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1.
Toxicol Appl Pharmacol ; 409: 115285, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33069749

RESUMO

The OX40 receptor plays a crucial co-stimulatory role in T effector cell survival, expansion, cytokine production, and cytotoxicity to tumor cells; therefore, OX40 agonists are being evaluated as anti-cancer immunotherapies, especially in combination with checkpoint inhibitors. To support clinical development of BMS-986178 (an OX40 agonist antibody), two repeat-dose toxicity studies were conducted in cynomolgus monkeys. In the first study, BMS-986178 was administered intravenously (IV) once weekly for one month at doses from 30 to 120 mg/kg. BMS-986178 was well tolerated; surprisingly, immune function was suppressed rather than increased based on pharmacodynamic (PD) and flow cytometry readouts (e.g. T-cell dependent antibody response [TDAR]). To determine whether immune suppression was due to a bi-phasic response, a follow-up study was conducted at lower doses (1 and 10 mg/kg). Although receptor engagement was confirmed, immune function was still suppressed at both doses. In addition, treatment-emergent anti-drug antibodies (ADAs) at 1 mg/kg resulted in hypersensitivity reactions and reduced BMS-986178 exposure after repeated dosing, which precluded a full PD assessment at this dose. In conclusion, BMS-986178 was clinically well-tolerated by monkeys at weekly IV doses from 10 to 120 mg/kg (AUC[0-168] ≤ 712,000 µg●h/mL). However, despite target engagement, PD assays and other immune endpoints demonstrated immune suppression, not stimulation. Due to the inverted immune response at higher doses and the onset of ADAs, additional repeat-dose toxicity studies of BMS-986178 in monkeys (that would typically be required to support Phase 3 clinical trials and registration) would not add value for human safety assessment.


Assuntos
Anticorpos Monoclonais/imunologia , Imunidade/imunologia , Receptores OX40/imunologia , Linfócitos T/imunologia , Animais , Feminino , Seguimentos , Humanos , Imunoterapia/métodos , Macaca fascicularis , Masculino
2.
Chem Res Toxicol ; 33(1): 95-118, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31625720

RESUMO

Unpredicted human safety events in clinical trials for new drugs are costly in terms of human health and money. The drug discovery industry attempts to minimize those events with diligent preclinical safety testing. Current standard practices are good at preventing toxic compounds from being tested in the clinic; however, false negative preclinical toxicity results are still a reality. Continual improvement must be pursued in the preclinical realm. Higher-quality therapies can be brought forward with more information about potential toxicities and associated mechanisms. The zebrafish model is a bridge between in vitro assays and mammalian in vivo studies. This model is powerful in its breadth of application and tractability for research. In the past two decades, our understanding of disease biology and drug toxicity has grown significantly owing to thousands of studies on this tiny vertebrate. This Review summarizes challenges and strengths of the model, discusses the 3Rs value that it can deliver, highlights translatable and untranslatable biology, and brings together reports from recent studies with zebrafish focusing on new drug discovery toxicology.


Assuntos
Descoberta de Drogas , Modelos Animais , Testes de Toxicidade/métodos , Peixe-Zebra , Alternativas ao Uso de Animais , Animais , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Embrião não Mamífero
3.
Development ; 143(14): 2641-50, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27317804

RESUMO

The zebrafish pineal complex consists of four cell types (rod and cone photoreceptors, projection neurons and parapineal neurons) that are derived from a single pineal complex anlage. After specification, parapineal neurons migrate unilaterally away from the rest of the pineal complex whereas rods, cones and projection neurons are non-migratory. The transcription factor Tbx2b is important for both the correct number and migration of parapineal neurons. We find that two additional transcription factors, Flh and Nr2e3, negatively regulate parapineal formation. Flh induces non-migratory neuron fates and limits the extent of parapineal specification, in part by activation of Nr2e3 expression. Tbx2b is positively regulated by Flh, but opposes Flh action during specification of parapineal neurons. Loss of parapineal neuron specification in Tbx2b-deficient embryos can be partially rescued by loss of Nr2e3 or Flh function; however, parapineal migration absolutely requires Tbx2b activity. We conclude that cell specification and migration in the pineal complex are regulated by a network of at least three transcription factors.


Assuntos
Linhagem da Célula/genética , Movimento Celular/genética , Redes Reguladoras de Genes , Glândula Pineal/citologia , Glândula Pineal/embriologia , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Padronização Corporal , Contagem de Células , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Habenula/embriologia , Habenula/metabolismo , Larva/metabolismo , Mosaicismo , Mutação/genética , Neurônios/citologia , Neurônios/metabolismo , Glândula Pineal/inervação , Glândula Pineal/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/citologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Fatores de Transcrição/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
4.
Development ; 141(7): 1572-9, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24598158

RESUMO

Left-right (L/R) asymmetries in the brain are thought to underlie lateralised cognitive functions. Understanding how neuroanatomical asymmetries are established has been achieved through the study of the zebrafish epithalamus. Morphological symmetry in the epithalamus is broken by leftward migration of the parapineal, which is required for the subsequent elaboration of left habenular identity; the habenular nuclei flank the midline and show L/R asymmetries in marker expression and connectivity. The Nodal target pitx2c is expressed in the left epithalamus, but nothing is known about its role during the establishment of asymmetry in the brain. We show that abrogating Pitx2c function leads to the right habenula adopting aspects of left character, and to an increase in parapineal cell numbers. Parapineal ablation in Pitx2c loss of function results in right habenular isomerism, indicating that the parapineal is required for the left character detected in the right habenula in this context. Partial parapineal ablation in the absence of Pitx2c, however, reduces the number of parapineal cells to wild-type levels and restores habenular asymmetry. We provide evidence suggesting that antagonism between Nodal and Pitx2c activities sets an upper limit on parapineal cell numbers. We conclude that restricting parapineal cell number is crucial for the correct elaboration of epithalamic asymmetry.


Assuntos
Padronização Corporal/genética , Habenula/embriologia , Glândula Pineal/embriologia , Fatores de Transcrição/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Contagem de Células , Embrião não Mamífero , Epitálamo/citologia , Epitálamo/embriologia , Habenula/citologia , Proteína Nodal/fisiologia , Tamanho do Órgão/genética , Glândula Pineal/citologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
5.
Development ; 140(2): 323-32, 2013 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-23250206

RESUMO

Left-right (L-R) asymmetries in neuroanatomy exist throughout the animal kingdom, with implications for function and behavior. The molecular mechanisms that control formation of such asymmetries are beginning to be understood. Significant progress has been made by studying the zebrafish parapineal organ, a group of neurons on the left side of the epithalamus. Parapineal cells arise from the medially located pineal complex anlage and migrate to the left side of the brain. We have found that Fgf8a regulates a fate decision among anterior pineal complex progenitors that occurs just prior to the initiation of leftward migration. Cell fate analysis shows that in the absence of Fgf8a a subset of cells in the anterior pineal complex anlage differentiate as cone photoreceptors rather than parapineal neurons. Fgf8a acts permissively to promote parapineal fate in conjunction with the transcription factor Tbx2b, but might also block cone photoreceptor fate. We conclude that this subset of anterior pineal complex precursors, which normally become parapineal cells, are bipotential and require Fgf8a to maintain parapineal identity and/or prevent cone identity.


Assuntos
Encéfalo/embriologia , Encéfalo/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Neurônios/metabolismo , Glândula Pineal/embriologia , Proteínas de Peixe-Zebra/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Inibidores Enzimáticos/farmacologia , Epitálamo/metabolismo , Proteínas de Choque Térmico/metabolismo , Hibridização In Situ , Microscopia de Fluorescência/métodos , Mutação , Transdução de Sinais , Peixe-Zebra
6.
Dev Biol ; 395(1): 144-53, 2014 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-25173875

RESUMO

We describe a method for isolating RNA suitable for high-throughput RNA sequencing (RNA-seq) from small numbers of fluorescently labeled cells isolated from live zebrafish (Danio rerio) embryos without using costly, commercially available columns. This method ensures high cell viability after dissociation and suspension of cells and gives a very high yield of intact RNA. We demonstrate the utility of our new protocol by isolating RNA from fluorescence activated cell sorted (FAC sorted) pineal complex neurons in wild-type and tbx2b knockdown embryos at 24 hours post-fertilization. Tbx2b is a transcription factor required for pineal complex formation. We describe a bioinformatics pipeline used to analyze differential expression following high-throughput sequencing and demonstrate the validity of our results using in situ hybridization of differentially expressed transcripts. This protocol brings modern transcriptome analysis to the study of small cell populations in zebrafish.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Glândula Pineal/metabolismo , Análise de Sequência de RNA/métodos , Transcriptoma/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Hibridização In Situ , Glândula Pineal/citologia , Glândula Pineal/embriologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas com Domínio T/genética , Fatores de Tempo , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genética
7.
Dev Biol ; 385(1): 13-22, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-24184636

RESUMO

In the developing brain, the production of neurons from multipotent precursors must be carefully regulated in order to generate the appropriate numbers of various differentiated neuronal types. Inductive signals from extrinsic elements such as growth factors need to be integrated with timely expression of intrinsic elements such as transcription factors that define the competence of the cell. The transcriptional Mediator complex offers a mechanism to coordinate the timing and levels of intrinsic and extrinsic influences by acting as a rapid molecular switch for transcription of poised RNA pol II. The epithalamus is a highly conserved region of the vertebrate brain that differentiates early and rapidly in the zebrafish. It includes the pineal and parapineal organs and the habenular nuclei. Mutation of the Mediator complex subunit Med12 impairs the specification of habenular and parapineal neurons and causes a loss of differentiation in pineal neurons and photoreceptors. Although FGF ligands and transcription factors for parapineal and photoreceptor development are still expressed in the pineal complex of med12 mutants, FGF signaling is impaired and transcription factor expression is reduced and/or delayed. We find that the timely expression of one of these transcription factors, tbx2b, is controlled by Med12 and is vital for parapineal specification. We propose that the Mediator complex is responsible for subtle but significant changes in transcriptional timing and amplitude that are essential for coordinating the development of neurons in the epithalamus.


Assuntos
Epitálamo/embriologia , Complexo Mediador/metabolismo , Células-Tronco Neurais/metabolismo , Proteínas com Domínio T/biossíntese , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Diferenciação Celular , Epitálamo/anormalidades , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Habenula/anormalidades , Habenula/embriologia , Complexo Mediador/genética , Glândula Pineal/anormalidades , Glândula Pineal/embriologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Transdução de Sinais , Transcrição Gênica , Ativação Transcricional , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética
8.
Dev Biol ; 374(2): 333-44, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-23201575

RESUMO

Differences between the left and right sides of the brain are present in many animal species. For instance, in humans the left cerebral hemisphere is largely responsible for language and tool use and the right for processing spatial information. Zebrafish have prominent left-right asymmetries in their epithalamus that have been associated with differential left and right eye use and navigational behavior. In wild-type (WT) zebrafish embryos, Nodal pathway genes are expressed in the left side of the pineal anlage. Shortly thereafter, a parapineal organ forms to the left of the pineal. The parapineal organ causes differences in gene expression, neuropil density, and connectivity of the left and right habenula nuclei. In embryos that have an open neural tube, such as embryos that are deficient in Nodal signaling or the cell adhesion protein N-cadherin, the left and right sides of the developing epithalamus remain separated from one another. We find that the brains of these embryos often become left isomerized: both sides of the brain develop morphology and gene expression patterns that are characteristic of the left side. However, other aspects of epithalamic development, such as differentiation of specific neuronal cell types, are intact. We propose that there is a mechanism in embryos with closed neural tubes that prevents both sides from developing like the left side. This mechanism fails when the two sides of the epithalamus are widely separated from one another, suggesting that it is dependent upon a signaling protein with limited range.


Assuntos
Epitálamo/fisiologia , Tubo Neural/fisiologia , Proteína Nodal/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Caderinas/genética , Caderinas/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Epitálamo/embriologia , Epitálamo/metabolismo , Lateralidade Funcional/genética , Lateralidade Funcional/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Habenula/embriologia , Habenula/metabolismo , Humanos , Hibridização In Situ , Mutação , Tubo Neural/embriologia , Tubo Neural/metabolismo , Proteína Nodal/genética , Proteína Nodal/metabolismo , Glândula Pineal/embriologia , Glândula Pineal/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
9.
Development ; 137(5): 681-91, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20147373

RESUMO

Brain asymmetries are thought to increase neural processing capacity and to prevent interhemispheric conflict. In order to develop asymmetrically, neurons must be specified along the left-right axis, assigned left-side versus right-side identities and differentiate appropriately. In C. elegans and zebrafish, the cellular and molecular mechanisms that lead to neural asymmetries have recently come to light. Here, we consider recent insights into the mechanisms involved in asymmetrical neural development in these two species. Although the molecular details are divergent, both organisms use iterative cell-cell communication to establish left-right neuronal identity.


Assuntos
Padronização Corporal/fisiologia , Encéfalo/embriologia , Caenorhabditis elegans/embriologia , Peixe-Zebra/embriologia , Animais , Padronização Corporal/genética , Embrião não Mamífero , Lateralidade Funcional/genética , Lateralidade Funcional/fisiologia , Junções Comunicantes/genética , Junções Comunicantes/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Modelos Biológicos
10.
Nat Genet ; 30(1): 117-21, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11753388

RESUMO

The photoneuroendocrine system translates environmental light conditions into the circadian production of endocrine and neuroendocrine signals. Central to this process is the pineal organ, which has a conserved role in the cyclical synthesis and release of melatonin to influence sleep patterns and seasonal reproduction. In lower vertebrates, the pineal organ contains photoreceptors whose activity entrains an endogenous circadian clock and regulates transcription in pinealocytes. In mammals, pineal function is influenced by retinal photoreceptors that project to the suprachiasmatic nucleus-the site of the endogenous circadian clock. A multisynaptic pathway then relays information about circadian rhythmicity and photoperiod to the pineal organ. The gene cone rod homeobox (crx), a member of the orthodenticle homeobox (otx) family, is thought to regulate pineal circadian activity. In the mouse, targeted inactivation of Crx causes a reduction in pineal gene expression and attenuated entrainment to light/dark cycles. Here we show that crx and otx5 orthologs are expressed in both the pineal organ and the asymmetrically positioned parapineal of larval zebrafish. Circadian gene expression is unaffected by a reduction in Crx expression but is inhibited specifically by depletion of Otx5. Our results indicate that Otx5 rather than Crx regulates genes that show circadian expression in the zebrafish pineal complex.


Assuntos
Ritmo Circadiano/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/fisiologia , Glândula Pineal/fisiologia , Proteínas de Peixe-Zebra , Peixe-Zebra/fisiologia , Sequência de Aminoácidos , Animais , Proteínas do Olho/biossíntese , Proteínas do Olho/genética , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Larva , Camundongos , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/farmacologia , Fatores de Transcrição Otx , Células Fotorreceptoras/efeitos da radiação , Glândula Pineal/efeitos da radiação , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , Retina/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Transativadores/biossíntese , Transativadores/genética , Transativadores/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Xenopus laevis/genética , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
11.
J Neurosci ; 31(27): 9869-78, 2011 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-21734278

RESUMO

Studies of the zebrafish epithalamus have provided recent insights into the development of left-right brain asymmetry, which is crucial to normal human brain function. The habenular nuclei of zebrafish are robustly asymmetric, with dense elaboration of neuropil only in the left lateral subnucleus. Because this feature is tightly correlated with asymmetric expression of K(+) channel tetramerization domain-containing proteins 12.1 and 12.2 (Kctd12.1/12.2), we screened for Kctd12.1-interacting proteins to identify molecular mechanisms leading to neuropil asymmetry, and uncovered a novel interaction between Kctd12.1 and Unc-51-like kinase 2 (Ulk2). We show here that knockdown of Ulk2 or overexpression of Kctd12 proteins reduces asymmetric neuropil elaboration. Conversely, overexpression of Ulk2 or mutation of kctd12 genes causes excess neuropil elaboration. We conclude that Ulk2 activity promotes neuropil elaboration while Kctd12 proteins limit Ulk2 activity asymmetrically. This work describes a regulatory mechanism for neuronal process extension that may be conserved in other developmental contexts in addition to the epithalamus.


Assuntos
Lateralidade Funcional/fisiologia , Habenula/citologia , Inibição Psicológica , Neurópilo/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Alquilantes/farmacologia , Animais , Animais Geneticamente Modificados , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Etilnitrosoureia/farmacologia , Lateralidade Funcional/efeitos dos fármacos , Lateralidade Funcional/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Fluorescência Verde/genética , Habenula/efeitos dos fármacos , Habenula/crescimento & desenvolvimento , Habenula/lesões , Imunoprecipitação , Larva , Mutação/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurópilo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/metabolismo , Tubulina (Proteína)/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
12.
Dev Biol ; 360(1): 44-57, 2011 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-21945073

RESUMO

The dorsal habenular nuclei (Dh) of the zebrafish are characterized by significant left-right differences in gene expression, anatomy, and connectivity. Notably, the lateral subnucleus of the Dh (LsDh) is larger on the left side of the brain than on the right, while the medial subnucleus (MsDh) is larger on the right compared to the left. A screen for mutations that affect habenular laterality led to the identification of the sec61a-like 1(sec61al1) gene. In sec61al1(c163) mutants, more neurons in the LsDh and fewer in the MsDh develop on both sides of the brain. Generation of neurons in the LsDh occurs more rapidly and continues for a longer time period in mutants than in WT. Expression of Nodal pathway genes on the left side of the embryos is unaffected in mutants, as is the left sided placement of the parapineal organ, which promotes neurogenesis in the LsDh of WT embryos. Ultrastructural analysis of the epithalamus indicates that ventricular precursor cells, which form an epithelium in WT embryos, lose apical-basal polarity in sec61al1(c163) mutants. Our results show that in the absence of sec61al1, an excess of precursor cells for the LsDh exit the ventricular region and differentiate, resulting in formation of bilaterally symmetric habenular nuclei.


Assuntos
Habenula/embriologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Animais , Sequência de Bases , Padronização Corporal/genética , Padronização Corporal/fisiologia , Primers do DNA/genética , Retículo Endoplasmático/fisiologia , Habenula/citologia , Habenula/fisiologia , Modelos Neurológicos , Mutação , Neurogênese/genética , Neurogênese/fisiologia , Neurônios/citologia , Ligantes da Sinalização Nodal/genética , Ligantes da Sinalização Nodal/fisiologia , Fenótipo , Canais de Translocação SEC , Peixe-Zebra/genética , Peixe-Zebra/fisiologia
13.
Dev Biol ; 358(1): 251-61, 2011 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-21840306

RESUMO

The formation of the embryonic brain requires the production, migration, and differentiation of neurons to be timely and coordinated. Coupling to the photoperiod could synchronize the development of neurons in the embryo. Here, we consider the effect of light and melatonin on the differentiation of embryonic neurons in zebrafish. We examine the formation of neurons in the habenular nuclei, a paired structure found near the dorsal surface of the brain adjacent to the pineal organ. Keeping embryos in constant darkness causes a temporary accumulation of habenular precursor cells, resulting in late differentiation and a long-lasting reduction in neuronal processes (neuropil). Because constant darkness delays the accumulation of the neurendocrine hormone melatonin in embryos, we looked for a link between melatonin signaling and habenular neurogenesis. A pharmacological block of melatonin receptors delays neurogenesis and reduces neuropil similarly to constant darkness, while addition of melatonin to embryos in constant darkness restores timely neurogenesis and neuropil. We conclude that light and melatonin schedule the differentiation of neurons and the formation of neural processes in the habenular nuclei.


Assuntos
Diferenciação Celular/fisiologia , Habenula/citologia , Luz , Melatonina/metabolismo , Neurogênese/fisiologia , Neurônios/fisiologia , Peixe-Zebra/embriologia , Animais , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Regulação da Expressão Gênica/efeitos da radiação , Habenula/fisiologia , Hibridização In Situ , Fotoperíodo , Receptores de Melatonina/genética , Receptores de Melatonina/metabolismo
14.
Proc Natl Acad Sci U S A ; 106(6): 2023-8, 2009 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-19179291

RESUMO

The vertebrate rod and cone photoreceptors are highly specialized sensory neurons that transduce light into the chemical and electrical signals of the nervous system. Although the physiological properties of cones and rods are well known, only a handful of genes have been identified that regulate the specification of photoreceptor subtypes. Taking advantage of the mosaic organization of photoreceptors in zebrafish, we report the isolation of a mutation resulting in a unique change in photoreceptor cell fate. Mutation of the lots-of-rods (lor) locus results in a near one-for-one transformation of UV-cone precursors into rods. The transformed cells exhibit morphological characteristics and a gene-expression pattern typical of rods, but differentiate in a temporal and spatial pattern consistent with UV-cone development. In mutant larvae and adults, the highly ordered photoreceptor mosaic is maintained and degeneration is not observed, suggesting that lor functions after the specification of the other photoreceptor subtypes. In genetic chimeras, lor functions cell-autonomously in the specification of photoreceptor cell fate. Linkage analysis and genetic-complementation testing indicate that lor is an allele of tbx2b/fby (from beyond). fby was identified by a pineal complex phenotype, and carries a nonsense mutation in the T-box domain of the tbx2b transcription factor. Homozygous fby mutant larvae and lor/fby transheterozygotes also display the lots-of-rods phenotype. Based upon these data, we propose a previously undescribed function for tbx2b in photoreceptor cell precursors, to promote the UV cone fate by repressing the rod differentiation pathway.


Assuntos
Diferenciação Celular , Células Fotorreceptoras de Invertebrados/citologia , Retina/crescimento & desenvolvimento , Proteínas com Domínio T/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Códon sem Sentido , Embrião não Mamífero , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Bastonetes/citologia , Proteínas com Domínio T/genética , Raios Ultravioleta , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
15.
Semin Cell Dev Biol ; 20(4): 491-7, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19084075

RESUMO

The human brain exhibits notable asymmetries. Little is known about these symmetry deviations; however scientists are beginning to understand them by employing the lateralized zebrafish epithalamus as a model. The zebrafish epithalamus consists of the pineal and parapineal organs and paired habenular nuclei located bilateral to the pineal complex. While zebrafish pineal and parapineal organs arise from a common population of cells, parapineal cells undergo a separate program that allows them to migrate left of the pineal anlage. Studying the processes that lead to brain laterality in zebrafish will allow a better understanding of how human brain laterality is established.


Assuntos
Encéfalo/embriologia , Epitálamo/embriologia , Lateralidade Funcional , Animais , Desenvolvimento Embrionário , Peixe-Zebra
16.
Trends Neurosci ; 26(6): 308-13, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12798600

RESUMO

How the brain becomes lateralized is poorly understood. By contrast, much is known about molecular cues that specify the left-right axis of the body, fashioning the asymmetric morphology and positioning of the visceral organs. In zebrafish, the Nodal signaling pathway functions in visceral asymmetry and also in the embryonic brain, to bias laterality of the epithalamus. Formation of an asymmetric pineal complex differentially influences adjacent diencephalic nuclei, the left and right habenulae, which acquire distinctive molecular and cellular features. Results from the genetically tractable zebrafish system provide a promising entry point for exploring how left-right biases are established and propagated in the developing vertebrate brain.


Assuntos
Padronização Corporal/genética , Epitálamo/crescimento & desenvolvimento , Lateralidade Funcional/fisiologia , Peixe-Zebra/crescimento & desenvolvimento , Animais , Padronização Corporal/fisiologia , Diencéfalo/crescimento & desenvolvimento , Diencéfalo/metabolismo , Epitálamo/metabolismo , Lateralidade Funcional/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Aprendizagem/fisiologia , Proteína Nodal , Organogênese/genética , Organogênese/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/genética , Peixe-Zebra/genética
17.
Zebrafish ; 13(5): 377-8, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27618129

RESUMO

For more than 60 years, zebrafish have been used in toxicological studies. Due to their transparency, genetic tractability, and compatibility with high-throughput screens, zebrafish embryos are uniquely suited to study the effects of pharmaceuticals and environmental insults on embryonic development, organ formation and function, and reproductive success. This special issue of Zebrafish highlights the ways zebrafish are used to investigate the toxic effects of endocrine disruptors, pesticides, and heavy metals.


Assuntos
Toxicologia , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/metabolismo , Animais , Modelos Animais , Peixe-Zebra/embriologia
18.
PLoS One ; 10(5): e0125980, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25950913

RESUMO

Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease such as simple steatosis, nonalcoholic steatohepatitis (NASH), cirrhosis and fibrosis. However, the molecular pathogenesis and genetic variations causing NAFLD are poorly understood. The high prevalence and incidence of NAFLD suggests that genetic variations on a large number of genes might be involved in NAFLD. To identify genetic variants causing inherited liver disease, we used zebrafish as a model system for a large-scale mutant screen, and adopted a whole genome sequencing approach for rapid identification of mutated genes found in our screen. Here, we report on a forward genetic screen of ENU mutagenized zebrafish. From 250 F2 lines of ENU mutagenized zebrafish during post-developmental stages (5 to 8 days post fertilization), we identified 19 unique mutant zebrafish lines displaying visual evidence of hepatomegaly and/or steatosis with no developmental defects. Histological analysis of mutants revealed several specific phenotypes, including common steatosis, micro/macrovesicular steatosis, hepatomegaly, ballooning, and acute hepatocellular necrosis. This work has identified multiple post-developmental mutants and establishes zebrafish as a novel animal model for post-developmental inherited liver disease.


Assuntos
Testes Genéticos/métodos , Mutação , Hepatopatia Gordurosa não Alcoólica/patologia , Peixe-Zebra/crescimento & desenvolvimento , Animais , Modelos Animais de Doenças , Etilnitrosoureia/toxicidade , Predisposição Genética para Doença , Humanos , Fígado/patologia , Hepatopatia Gordurosa não Alcoólica/genética , Peixe-Zebra/genética
19.
PLoS One ; 9(10): e110280, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25329151

RESUMO

The habenular nuclei of the limbic system regulate responses, such as anxiety, to aversive stimuli in the environment. The habenulae receive inputs from the telencephalon via elaborate dendrites that form in the center of the nuclei. The kinase Ulk2 positively regulates dendritogenesis on habenular neurons, and in turn is negatively regulated by the cytoplasmic protein Kctd12. Given that the habenulae are a nexus in the aversive response circuit, we suspected that incomplete habenular dendritogenesis would have profound implications for behavior. We find that Ulk2, which interacts with Kctd12 proteins via a small proline-serine rich domain, promotes branching and elaboration of dendrites. Loss of Kctd12 results in increased branching/elaboration and decreased anxiety. We conclude that fine-tuning of habenular dendritogenesis during development is essential for appropriate behavioral responses to negative stimuli.


Assuntos
Comportamento Animal , Proteínas de Transporte de Cátions/metabolismo , Dendritos/metabolismo , Habenula/citologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Ansiedade/metabolismo , Ansiedade/patologia , Meio Ambiente , Técnicas de Silenciamento de Genes , Habenula/crescimento & desenvolvimento , Proteínas Serina-Treonina Quinases , Estrutura Terciária de Proteína , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética
20.
Neural Dev ; 9: 20, 2014 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-25212830

RESUMO

BACKGROUND: The conserved habenular nuclei function as a relay system connecting the forebrain with the brain stem. They play crucial roles in various cognitive behaviors by modulating cholinergic, dopaminergic and serotonergic activities. Despite the renewed interest in this conserved forebrain region because of its importance in regulating aversion and reward behaviors, the formation of the habenular nuclei during embryogenesis is poorly understood due to their small size and deep location in the brain, as well as the lack of known markers for habenular progenitors. In zebrafish, the bilateral habenular nuclei are subdivided into dorsal and ventral compartments, are particularly large and found on the dorsal surface of the brain, which facilitates the study of their development. RESULTS: Here we examine the expression of a homeodomain transcription factor, dbx1b, and its potential to serve as an early molecular marker of dorsal habenular progenitors. Detailed spatiotemporal expression profiles demonstrate that the expression domain of dbx1b correlates with the presumptive habenular region, and dbx1b-expressing cells are proliferative along the ventricle. A lineage-tracing experiment using the Cre-lox system confirms that all or almost all dorsal habenular neurons are derived from dbx1b-expressing cells. In addition, mutant analysis and pharmacological treatments demonstrate that both initiation and maintenance of dbx1b expression requires precise regulation by fibroblast growth factor (FGF) signaling. CONCLUSIONS: We provide clear evidence in support of dbx1b marking the progenitor populations that give rise to the dorsal habenulae. In addition, the expression of dbx1b in the dorsal diencephalon is tightly controlled by FGF signaling.


Assuntos
Habenula/embriologia , Habenula/metabolismo , Proteínas de Homeodomínio/metabolismo , Neurônios/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Fatores de Crescimento de Fibroblastos/metabolismo , Peixe-Zebra
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