controlled by haploinsufficiency of two or more 16p11.2 genes. To identify interacting 16p11.2 genes, we used a pairwise partial loss of function antisense screen for embryonic brain morphology, using the accessible zebrafish model. fam57ba, encoding a ceramide synthase, was identified as interacting with the doc2a gene, encoding a calcium-sensitive exocytosis regulator, a genetic interaction not previously described. Using genetic mutants, we demonstrated that doc2a+/- fam57ba+/- double heterozygotes show hyperactivity and increased seizure susceptibility relative to wild-type or single doc2a-/- or fam57ba-/- mutants. Additionally, doc2a+/- fam57ba+/- double heterozygotes demonstrate the increased body length and head size. Single doc2a+/- and fam57ba+/- heterozygotes do not show a body size increase; however, fam57ba-/- homozygous mutants show a strongly increased head size and body length, suggesting a greater contribution from fam57ba to the haploinsufficient interaction between doc2a and fam57ba. The doc2a+/- fam57ba+/- interaction has not been reported before, nor has any 16p11.2 gene previously been linked to increased body size. These findings demonstrate that one pair of 16p11.2 homologs can regulate both brain and body phenotypes that are reflective of those in people with 16p11.2 deletion. Together, these findings suggest that dysregulation of ceramide pathways and calcium sensitive exocytosis underlies seizures and large body size associated with 16p11.2 homologs in zebrafish. The data inform consideration of mechanisms underlying human 16p11.2 deletion symptoms.
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Transtorno Autístico/genética , Transtorno Autístico/metabolismo , Proteínas de Ligação ao Cálcio/genética , Transtornos Cromossômicos/genética , Transtornos Cromossômicos/metabolismo , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Proteínas do Tecido Nervoso/genética , Oxirredutases/genética , Animais , Animais Geneticamente Modificados , Tamanho Corporal/genética , Encéfalo/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Deleção Cromossômica , Cromossomos Humanos Par 16/genética , Cromossomos Humanos Par 16/metabolismo , Variações do Número de Cópias de DNA , Modelos Animais de Doenças , Exocitose/genética , Humanos , Proteínas do Tecido Nervoso/metabolismo , Oxirredutases/metabolismo , Fenótipo , Convulsões/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
A mouth is present in all animals, and comprises an opening from the outside into the oral cavity and the beginnings of the digestive tract to allow eating. This review focuses on the earliest steps in mouth formation. In the first half, we conclude that the mouth arose once during evolution. In all animals, the mouth forms from ectoderm and endoderm. A direct association of oral ectoderm and digestive endoderm is present even in triploblastic animals, and in chordates, this region is known as the extreme anterior domain (EAD). Further support for a single origin of the mouth is a conserved set of genes that form a 'mouth gene program' including foxA and otx2. In the second half of this review, we discuss steps involved in vertebrate mouth formation, using the frog Xenopus as a model. The vertebrate mouth derives from oral ectoderm from the anterior neural ridge, pharyngeal endoderm and cranial neural crest (NC). Vertebrates form a mouth by breaking through the body covering in a precise sequence including specification of EAD ectoderm and endoderm as well as NC, formation of a 'pre-mouth array,' basement membrane dissolution, stomodeum formation, and buccopharyngeal membrane perforation. In Xenopus, the EAD is also a craniofacial organizer that guides NC, while reciprocally, the NC signals to the EAD to elicit its morphogenesis into a pre-mouth array. Human mouth anomalies are prevalent and are affected by genetic and environmental factors, with understanding guided in part by use of animal models. WIREs Dev Biol 2017, 6:e275. doi: 10.1002/wdev.275 For further resources related to this article, please visit the WIREs website.
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Boca/embriologia , Animais , Ectoderma/citologia , Ectoderma/embriologia , Endoderma/citologia , Endoderma/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Boca/citologia , Crista Neural/citologia , Crista Neural/embriologia , Xenopus/embriologiaRESUMO
Advances in genome-editing technology have made creation of zebrafish mutant lines accessible to the community. Experimental validation of protein knockout is a critical step in verifying null mutants, but this can be a difficult task. Absence of protein can be confirmed by Western blotting; however, this approach requires target-specific antibodies that are generally not available for zebrafish proteins. We address this issue using in vitro translation assays, a fast and standard procedure that can be easily implemented.
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Animais Geneticamente Modificados/metabolismo , Sistema Livre de Células , Técnicas de Inativação de Genes/métodos , Biossíntese de Proteínas , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados/genética , Edição de Genes , Mutação , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
The mouth arises from the extreme anterior domain (EAD), a region where the ectoderm and endoderm are directly juxtaposed. Here, we identify a "pre-mouth array" in Xenopus that forms soon after the cranial neural crest has migrated to lie on either side of the EAD. Initially, EAD ectoderm comprises a wide and short epithelial mass that becomes narrow and tall with cells and nuclei changing shape, a characteristic of convergent extension. The resulting two rows of cells-the pre-mouth array-later split down the midline to surround the mouth opening. Neural crest is essential for convergent extension and likely signals to the EAD through the Wnt/planar cell polarity (PCP) pathway. Fzl7 receptor is locally required in EAD ectoderm, while Wnt11 ligand is required more globally. Indeed, heterologous cells expressing Wnt11 can elicit EAD convergent extension. The study reveals a precise cellular mechanism that positions and contributes to the future mouth.
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Polaridade Celular/fisiologia , Boca/metabolismo , Crista Neural/metabolismo , Proteínas Wnt/metabolismo , Animais , Ectoderma/metabolismo , Endoderma/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismoRESUMO
BACKGROUND: Cerebrospinal fluid (CSF) contained within the brain ventricles contacts neuroepithelial progenitor cells during brain development. Dynamic properties of CSF movement may limit locally produced factors to specific regions of the developing brain. However, there is no study of in vivo CSF dynamics between ventricles in the embryonic brain. We address CSF movement using the zebrafish larva, during the major period of developmental neurogenesis. METHODS: CSF movement was monitored at two stages of zebrafish development: early larva [pharyngula stage; 27-30 h post-fertilization (hpf)] and late larva (hatching period; 51-54 hpf) using photoactivatable Kaede protein to calculate average maximum CSF velocity between ventricles. Potential roles for heartbeat in early CSF movement were investigated using tnnt2a mutant fish (tnnt2a (-/-)) and chemical [2,3 butanedione monoxime (BDM)] treatment. Cilia motility was monitored at these stages using the Tg(ßact:Arl13b-GFP) transgenic fish line. RESULTS: In wild-type early larva there is net CSF movement from the telencephalon to the combined diencephalic/mesencephalic superventricle. This movement directionality reverses at late larval stage. CSF moves directionally from diencephalic to rhombencephalic ventricles at both stages examined, with minimal movement from rhombencephalon to diencephalon. Directional movement is partially dependent on heartbeat, as indicated in assays of tnnt2a (-/-) fish and after BDM treatment. Brain cilia are immotile at the early larval stage. CONCLUSION: These data demonstrate directional movement of the embryonic CSF in the zebrafish model during the major period of developmental neurogenesis. A key conclusion is that CSF moves preferentially from the diencephalic into the rhombencephalic ventricle. In addition, the direction of CSF movement between telencephalic and diencephalic ventricles reverses between the early and late larval stages. CSF movement is partially dependent on heartbeat. At early larval stage, the absence of motile cilia indicates that cilia likely do not direct CSF movement. These data suggest that CSF components may be compartmentalized and could contribute to specialization of the early brain. In addition, CSF movement may also provide directional mechanical signaling.
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Ventrículos Cerebrais/embriologia , Ventrículos Cerebrais/fisiologia , Líquido Cefalorraquidiano/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Cílios/fisiologia , Diencéfalo/embriologia , Diencéfalo/fisiologia , Coração/embriologia , Coração/fisiologia , Hidrodinâmica , Microscopia Confocal , Movimento , Rombencéfalo/embriologia , Telencéfalo/embriologia , Telencéfalo/fisiologia , Troponina T/genética , Troponina T/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Cerebrospinal fluid (CSF) includes conserved factors whose function is largely unexplored. To assess the role of CSF during embryonic development, CSF was repeatedly drained from embryonic zebrafish brain ventricles soon after their inflation. Removal of CSF increased cell death in the diencephalon, indicating a survival function. Factors within the CSF are required for neuroepithelial cell survival as injected mouse CSF but not artificial CSF could prevent cell death after CSF depletion. Mass spectrometry analysis of the CSF identified retinol binding protein 4 (Rbp4), which transports retinol, the precursor to retinoic acid (RA). Consistent with a role for Rbp4 in cell survival, inhibition of Rbp4 or RA synthesis increased neuroepithelial cell death. Conversely, ventricle injection of exogenous human RBP4 plus retinol, or RA alone prevented cell death after CSF depletion. Zebrafish rbp4 is highly expressed in the yolk syncytial layer, suggesting Rbp4 protein and retinol/RA precursors can be transported into the CSF from the yolk. In accord with this suggestion, injection of human RBP4 protein into the yolk prevents neuroepithelial cell death in rbp4 loss-of-function embryos. Together, these data support the model that Rbp4 and RA precursors are present within the CSF and used for synthesis of RA, which promotes embryonic neuroepithelial survival.
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Retinoides/metabolismo , Proteínas Plasmáticas de Ligação ao Retinol/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Morte Celular , Sobrevivência Celular , Ventrículos Cerebrais/metabolismo , Tretinoína/líquido cefalorraquidianoRESUMO
The treatment of psychiatric disorders presents three major challenges to the research and clinical community: defining a genotype associated with a disorder, characterizing the molecular pathology of each disorder and developing new therapies. This Review addresses how cellular and animal systems can help to meet these challenges, with an emphasis on the role of the zebrafish. Genetic changes account for a large proportion of psychiatric disorders and, as gene variants that predispose to psychiatric disease are beginning to be identified in patients, these are tractable for study in cellular and animal systems. Defining cellular and molecular criteria associated with each disorder will help to uncover causal physiological changes in patients and will lead to more objective diagnostic criteria. These criteria should also define co-morbid pathologies within the nervous system or in other organ systems. The definition of genotypes and of any associated pathophysiology is integral to the development of new therapies. Cell culture-based approaches can address these challenges by identifying cellular pathology and by high-throughput screening of gene variants and potential therapeutics. Whole-animal systems can define the broadest function of disorder-associated gene variants and the organismal impact of candidate medications. Given its evolutionary conservation with humans and its experimental tractability, the zebrafish offers several advantages to psychiatric disorder research. These include assays ranging from molecular to behavioural, and capability for chemical screening. There is optimism that the multiple approaches discussed here will link together effectively to provide new diagnostics and treatments for psychiatric patients.
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Transtornos Mentais/etiologia , Transtornos Mentais/terapia , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Evolução Biológica , Caenorhabditis elegans , Técnicas de Cultura de Células , Modelos Animais de Doenças , Drosophila melanogaster , Humanos , Camundongos , Fenótipo , Pesquisa Translacional Biomédica , Peixe-Zebra/genéticaRESUMO
Mental health disorders are notoriously difficult to diagnose and treat for a variety of reasons, including genetic heterogeneity, comorbidities, and qualitative diagnostic criteria. Discovery of the molecular pathology underlying these disorders is crucial to the development of quantitative biomarkers and novel therapeutics. In this review, we discuss contributions to the mental health field of different cellular and whole-animal approaches in characterizing psychiatric genetics and molecular pathology. These approaches include mammalian cell and neuronal culture, cerebral organoids, induced pluripotent stem cells, and the whole-animal models of nematodes, flies, mollusks, frogs, mice, and zebrafish, on the last of which we place extra emphasis. Integrative use of these cellular and animal systems in a complementary and informative fashion maximizes the potential contributions to the mental health field as a whole.
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Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Transtornos Mentais/genética , Transtornos Mentais/patologia , Animais , Aplysia/genética , Comportamento Animal/fisiologia , Caenorhabditis elegans/genética , Células Cultivadas , Drosophila melanogaster/genética , Descoberta de Drogas , Regulação da Expressão Gênica , Humanos , Transtornos Mentais/tratamento farmacológico , Camundongos , Neurônios/patologia , Células-Tronco Pluripotentes/patologia , Células-Tronco Pluripotentes/fisiologia , Xenopus laevis/genética , Peixe-ZebraRESUMO
The extreme anterior domain (EAD) is a conserved embryonic region that includes the presumptive mouth. We show that the Kinin-Kallikrein pathway is active in the EAD and necessary for craniofacial development in Xenopus and zebrafish. The mouth failed to form and neural crest (NC) development and migration was abnormal after loss of function (LOF) in the pathway genes kng, encoding Bradykinin (xBdk), carboxypeptidase-N (cpn), which cleaves Bradykinin, and neuronal nitric oxide synthase (nNOS). Consistent with a role for nitric oxide (NO) in face formation, endogenous NO levels declined after LOF in pathway genes, but these were restored and a normal face formed after medial implantation of xBdk-beads into LOF embryos. Facial transplants demonstrated that Cpn function from within the EAD is necessary for the migration of first arch cranial NC into the face and for promoting mouth opening. The study identifies the EAD as an essential craniofacial organizer acting through Kinin-Kallikrein signaling.
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Sistema Calicreína-Cinina , Boca/embriologia , Crista Neural/embriologia , Transdução de Sinais , Animais , Bradicinina/metabolismo , Carboxipeptidases/metabolismo , Crista Neural/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Xenopus , Peixe-ZebraRESUMO
Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.
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Transplante de Face/métodos , Animais , Ossos Faciais/embriologia , Ossos Faciais/crescimento & desenvolvimento , Feminino , Desenvolvimento Maxilofacial/fisiologia , Modelos Animais , Crista Neural/embriologia , Crista Neural/crescimento & desenvolvimento , Crânio/embriologia , Crânio/crescimento & desenvolvimento , Xenopus laevisRESUMO
Poly(A) tails enhance the stability and translation of most eukaryotic messenger RNAs, but difficulties in globally measuring poly(A)-tail lengths have impeded greater understanding of poly(A)-tail function. Here we describe poly(A)-tail length profiling by sequencing (PAL-seq) and apply it to measure tail lengths of millions of individual RNAs isolated from yeasts, cell lines, Arabidopsis thaliana leaves, mouse liver, and zebrafish and frog embryos. Poly(A)-tail lengths were conserved between orthologous mRNAs, with mRNAs encoding ribosomal proteins and other 'housekeeping' proteins tending to have shorter tails. As expected, tail lengths were coupled to translational efficiencies in early zebrafish and frog embryos. However, this strong coupling diminished at gastrulation and was absent in non-embryonic samples, indicating a rapid developmental switch in the nature of translational control. This switch complements an earlier switch to zygotic transcriptional control and explains why the predominant effect of microRNA-mediated deadenylation concurrently shifts from translational repression to mRNA destabilization.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Poli A/análise , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Animais , Arabidopsis/genética , Sequência de Bases , Linhagem Celular , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Gastrulação/genética , Humanos , Fígado/metabolismo , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Genéticos , Folhas de Planta/genética , Poli A/genética , Estabilidade de RNA/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Análise de Sequência de RNA , Especificidade da Espécie , Transcrição Gênica , Xenopus/embriologia , Xenopus/genética , Leveduras/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Zigoto/metabolismoRESUMO
Cerebrospinal fluid (CSF) is a protein rich fluid contained within the brain ventricles. It is present during early vertebrate embryonic development and persists throughout life. Adult CSF is thought to cushion the brain, remove waste, and carry secreted molecules(1,2). In the adult and older embryo, the majority of CSF is made by the choroid plexus, a series of highly vascularized secretory regions located adjacent to the brain ventricles(3-5). In zebrafish, the choroid plexus is fully formed at 144 hours post fertilization (hpf)(6). Prior to this, in both zebrafish and other vertebrate embryos including mouse, a significant amount of embryonic CSF (eCSF) is present . These data and studies in chick suggest that the neuroepithelium is secretory early in development and may be the major source of eCSF prior to choroid plexus development(7). eCSF contains about three times more protein than adult CSF, suggesting that it may have an important role during development(8,9). Studies in chick and mouse demonstrate that secreted factors in the eCSF, fluid pressure, or a combination of these, are important for neurogenesis, gene expression, cell proliferation, and cell survival in the neuroepithelium(10-20). Proteomic analyses of human, rat, mouse, and chick eCSF have identified many proteins that may be necessary for CSF function. These include extracellular matrix components, apolipoproteins, osmotic pressure regulating proteins, and proteins involved in cell death and proliferation(21-24). However, the complex functions of the eCSF are largely unknown. We have developed a method for removing eCSF from zebrafish brain ventricles, thus allowing for identification of eCSF components and for analysis of the eCSF requirement during development. Although more eCSF can be collected from other vertebrate systems with larger embryos, eCSF can be collected from the earliest stages of zebrafish development, and under genetic or environmental conditions that lead to abnormal brain ventricle volume or morphology. Removal and collection of eCSF allows for mass spectrometric analysis, investigation of eCSF function, and reintroduction of select factors into the ventricles to assay their function. Thus the accessibility of the early zebrafish embryo allows for detailed analysis of eCSF function during development.