RESUMO
Thousands of long intervening noncoding RNAs (lincRNAs) have been identified in mammals. To better understand the evolution and functions of these enigmatic RNAs, we used chromatin marks, poly(A)-site mapping and RNA-Seq data to identify more than 550 distinct lincRNAs in zebrafish. Although these shared many characteristics with mammalian lincRNAs, only 29 had detectable sequence similarity with putative mammalian orthologs, typically restricted to a single short region of high conservation. Other lincRNAs had conserved genomic locations without detectable sequence conservation. Antisense reagents targeting conserved regions of two zebrafish lincRNAs caused developmental defects. Reagents targeting splice sites caused the same defects and were rescued by adding either the mature lincRNA or its human or mouse ortholog. Our study provides a roadmap for identification and analysis of lincRNAs in model organisms and shows that lincRNAs play crucial biological roles during embryonic development with functionality conserved despite limited sequence conservation.
Assuntos
Desenvolvimento Embrionário , Evolução Molecular , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Vertebrados/embriologia , Vertebrados/genéticaRESUMO
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.
Assuntos
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
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
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
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.
Assuntos
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
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.
Assuntos
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
MicroRNAs (miRNAs) are small noncoding RNAs that direct post-transcriptional repression of protein-coding genes. In vertebrates, each highly conserved miRNA typically regulates hundreds of target mRNAs. However, the precise relationship between expression of the miRNAs and that of their targets has remained unclear, in part because of the scarcity of quantitative expression data at cellular resolution. Here we report quantitative analyses of mRNA levels in miRNA-expressing cells of the zebrafish embryo, capturing entire miRNA expression domains, purified to cellular resolution using fluorescent-activated cell sorting (FACS). Focus was on regulation by miR-206 and miR-133 in the developing somites and miR-124 in the developing central nervous system. Comparison of wild-type embryos and those lacking miRNAs revealed predicted targets that responded to the miRNAs and distinguished miRNA-mediated mRNA destabilization from other regulatory effects. For all three miRNAs examined, expression of the miRNAs and that of their predicted targets usually overlapped. A few targets were expressed at higher levels in miRNA-expressing cells than in the rest of the embryo, demonstrating that miRNA-mediated repression can act in opposition to other regulatory processes. However, for most targets expression was lower in miRNA-expressing cells than in the rest of the embryo, indicating that miRNAs usually operate in concert with the other regulatory machinery of the cell.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Sequência Conservada/genética , Ribonuclease III/genética , Peixe-Zebra/genéticaRESUMO
The post-transcriptional fate of messenger RNAs (mRNAs) is largely dictated by their 3' untranslated regions (3' UTRs), which are defined by cleavage and polyadenylation (CPA) of pre-mRNAs. We used poly(A)-position profiling by sequencing (3P-seq) to map poly(A) sites at eight developmental stages and tissues in the zebrafish. Analysis of over 60 million 3P-seq reads substantially increased and improved existing 3' UTR annotations, resulting in confidently identified 3' UTRs for >79% of the annotated protein-coding genes in zebrafish. mRNAs from most zebrafish genes undergo alternative CPA, with those from more than a thousand genes using different dominant 3' UTRs at different stages. These included one of the poly(A) polymerase genes, for which alternative CPA reinforces its repression in the ovary. 3' UTRs tend to be shortest in the ovaries and longest in the brain. Isoforms with some of the shortest 3' UTRs are highly expressed in the ovary, yet absent in the maternally contributed RNAs of the embryo, perhaps because their 3' UTRs are too short to accommodate a uridine-rich motif required for stability of the maternal mRNA. At 2 h post-fertilization, thousands of unique poly(A) sites appear at locations lacking a typical polyadenylation signal, which suggests a wave of widespread cytoplasmic polyadenylation of mRNA degradation intermediates. Our insights into the identities, formation, and evolution of zebrafish 3' UTRs provide a resource for studying gene regulation during vertebrate development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Poliadenilação , Peixe-Zebra/genética , Regiões 3' não Traduzidas , Animais , Evolução Molecular , Feminino , Genômica , Humanos , Anotação de Sequência Molecular , Organogênese/genética , Ovário/embriologia , Ovário/metabolismo , Poli A , Transcrição Gênica , Peixe-Zebra/embriologiaRESUMO
Formation of the vertebrate brain ventricles requires both production of cerebrospinal fluid (CSF), and its retention in the ventricles. The Na,K-ATPase is required for brain ventricle development, and we show here that this protein complex impacts three associated processes. The first requires both the alpha subunit (Atp1a1) and the regulatory subunit, Fxyd1, and leads to formation of a cohesive neuroepithelium, with continuous apical junctions. The second process leads to modulation of neuroepithelial permeability, and requires Atp1a1, which increases permeability with partial loss of function and decreases it with overexpression. In contrast, fxyd1 overexpression does not alter neuroepithelial permeability, suggesting that its activity is limited to neuroepithelium formation. RhoA regulates both neuroepithelium formation and permeability, downstream of the Na,K-ATPase. A third process, likely to be CSF production, is RhoA-independent, requiring Atp1a1, but not Fxyd1. Consistent with a role for Na,K-ATPase pump function, the inhibitor ouabain prevents neuroepithelium formation, while intracellular Na(+) increases after Atp1a1 and Fxyd1 loss of function. These data include the first reported role for Fxyd1 in the developing brain, and indicate that the Na,K-ATPase regulates three aspects of brain ventricle development essential for normal function: formation of a cohesive neuroepithelium, restriction of neuroepithelial permeability, and production of CSF.
Assuntos
Ventrículos Cerebrais/metabolismo , Embrião não Mamífero/metabolismo , Proteínas de Membrana/genética , Fosfoproteínas/genética , ATPase Trocadora de Sódio-Potássio/genética , Proteínas de Peixe-Zebra/genética , Animais , Permeabilidade da Membrana Celular , Ventrículos Cerebrais/efeitos dos fármacos , Ventrículos Cerebrais/embriologia , Líquido Cefalorraquidiano/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/embriologia , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Imuno-Histoquímica , Hibridização In Situ , Proteínas de Membrana/metabolismo , Mutação , Células Neuroepiteliais/metabolismo , Ouabaína/farmacologia , Fosfoproteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
We demonstrate that in the zebrafish hindbrain, cell shape, rhombomere morphogenesis and, unexpectedly, brain ventricle lumen expansion depend on the contractile state of the neuroepithelium. The hindbrain neural tube opens in a specific sequence, with initial separation along the midline at rhombomere boundaries, subsequent openings within rhombomeres and eventual coalescence of openings into the hindbrain ventricle lumen. A mutation in the myosin phosphatase regulator mypt1 results in a small ventricle due to impaired stretching of the surrounding neuroepithelium. Although initial hindbrain opening remains normal, mypt1 mutant rhombomeres do not undergo normal morphological progression. Three-dimensional reconstruction demonstrates cell shapes within rhombomeres and at rhombomere boundaries are abnormal in mypt1 mutants. Wild-type cell shape requires that surrounding cells are also wild type, whereas mutant cell shape is autonomously regulated. Supporting the requirement for regulation of myosin function during hindbrain morphogenesis, wild-type embryos show dynamic levels of phosphorylated myosin regulatory light chain (pMRLC). By contrast, mutants show continuously high pMRLC levels, with concentration of pMRLC and myosin II at the apical side of the epithelium, and myosin II and actin concentration at rhombomere boundaries. Brain ventricle lumen expansion, rhombomere morphology and cell shape are rescued by inhibition of myosin II function, indicating that each defect is a consequence of overactive myosin. We suggest that the epithelium must ;relax', via activity of myosin phosphatase, to allow for normal hindbrain morphogenesis and expansion of the brain ventricular lumen. Epithelial relaxation might be a widespread strategy to facilitate tube inflation in many organs.
Assuntos
Ventrículos Cerebrais/embriologia , Células Epiteliais/fisiologia , Fosfatase de Miosina-de-Cadeia-Leve/fisiologia , Rombencéfalo/embriologia , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Padronização Corporal/fisiologia , Movimento Celular/genética , Movimento Celular/fisiologia , Forma Celular/genética , Ventrículos Cerebrais/metabolismo , Embrião não Mamífero , Células Epiteliais/metabolismo , Modelos Biológicos , Morfogênese/fisiologia , Fosfatase de Miosina-de-Cadeia-Leve/genética , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Rombencéfalo/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/fisiologiaRESUMO
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.
Assuntos
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.
Assuntos
Fertilização in vitro , Fertilização , Animais , Xenopus laevis , Xenopus , LarvaRESUMO
Disc1 is a schizophrenia risk gene that engages multiple signaling pathways during neurogenesis and brain development. Using the zebrafish as a tool, we analyze the function of zebrafish Disc1 (zDisc1) at the earliest stages of brain and body development. We define a "tool" as a biological system that gives insight into mechanisms underlying a human disorder, although the system does not phenocopy the disorder. A zDisc1 peptide binds to GSK3ß, and zDisc1 directs early brain development and neurogenesis, by promoting ß-catenin-mediated Wnt signaling and inhibiting GSK3ß activity. zDisc1 loss-of-function embryos additionally display a convergence and extension phenotype, demonstrated by abnormal movement of dorsolateral cells during gastrulation, through changes in gene expression, and later through formation of abnormal, U-shaped muscle segments, and a truncated tail. These phenotypes are caused by alterations in the noncanonical Wnt pathway, via Daam and Rho signaling. The convergence and extension phenotype can be rescued by a dominant negative GSK3ß construct, suggesting that zDisc1 inhibits GSK3ß activity during noncanonical Wnt signaling. This is the first demonstration that Disc1 modulates the noncanonical Wnt pathway and suggests a previously unconsidered mechanism by which Disc1 may contribute to the etiology of neuropsychiatric disorders.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Via de Sinalização Wnt , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , beta Catenina/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Sítios de Ligação , Encéfalo/embriologia , Encéfalo/metabolismo , Sequência Conservada , Primers do DNA/genética , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Mutagênese , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Neurogênese/genética , Neurogênese/fisiologia , Oligodesoxirribonucleotídeos Antissenso/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Especificidade da Espécie , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genéticaRESUMO
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.
RESUMO
A unique feature of the vertebrate brain is the ventricular system, a series of connected cavities which are filled with cerebrospinal fluid (CSF) and surrounded by neuroepithelium. While CSF is critical for both adult brain function and embryonic brain development, neither development nor function of the brain ventricular system is fully understood. In this review, we discuss the mystery of why vertebrate brains have ventricles, and whence they originate. The brain ventricular system develops from the lumen of the neural tube, as the neuroepithelium undergoes morphogenesis. The molecular mechanisms underlying this ontogeny are described. We discuss possible functions of both adult and embryonic brain ventricles, as well as major brain defects that are associated with CSF and brain ventricular abnormalities. We conclude that vertebrates have taken advantage of their neural tube to form the essential brain ventricular system.
Assuntos
Ventrículos Cerebrais/embriologia , Ventrículos Cerebrais/fisiologia , Animais , Encéfalo/anatomia & histologia , Encéfalo/citologia , Encéfalo/embriologia , Encéfalo/fisiologia , Ventrículos Cerebrais/anatomia & histologia , Ventrículos Cerebrais/citologia , Líquido Cefalorraquidiano/metabolismo , Líquido Cefalorraquidiano/fisiologia , Humanos , Modelos Biológicos , Neurogênese/fisiologiaRESUMO
The biology of schizophrenia is complex with multiple hypotheses (dopamine, glutamate, neurodevelopmental) well supported to underlie the disease. Pathways centered on the risk factor "disrupted in schizophrenia 1" (DISC1) may be able to explain and unite these disparate hypotheses and will be the topic of this mini-symposium preview. Nearly a decade after its original identification at the center of a translocation breakpoint in a large Scottish family that was associated with major psychiatric disease, we are starting to obtain credible insights into its function and role in disease etiology. This preview will highlight a number of exciting areas of current DISC1 research that are revealing roles for DISC1 during normal brain development and also in the disease state. Together these different threads will provide a timely and exciting overview of the DISC1 field and its potential in furthering our understanding of psychiatric diseases and in developing new therapies.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Predisposição Genética para Doença , Transtornos Mentais/genética , Transtornos Mentais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Modelos Animais de Doenças , Humanos , Modelos Biológicos , Proteínas do Tecido Nervoso/genética , Fatores de Risco , Transdução de Sinais/genética , Sinapses/genética , Sinapses/metabolismoRESUMO
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
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.
RESUMO
The zic1 gene plays an important role in early patterning of the Xenopus neurectoderm. While Zic1 does not act as a neural inducer, it synergizes with the neural inducing factor Noggin to activate expression of posterior neural genes, including the midbrain/hindbrain boundary marker engrailed-2. Since the Drosophila homologue of zic1, odd-paired (opa), regulates expression of the wingless and engrailed genes and since Wnt proteins posteriorize neural tissue in Xenopus, we asked whether Xenopus Zic1 acted through the Wnt pathway. Using Wnt signaling inhibitors, we demonstrate that an active Wnt pathway is required for activation of en-2 expression by zic1. Consistent with this result, Zic1 induces expression of several wnt genes, including wnt1, wnt4 and wnt8b. wnt1 gene expression activates expression of engrailed in various organisms, including Xenopus, as demonstrated here. Together, our data suggest that zic1 is an upstream regulator of several wnt genes and that the regulatory relationships between opa, wingless and engrailed seen in Drosophila are also present in vertebrates.