RESUMO
Basolateral amygdala (BLA) projects widely across the macaque frontal cortex, and amygdalo-frontal projections are critical for appropriate emotional responding and decision making. While it is appreciated that single BLA neurons branch and project to multiple areas in frontal cortex, the organization and frequency of this branching has yet to be fully characterized. Here, we determined the projection patterns of more than 3,000 macaque BLA neurons. We found that one-third of BLA neurons had two or more distinct projection targets in frontal cortex and subcortical structures. The patterns of single BLA neuron projections to multiple areas were organized into repeating motifs that targeted distinct sets of areas in medial and ventral frontal cortex, indicative of separable BLA networks. Our findings begin to reveal the rich structure of single-neuron connections in the non-human primate brain, providing a neuroanatomical basis for the role of BLA in coordinating brain-wide responses to valent stimuli.
Assuntos
Complexo Nuclear Basolateral da Amígdala , Animais , Complexo Nuclear Basolateral da Amígdala/fisiologia , Macaca , Vias Neurais/fisiologia , Lobo Frontal , Neurônios/fisiologia , Córtex Pré-Frontal/fisiologiaRESUMO
Neurons in the cortex are heterogenous, sending diverse axonal projections to multiple brain regions. Unraveling the logic of these projections requires single-neuron resolution. Although a growing number of techniques have enabled high-throughput reconstruction, these techniques are typically limited to dozens or at most hundreds of neurons per brain, requiring that statistical analyses combine data from different specimens. Here we present axonal BARseq, a high-throughput approach based on reading out nucleic acid barcodes using in situ RNA sequencing, which enables analysis of even densely labeled neurons. As a proof of principle, we have mapped the long-range projections of >8000 mouse primary auditory cortex neurons from a single brain. We identified major cell types based on projection targets and axonal trajectory. The large sample size enabled us to systematically quantify the projections of intratelencephalic (IT) neurons, and revealed that individual IT neurons project to different layers in an area-dependent fashion. Axonal BARseq is a powerful technique for studying the heterogeneity of single neuronal projections at high throughput within individual brains.
RESUMO
The basolateral amygdala (BLA) projects widely across the macaque frontal cortex1-4, and amygdalo-frontal projections are critical for optimal emotional responding5 and decision-making6. Yet, little is known about the single-neuron architecture of these projections: namely, whether single BLA neurons project to multiple parts of the frontal cortex. Here, we use MAPseq7 to determine the projection patterns of over 3000 macaque BLA neurons. We found that one-third of BLA neurons have two or more distinct targets in parts of frontal cortex and of subcortical structures. Further, we reveal non-random structure within these branching patterns such that neurons with four targets are more frequently observed than those with two or three, indicative of widespread networks. Consequently, these multi-target single neurons form distinct networks within medial and ventral frontal cortex consistent with their known functions in regulating mood and decision-making. Additionally, we show that branching patterns of single neurons shape functional networks in the brain as assessed by fMRI-based functional connectivity. These results provide a neuroanatomical basis for the role of the BLA in coordinating brain-wide responses to valent stimuli8 and highlight the importance of high-resolution neuroanatomical data for understanding functional networks in the brain.
RESUMO
Forelimb-related areas of the motor cortex communicate directly to downstream areas in the brainstem and spinal cord via axons that project to and through the pyramidal tract (PT). To better understand the diversity of the brainstem branching patterns of these pyramidal tract projections, we used MAPseq, a molecular barcode technique for population-scale sampling with single-axon resolution. In experiments using mice of both sexes, we first confirmed prior results demonstrating the basic efficacy of axonal barcode identification of primary motor cortex (M1) PT-type axons, including corticobulbar (CBULB) and corticospinal (CSPI) subclasses. We then used multiplexed MAPseq to analyze projections from M1 and M2 (caudal and rostral forelimb areas). The four basic axon subclasses comprising these projections (M1-CSPI, M1-CBULB, M2-CSPI, M2-CBULB) showed a complex mix of differences and similarities in their brainstem projection profiles. This included relatively abundant branching by all classes in the dorsal midbrain, by M2 subclasses in the pons, and by CSPI subclasses in the dorsal medulla. Cluster analysis showed graded distributions of the basic subclasses within the PT class. Clusters were of diversely mixed subclass composition and showed distinct rostrocaudal and/or dorsomedial projection biases. Exemplifying these patterns was a subcluster likely enriched in corticocuneate branches. Overall, the results indicate high yet systematic PT axon diversity at the level of brainstem branching patterns; projections of M1 and M2 appear qualitatively similar, yet with quantitative differences in subclasses and clusters.SIGNIFICANCE STATEMENT Axons of the PT class of cortical projection neurons, which includes corticospinal and corticobulbar neurons, anatomically link motor cortex to brainstem and spinal cord circuits. Both of these subclasses can form branches to brainstem destinations along the way, but the extent and diversity of these branching patterns is incompletely understood. Here, we used MAPseq to tag PT axons with individual molecular barcodes for high-throughput quantification of branching patterns across the brainstem. The results reveal diverse, complex, yet systematic branching patterns of corticospinal and corticobulbar neurons arising from two motor cortex areas, M1 and M2.
Assuntos
Córtex Motor , Tratos Piramidais , Feminino , Masculino , Camundongos , Animais , Tratos Piramidais/fisiologia , Axônios/fisiologia , Membro Anterior , Córtex Motor/fisiologia , Extremidade SuperiorRESUMO
In most sensory modalities, neuronal connectivity reflects behaviorally relevant stimulus features, such as spatial location, orientation, and sound frequency. By contrast, the prevailing view in the olfactory cortex, based on the reconstruction of dozens of neurons, is that connectivity is random. Here, we used high-throughput sequencing-based neuroanatomical techniques to analyze the projections of 5,309 mouse olfactory bulb and 30,433 piriform cortex output neurons at single-cell resolution. Surprisingly, statistical analysis of this much larger dataset revealed that the olfactory cortex connectivity is spatially structured. Single olfactory bulb neurons targeting a particular location along the anterior-posterior axis of piriform cortex also project to matched, functionally distinct, extra-piriform targets. Moreover, single neurons from the targeted piriform locus also project to the same matched extra-piriform targets, forming triadic circuit motifs. Thus, as in other sensory modalities, olfactory information is routed at early stages of processing to functionally diverse targets in a coordinated manner.
Assuntos
Córtex Olfatório , Condutos Olfatórios , Camundongos , Animais , Bulbo Olfatório , Neurônios/fisiologia , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
Functional circuits consist of neurons with diverse axonal projections and gene expression. Understanding the molecular signature of projections requires high-throughput interrogation of both gene expression and projections to multiple targets in the same cells at cellular resolution, which is difficult to achieve using current technology. Here, we introduce BARseq2, a technique that simultaneously maps projections and detects multiplexed gene expression by in situ sequencing. We determined the expression of cadherins and cell-type markers in 29,933 cells and the projections of 3,164 cells in both the mouse motor cortex and auditory cortex. Associating gene expression and projections in 1,349 neurons revealed shared cadherin signatures of homologous projections across the two cortical areas. These cadherins were enriched across multiple branches of the transcriptomic taxonomy. By correlating multigene expression and projections to many targets in single neurons with high throughput, BARseq2 provides a potential path to uncovering the molecular logic underlying neuronal circuits.
Assuntos
Córtex Auditivo/metabolismo , Mapeamento Encefálico/métodos , Processamento Eletrônico de Dados/métodos , Redes Reguladoras de Genes/genética , Córtex Motor/metabolismo , Animais , Córtex Auditivo/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Córtex Motor/química , Vias Neurais/química , Vias Neurais/metabolismoRESUMO
Which neural circuits undergo synaptic changes when an animal learns? Although it is widely accepted that changes in synaptic strength underlie many forms of learning and memory, it remains challenging to connect changes in synaptic strength at specific neural pathways to specific behaviors and memories. Here we introduce SYNPLA (synaptic proximity ligation assay), a synapse-specific, high-throughput, and potentially brain-wide method capable of detecting circuit-specific learning-induced synaptic plasticity.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Aprendizagem/fisiologia , Plasticidade Neuronal/fisiologia , Mapeamento de Interação de Proteínas/métodos , Sinapses , Animais , Córtex Auditivo/química , Córtex Auditivo/citologia , Córtex Auditivo/metabolismo , Células Cultivadas , Condicionamento Psicológico/fisiologia , Corpos Geniculados/química , Corpos Geniculados/citologia , Corpos Geniculados/metabolismo , Hipocampo/química , Hipocampo/citologia , Hipocampo/metabolismo , Camundongos , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Ratos , Sinapses/química , Sinapses/metabolismoRESUMO
Understanding neural circuits requires deciphering interactions among myriad cell types defined by spatial organization, connectivity, gene expression, and other properties. Resolving these cell types requires both single-neuron resolution and high throughput, a challenging combination with conventional methods. Here, we introduce barcoded anatomy resolved by sequencing (BARseq), a multiplexed method based on RNA barcoding for mapping projections of thousands of spatially resolved neurons in a single brain and relating those projections to other properties such as gene or Cre expression. Mapping the projections to 11 areas of 3,579 neurons in mouse auditory cortex using BARseq confirmed the laminar organization of the three top classes (intratelencephalic [IT], pyramidal tract-like [PT-like], and corticothalamic [CT]) of projection neurons. In depth analysis uncovered a projection type restricted almost exclusively to transcriptionally defined subtypes of IT neurons. By bridging anatomical and transcriptomic approaches at cellular resolution with high throughput, BARseq can potentially uncover the organizing principles underlying the structure and formation of neural circuits.
Assuntos
Córtex Auditivo/metabolismo , Rede Nervosa/metabolismo , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Mapeamento Encefálico , Humanos , Integrases/genética , Camundongos , Neuritos/metabolismo , Células Piramidais/metabolismo , Tratos Piramidais/metabolismoRESUMO
Myc is a pleiotropic transcription factor that is involved in many cellular activities relevant to carcinogenesis, including hepatocarcinogenesis. The zebrafish has been increasingly used to model human diseases and it is particularly valuable in helping to identify common and conserved molecular mechanisms in vertebrates. Here we generated a liver tumor model in transgenic zebrafish by liver-specific expression of mouse Myc using a Tet-On system. Dosage-dependent induction of Myc expression specifically in the liver was observed in our Myc transgenic zebrafish, TO(Myc), and the elevated Myc expression caused liver hyperplasia, which progressed to hepatocellular adenoma and carcinoma with prolonged induction. Next generation sequencing-based transcriptomic analyses indicated that ribosome proteins were overwhelmingly upregulated in the Myc-induced liver tumors. Cross-species analyses showed that the zebrafish Myc model correlated well with Myc transgenic mouse models for liver cancers. The Myc-induced zebrafish liver tumors also possessed molecular signatures highly similar to human those of hepatocellular carcinoma. Finally, we found that a small Myc target gene set of 16 genes could be used to identify liver tumors due to Myc upregulation. Thus, our zebrafish model demonstrated the conserved role of Myc in promoting hepatocarcinogenesis in all vertebrate species.
Assuntos
Perfilação da Expressão Gênica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Mamíferos/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , Camundongos , Camundongos Transgênicos , Estadiamento de Neoplasias , Proteínas Proto-Oncogênicas c-myc/genética , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Tetraciclina/farmacologiaRESUMO
Connectivity determines the function of neural circuits. Historically, circuit mapping has usually been viewed as a problem of microscopy, but no current method can achieve high-throughput mapping of entire circuits with single neuron precision. Here we describe a novel approach to determining connectivity. We propose BOINC ("barcoding of individual neuronal connections"), a method for converting the problem of connectivity into a form that can be read out by high-throughput DNA sequencing. The appeal of using sequencing is that its scale--sequencing billions of nucleotides per day is now routine--is a natural match to the complexity of neural circuits. An inexpensive high-throughput technique for establishing circuit connectivity at single neuron resolution could transform neuroscience research.
Assuntos
Conectoma , Animais , Mapeamento Encefálico/métodos , Humanos , Vias Neurais/fisiologia , Neurônios/fisiologia , Análise de Sequência de DNA/métodosRESUMO
BACKGROUND & AIMS: Liver cancer, mainly hepatocellular carcinoma, is a major malignancy and currently there are no effective treatment protocols due to insufficient understanding of hepatocarcinogenesis. As a potentially high-throughput and cost-effective experimental model, the zebrafish is increasingly recognized for disease studies. Here, we aim at using the zebrafish to generate a convenient hepatocellular carcinoma model. METHODS: Using the Tet-on system for liver-specific expression of fish oncogene xmrk, a hyperactive version of epidermal growth factor receptor homolog, we have generated transgenic zebrafish with inducible development of liver cancer. RESULTS: Liver tumors were rapidly induced with 100% penetrance in both juvenile and adult xmrk transgenic fish. Histological examination indicated that they all showed features of hepatocellular carcinoma. The induced liver tumors regressed rapidly upon inducer withdrawal. During the tumor induction stage, we detected increased cell proliferation and activation of Xmrk downstream targets Erk and Stat5, which were important for liver tumorigenesis as proved by inhibition experiments. When tumors regressed, there were decreased phosphorylated Erk and Stat5 accompanied with an increase in apoptosis. CONCLUSIONS: Our zebrafish model demonstrates the potential of a hyperactivated epidermal growth factor receptor pathway in initiating heptocarcinogenesis. It provides clear evidence for the requirement of only a single oncogene for HCC initiation and maintenance and is thus a convenient model for further investigation of oncogene addiction and future anti-cancer drug screening.
Assuntos
Proteínas de Peixes/genética , Neoplasias Hepáticas Experimentais/genética , Oncogenes , Receptores Proteína Tirosina Quinases/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Doxiciclina/administração & dosagem , Feminino , Expressão Gênica , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Sistema de Sinalização das MAP Quinases , Masculino , Oncogenes/efeitos dos fármacos , Proteínas Recombinantes/genética , Fator de Transcrição STAT5/metabolismoRESUMO
Although the underlying molecular mechanism of hepatocellular carcinoma remains unclear, signalling pathways essential in cell survival and growth are altered, including the Raf-MEK-MAPK pathway. This pathway can be activated by hepatitis B or C virus infections and the ectopic expression of the Raf-1 oncogene is frequently seen in hepatocellular carcinomas. In addition, the Raf-MEK-MAPK pathway was also shown to be deregulated in zebrafish liver tumours. Based on the genetic conservation between zebrafish and human liver tumours, the zebrafish was used as an animal model to better understand the molecular basis of hepatocellular carcinoma. Here we establish an inducible oncogenic zebrafish cell model, in which oncogenic human Raf-1(ΔRaf1) can be post-transcriptionally activated in zebrafish liver cells by administration of 4-hydroxytamoxifen (4HT). The ΔRaf1 activation resulted in the hyperactivation of the zebrafish MEK-ERK cascade, promoted cell growth and proliferation, and inhibited apoptosis. The mitogenic transformation of the ZFL-ΔRaf1-ER cells was confirmed by in vivo allo-transplantation and in silico microarray analyses. Gene expression profiling of cells treated with 4HT and a MEK-inhibitor identified a Raf-MEK-dependent signature set. This transcriptome response was compared to zebrafish and human liver cancer transcriptomes. We identified, and validated by quantitative PCR, a set of genes transcriptionally regulated by hyperactive MAPK signalling in ZFL-ΔRaf1-ER cells, zebrafish liver tumours and human liver tumours, suggesting that the in vitro zebrafish liver cell model can be used for further study of the molecular basis of human hepatocellular carcinoma. The molecular targeting of the commonly regulated hepatocellular carcinoma genes using the ZFL-ΔRaf1-ER cell model can be applied for high-throughput preclinical target discovery.
Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas Experimentais/genética , 9,10-Dimetil-1,2-benzantraceno , Animais , Carcinógenos , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Transplante de Neoplasias , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Transplante Heterólogo , Células Tumorais Cultivadas , Peixe-ZebraRESUMO
BACKGROUND: DNA methylation is an epigenetic mechanism associated with regulation of gene expression and it is modulated during chemical carcinogenesis. The zebrafish is increasingly employed as a human disease model; however there is a lack of information on DNA methylation in zebrafish and during fish tumorigenesis. RESULTS: A novel CpG island tiling array containing 44,000 probes, in combination with immunoprecipitation of methylated DNA, was used to achieve the first comprehensive methylation profiling of normal adult zebrafish liver. DNA methylation alterations were detected in zebrafish liver tumors induced by the environmental carcinogen 7, 12-dimethylbenz(a)anthracene. Genes significantly hypomethylated in tumors were associated particularly with proliferation, glycolysis, transcription, cell cycle, apoptosis, growth and metastasis. Hypermethylated genes included those associated with anti-angiogenesis and cellular adhesion. Of 49 genes that were altered in expression within tumors, and which also had appropriate CpG islands and were co-represented on the tiling array, approximately 45% showed significant changes in both gene expression and methylation. CONCLUSION: The functional pathways containing differentially methylated genes in zebrafish hepatocellular carcinoma have also been reported to be aberrantly methylated during tumorigenesis in humans. These findings increase the confidence in the use of zebrafish as a model for human cancer in addition to providing the first comprehensive mapping of DNA methylation in the normal adult zebrafish liver.
Assuntos
Carcinoma Hepatocelular/induzido quimicamente , Metilação de DNA , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/genética , Fígado/fisiologia , Regiões Promotoras Genéticas , Peixe-Zebra/genética , 9,10-Dimetil-1,2-benzantraceno , Animais , Carcinógenos , Ilhas de CpG , Modelos Animais de Doenças , Expressão Gênica , Regulação Neoplásica da Expressão GênicaRESUMO
Walleye dermal sarcoma (WDS) is a benign tumor of walleye fish that develops and completely regresses seasonally. The retrovirus associated with this disease, walleye dermal sarcoma virus, encodes three accessory genes, two of which, rv-cyclin (orfA) and orfb, are thought to play a role in tumor development. In this study, we attempted to recapitulate WDS development by expressing rv-cyclin in chimeric and stable transgenic zebrafish. Six stable transgenic lines expressing rv-cyclin from the constitutive CMVtk promoter were generated. Immunohistochemistry and quantitative reverse transcriptase polymerase chain reaction demonstrate that rv-cyclin is widely expressed in different tissues in these fish. These lines were viable and histologically normal for up to 2 years. No increase in tumors or tissue proliferation was observed following N-ethyl N-nitrosourea exposure or following tail wounding and subsequent tissue regeneration compared to controls. These data indicate that rv-cyclin is not independently sufficient for tumor induction in zebrafish.
Assuntos
Animais Geneticamente Modificados/metabolismo , Epsilonretrovirus/genética , Doenças dos Peixes/metabolismo , Sarcoma/veterinária , Neoplasias Cutâneas/veterinária , Peixe-Zebra/genética , Animais , Proliferação de Células , Doenças dos Peixes/patologia , Doenças dos Peixes/virologia , Regulação Viral da Expressão Gênica , Técnicas de Transferência de Genes , Genes Virais , Regeneração/genética , Sarcoma/metabolismo , Sarcoma/patologia , Sarcoma/virologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/virologia , Cauda/lesões , Cauda/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Peixe-Zebra/metabolismoRESUMO
A retrovirus homologue gene of cellular cyclin D1, walleye dermal sarcoma virus rv-cyclin gene (orf A or rv-cyclin), was expressed in the livers of zebrafish under the control of liver fatty acid-binding protein (lfabp) promoter. To prevent possible fatality caused by overexpression of the oncogene, the GAL4/upstream activation sequence (GAL4/UAS) system was used to maintain the transgenic lines. Thus, both GAL4-activator [Tg(lfabp:GAL4)] and UAS-effector [Tg(UAS:rvcyclin)] lines were generated, and the rv-cyclin gene was activated in the liver after crossing these two lines. Since no obvious neoplasia phenotypes were observed in the double-transgenic line, cancer susceptibility of the transgenic fish expressing rv-cyclin was tested by carcinogen treatment. Unexpectedly, transgenic fish expressing rv-cyclin gene (rvcyclin+) were more resistant to the carcinogen than siblings not expressing this gene (rvcyclin-). Lower incidences of multiple and malignant liver tumors were observed in rvcyclin+ than in rvcyclin- fish, and the liver tumors in the rvcyclin+ group appeared later and were less malignant. These results suggest that expression of rv-cyclin protects the fish liver from carcinogen damage and delays onset of malignancy. These findings indicate that transgenic fish models are powerful systems for investigating mechanisms of inhibition and regression of liver tumors.
Assuntos
Animais Geneticamente Modificados/genética , Epsilonretrovirus/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas Experimentais/genética , Peixe-Zebra/genética , Adenoma de Células Hepáticas/genética , Adenoma de Células Hepáticas/metabolismo , Adenoma de Células Hepáticas/patologia , Animais , Animais Geneticamente Modificados/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Genes Supressores de Tumor , Genes Virais , Fígado/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Peixe-Zebra/metabolismoRESUMO
A stable Tg(UAS:GFP) zebrafish line was generated and crossed with Tg(hsp70:GAL4) line, in which the GAL4 gene is under the control of an inducible zebrafish promoter derived from the heat shock 70 protein gene (hsp70). The dynamic green fluorescent protein (GFP) expression in early zebrafish embryos in the GAL4/UAS binary system was then investigated. We found that, at early developmental stages, expression of GFP effector gene was restricted and required a long recovery time to reach a detectable level. At later developmental stage (after 2 days postfertilization), GFP could be activated in multiple tissues in a shorter time, apparently due to a higher level of GAL4 messenger RNA induction. It appears that the type of tissues expressing GFP was dependent on whether they had been developed at the time of heat shock. Therefore, the delayed and restricted transgene expression should be taken into consideration when GAL4/UAS system is used to study transgene expression in early developmental stages.
Assuntos
Animais Geneticamente Modificados/fisiologia , Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Fluorescência Verde/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Fatores de Transcrição/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/fisiologia , Animais , Ativação Transcricional/genéticaRESUMO
Although the transgenic technology has been successfully used to generate fluorescent zebrafish and medaka for ornamental purposes, the practicability of the technology has not been demonstrated in other ornamental fish species. In the present study, we have tested the transgenic technology in a bona fide ornamental fish species, the white skirt tetra (Gymnocorymbus ternetzi). First, its embryonic development was briefly described. Second, we successfully introduced an rfp (red fluorescent protein) gene construct driven by a strong muscle-specific mylz2 promoter from the zebrafish into the white skirt tetra and demonstrated muscle-specific expression of the RFP reporter protein. Importantly, the vivid red fluorescent color was prominently visible in adult transgenic founders under the normal daylight, like the currently marketed red fluorescent transgenic zebrafish. Thus, our current study demonstrated the feasibility of using the well-characterized zebrafish mylz2 promoters to produce useful fluorescent ornamental fish in other fish species by the transgenic technology.
Assuntos
Peixes/genética , Peixes/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Células Musculares/metabolismo , Animais , Animais Geneticamente Modificados , Miosinas Cardíacas/genética , Feminino , Peixes/embriologia , Masculino , Cadeias Leves de Miosina/genética , Regiões Promotoras Genéticas , Proteína Vermelha FluorescenteRESUMO
The zebrafish (Danio rerio) has been long advocated as a model for cancer research, but little is known about the real molecular similarities between zebrafish and human tumors. Comparative analysis of microarray data from zebrafish liver tumors with those from four human tumor types revealed molecular conservation at various levels between fish and human tumors. This approach provides a useful strategy for identifying an expression signature that is strongly associated with a disease phenotype.