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1.
Dis Model Mech ; 12(9)2019 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-31481433

RESUMO

Diabetes is associated with dysfunction of the neurovascular unit, although the mechanisms of this are incompletely understood and currently no treatment exists to prevent these negative effects. We previously found that the nitric oxide (NO) donor sodium nitroprusside (SNP) prevents the detrimental effect of glucose on neurovascular coupling in zebrafish. We therefore sought to establish the wider effects of glucose exposure on both the neurovascular unit and on behaviour in zebrafish, and the ability of SNP to prevent these. We incubated 4-days post-fertilisation (dpf) zebrafish embryos in 20 mM glucose or mannitol for 5 days until 9 dpf, with or without 0.1 mM SNP co-treatment for 24 h (8-9 dpf), and quantified vascular NO reactivity, vascular mural cell number, expression of a klf2a reporter, glial fibrillary acidic protein (GFAP) and transient receptor potential cation channel subfamily V member 4 (TRPV4), as well as spontaneous neuronal activation at 9 dpf, all in the optic tectum. We also assessed the effect on light/dark preference and locomotory characteristics during free-swimming studies. We find that glucose exposure significantly reduced NO reactivity, klf2a reporter expression, vascular mural cell number and TRPV4 expression, while significantly increasing spontaneous neuronal activation and GFAP expression (all in the optic tectum). Furthermore, when we examined larval behaviour, we found that glucose exposure significantly altered light/dark preference and high and low speed locomotion while in light. Co-treatment with SNP reversed all these molecular and behavioural effects of glucose exposure. Our findings comprehensively describe the negative effects of glucose exposure on the vascular anatomy, molecular phenotype and function of the optic tectum, and on whole-organism behaviour. We also show that SNP or other NO donors may represent a therapeutic strategy to ameliorate the complications of diabetes on the neurovascular unit.This article has an associated First Person interview with the first author of the paper.

2.
EMBO Rep ; 20(8): e47047, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31379129

RESUMO

We identify a novel endothelial membrane behaviour in transgenic zebrafish. Cerebral blood vessels extrude large transient spherical structures that persist for an average of 23 min before regressing into the parent vessel. We term these structures "kugeln", after the German for sphere. Kugeln are only observed arising from the cerebral vessels and are present as late as 28 days post fertilization. Kugeln do not communicate with the vessel lumen and can form in the absence of blood flow. They contain little or no cytoplasm, but the majority are highly positive for nitric oxide reactivity. Kugeln do not interact with brain lymphatic endothelial cells (BLECs) and can form in their absence, nor do they perform a scavenging role or interact with macrophages. Inhibition of actin polymerization, Myosin II, or Notch signalling reduces kugel formation, while inhibition of VEGF or Wnt dysregulation (either inhibition or activation) increases kugel formation. Kugeln represent a novel Notch-dependent NO-containing endothelial organelle restricted to the cerebral vessels, of currently unknown function.

3.
Glia ; 67(7): 1401-1411, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30924555

RESUMO

Cell shape is critical for the proper function of every cell in every tissue in the body. This is especially true for the highly morphologically diverse neural and glia cells of the central nervous system. The molecular processes by which these, or indeed any, cells gain their particular cell-specific morphology remain largely unexplored. To identify the genes involved in the morphogenesis of the principal glial cell type in the vertebrate retina, the Müller glia (MG), we used genomic and CRISPR based strategies in zebrafish (Danio rerio). We identified 41 genes involved in various aspects of MG cell morphogenesis and revealed a striking concordance between the sequential steps of anatomical feature addition and the expression of cohorts of functionally related genes that regulate these steps. We noted that the many of the genes preferentially expressed in zebrafish MG showed conservation in glia across species suggesting evolutionarily conserved glial developmental pathways.


Assuntos
Células Ependimogliais/fisiologia , Perfilação da Expressão Gênica/métodos , Morfogênese/fisiologia , Neurogênese/fisiologia , Neuroglia/fisiologia , Transcriptoma/fisiologia , Animais , Animais Geneticamente Modificados , Diferenciação Celular/fisiologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Peixe-Zebra
4.
Nat Commun ; 10(1): 732, 2019 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-30760708

RESUMO

Angiogenesis requires co-ordination of multiple signalling inputs to regulate the behaviour of endothelial cells (ECs) as they form vascular networks. Vascular endothelial growth factor (VEGF) is essential for angiogenesis and induces downstream signalling pathways including increased cytosolic calcium levels. Here we show that transmembrane protein 33 (tmem33), which has no known function in multicellular organisms, is essential to mediate effects of VEGF in both zebrafish and human ECs. We find that tmem33 localises to the endoplasmic reticulum in zebrafish ECs and is required for cytosolic calcium oscillations in response to Vegfa. tmem33-mediated endothelial calcium oscillations are critical for formation of endothelial tip cell filopodia and EC migration. Global or endothelial-cell-specific knockdown of tmem33 impairs multiple downstream effects of VEGF including ERK phosphorylation, Notch signalling and embryonic vascular development. These studies reveal a hitherto unsuspected role for tmem33 and calcium oscillations in the regulation of vascular development.


Assuntos
Sinalização do Cálcio , Células Endoteliais/metabolismo , Proteínas de Membrana/metabolismo , Neovascularização Fisiológica , Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Vasos Sanguíneos/embriologia , Vasos Sanguíneos/metabolismo , Embrião não Mamífero/irrigação sanguínea , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Retículo Endoplasmático/metabolismo , MAP Quinases Reguladas por Sinal Extracelular , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Membrana/genética , Fosforilação , Peixe-Zebra
5.
Front Cell Dev Biol ; 7: 12, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30805338

RESUMO

The zebrafish has emerged as an exciting vertebrate model to study different aspects of immune system development, particularly due to its transparent embryonic development, the availability of multiple fluorescent reporter lines, efficient genetic tools and live imaging capabilities. However, the study of immunity in zebrafish has largely been limited to early larval stages due to an incomplete knowledge of the full repertoire of immune cells and their specific markers, in particular, a lack of cell surface antibodies to detect and isolate such cells in living tissues. Here we focus on tissue resident or associated immunity beyond development, in the adult zebrafish. It is our view that, with our increasing knowledge and the development of improved tools and protocols, the adult zebrafish will be increasingly appreciated for offering valuable insights into the role of immunity in tissue repair and maintenance, in both health and disease throughout the lifecourse.

6.
J Dev Biol ; 6(1)2018 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-29615555

RESUMO

The zebrafish is an established model to study the development and function of visual neuronal circuits in vivo, largely due to their optical accessibility at embryonic and larval stages. In the past decade multiple experimental paradigms have been developed to study visually-driven behaviours, particularly those regulated by the optic tectum, the main visual centre in lower vertebrates. With few exceptions these techniques are limited to young larvae (7-9 days post-fertilisation, dpf). However, many forms of visually-driven behaviour, such as shoaling, emerge at later developmental stages. Consequently, there is a need for an experimental paradigm to image the visual system in zebrafish larvae beyond 9 dpf. Here, we show that using NBT:GCaMP3 line allows for imaging neuronal activity in the optic tectum in late stage larvae until at least 21 dpf. Utilising this line, we have characterised the receptive field properties of tectal neurons of the 2-3 weeks old fish in the cell bodies and the neuropil. The NBT:GCaMP3 line provides a complementary approach and additional opportunities to study neuronal activity in late stage zebrafish larvae.

7.
Curr Opin Neurobiol ; 47: 31-37, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28850820

RESUMO

Müller Glia (MG), the radial glia cells of the retina, have spectacular morphologies subserving their enormous functional complexity. As early as 1892, the great neuroanatomist Santiago Ramon y Cajal studied the morphological development of MG, defining several steps in their morphogenesis [1,2]. However, the molecular cues controlling these developmental steps remain poorly understood. As MG have roles to play in every cellular and plexiform layer, this review discusses our current understanding on how MG morphology may be linked to their function, including the developmental mechanisms involved in MG patterning and morphogenesis. Uncovering the mechanisms governing glial morphogenesis, using transcriptomics and imaging, may provide shed new light on the pathophysiology and treatment of human neurological disorders.


Assuntos
Células Ependimogliais/citologia , Retina/embriologia , Animais , Diferenciação Celular/fisiologia , Humanos , Morfogênese , Retina/citologia , Retina/crescimento & desenvolvimento
8.
Zebrafish ; 14(3): 284-286, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28027028

RESUMO

Glutamate is the major excitatory neurotransmitter in the brain. Its release and eventual recycling are key to rapid sustained neural activity. We have paired the gfap promoter region with the glutamate reporter molecule, iGluSnFR, to drive expression in glial cells throughout the nervous system. Tg(gfap:iGluSnFR) is expressed on the glial membrane of Müller glia cells in the retina, which rapidly respond to stimulation and the release of extracellular glutamate. As glial cells are associated with most, if not all, synapses, Tg(gfap:iGluSnFR) is a novel and exciting tool to measure neuronal activity and extracellular glutamate dynamics in many regions of the nervous system.


Assuntos
Proteínas de Escherichia coli/metabolismo , Ácido Glutâmico/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Sistema Nervoso/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/metabolismo , Regulação da Expressão Gênica , Sistema Nervoso/citologia , Neuroglia/citologia , Neuroglia/metabolismo , Regiões Promotoras Genéticas , Retina/citologia , Retina/metabolismo , Peixe-Zebra/crescimento & desenvolvimento
9.
PLoS Genet ; 12(4): e1005984, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27070787

RESUMO

The Notch pathway controls proliferation during development and in adulthood, and is frequently affected in many disorders. However, the genetic sensitivity and multi-layered transcriptional properties of the Notch pathway has made its molecular decoding challenging. Here, we address the complexity of Notch signaling with respect to proliferation, using the developing Drosophila CNS as model. We find that a Notch/Su(H)/E(spl)-HLH cascade specifically controls daughter, but not progenitor proliferation. Additionally, we find that different E(spl)-HLH genes are required in different neuroblast lineages. The Notch/Su(H)/E(spl)-HLH cascade alters daughter proliferation by regulating four key cell cycle factors: Cyclin E, String/Cdc25, E2f and Dacapo (mammalian p21CIP1/p27KIP1/p57Kip2). ChIP and DamID analysis of Su(H) and E(spl)-HLH indicates direct transcriptional regulation of the cell cycle genes, and of the Notch pathway itself. These results point to a multi-level signaling model and may help shed light on the dichotomous proliferative role of Notch signaling in many other systems.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sistema Nervoso Central/embriologia , Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Receptores Notch/metabolismo , Proteínas Repressoras/metabolismo , Animais , Sítios de Ligação/genética , Comunicação Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem da Célula , Proliferação de Células/genética , Ciclina E/metabolismo , Drosophila/genética , Drosophila/metabolismo , Fatores de Transcrição E2F/metabolismo , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Nucleares/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais , Fatores de Transcrição , Transcrição Genética/genética , Ativação Transcricional/genética
10.
J Cell Biol ; 210(7): 1075-83, 2015 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-26416961

RESUMO

To investigate the cellular basis of tissue integrity in a vertebrate central nervous system (CNS) tissue, we eliminated Müller glial cells (MG) from the zebrafish retina. For well over a century, glial cells have been ascribed a mechanical role in the support of neural tissues, yet this idea has not been specifically tested in vivo. We report here that retinas devoid of MG rip apart, a defect known as retinoschisis. Using atomic force microscopy, we show that retinas without MG have decreased resistance to tensile stress and are softer than controls. Laser ablation of MG processes showed that these cells are under tension in the tissue. Thus, we propose that MG act like springs that hold the neural retina together, finally confirming an active mechanical role of glial cells in the CNS.


Assuntos
Células Ependimogliais/metabolismo , Retina/metabolismo , Resistência à Tração/fisiologia , Peixe-Zebra/embriologia , Animais , Células Ependimogliais/ultraestrutura , Microscopia de Força Atômica/métodos , Retina/ultraestrutura , Peixe-Zebra/anatomia & histologia
11.
Dev Cell ; 30(2): 192-208, 2014 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-25073156

RESUMO

During central nervous system (CNS) development, progenitors typically divide asymmetrically, renewing themselves while budding off daughter cells with more limited proliferative potential. Variation in daughter cell proliferation has a profound impact on CNS development and evolution, but the underlying mechanisms remain poorly understood. We find that Drosophila embryonic neural progenitors (neuroblasts) undergo a programmed daughter proliferation mode switch, from generating daughters that divide once (type I) to generating neurons directly (type 0). This typeI>0 switch is triggered by activation of Dacapo (mammalian p21(CIP1)/p27(KIP1)/p57(Kip2)) expression in neuroblasts. In the thoracic region, Dacapo expression is activated by the temporal cascade (castor) and the Hox gene Antennapedia. In addition, castor, Antennapedia, and the late temporal gene grainyhead act combinatorially to control the precise timing of neuroblast cell-cycle exit by repressing Cyclin E and E2f. This reveals a logical principle underlying progenitor and daughter cell proliferation control in the Drosophila CNS.


Assuntos
Linhagem da Célula , Proliferação de Células , Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Neurais/citologia , Animais , Proteína do Homeodomínio de Antennapedia/genética , Proteína do Homeodomínio de Antennapedia/metabolismo , Ciclina E/genética , Ciclina E/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Drosophila/embriologia , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Genes de Troca , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/fisiologia , Neurogênese , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Genética
12.
Curr Opin Neurobiol ; 27: 68-74, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24637222

RESUMO

Recent advances suggest that there is a stochastic contribution to the proliferation and fate choice of retinal progenitors. How does this stochasticity fit with the progression of temporal competence and the transcriptional hierarchies that also influence cell division and cell fate in the developing retina? Where may stochasticity arise in the system and how do we make progress in this field when we may never fully explain the behavior of individual progenitor cells?


Assuntos
Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Modelos Neurológicos , Retina/citologia , Processos Estocásticos , Animais , Humanos
13.
Dev Biol ; 381(1): 276-85, 2013 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-23747543

RESUMO

During development of the mouse forebrain interneurons, the Dlx genes play a key role in a gene regulatory network (GRN) that leads to the GABAergic phenotype. Here, we have examined the regulatory relationships between the ascl1a, dlx, and gad1b genes in the zebrafish forebrain. Expression of ascl1a overlaps with dlx1a in the telencephalon and diencephalon during early forebrain development. The loss of Ascl1a function results in a loss of dlx expression, and subsequent losses of dlx5a and gad1b expression in the diencephalic prethalamus and hypothalamus. Loss of Dlx1a and Dlx2a function, and, to a lesser extent, of Dlx5a and Dlx6a, impairs gad1b expression in the prethalamus and hypothalamus. We conclude that dlx1a/2a act downstream of ascl1a but upstream of dlx5a/dlx6a and gad1b to activate GABAergic specification. This pathway is conserved in the diencephalon, but has diverged between mammals and teleosts in the telencephalon.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Glutamato Descarboxilase/fisiologia , Proteínas de Homeodomínio/fisiologia , Fatores de Transcrição/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/fisiologia , Animais , Diencéfalo/metabolismo , Neurônios GABAérgicos/metabolismo , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Hipotálamo/metabolismo , Interneurônios/metabolismo , Mutação , Fenótipo , Telencéfalo/metabolismo
14.
Cell Rep ; 3(2): 282-90, 2013 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-23416047

RESUMO

How synaptic neuropil is formed within the CNS is poorly understood. The retinal inner plexiform layer (IPL) is positioned between the cell bodies of amacrine cells (ACs) and retinal ganglion cells (RGCs). It consists of bipolar cell (BC) axon terminals that synapse on the dendrites of ACs and RGCs intermingled with projections from Müller glia (MG). We examined whether any of these cellular processes are specifically required for the formation of the IPL. Using genetic and pharmacological strategies, we eliminated RGCs, ACs, and MG individually or in combination. Even in the absence of all of these partner cells, an IPL-like neuropil consisting of only BC axon terminals still forms, complete with presynaptic specializations and sublaminar organization. Previous studies have shown that an IPL can form in the complete absence of BCs; therefore, we conclude that neither presynaptic nor postsynaptic processes are individually essential for the formation of this synaptic neuropil.


Assuntos
Neurópilo/citologia , Retina/ultraestrutura , Células Amácrinas/citologia , Células Amácrinas/patologia , Células Amácrinas/ultraestrutura , Animais , Animais Geneticamente Modificados , Embrião não Mamífero/metabolismo , Microscopia de Fluorescência , Microscopia de Vídeo , Neurônios/patologia , Neurópilo/patologia , Terminações Pré-Sinápticas/patologia , Retina/patologia , Células Bipolares da Retina/citologia , Células Bipolares da Retina/patologia , Células Bipolares da Retina/ultraestrutura , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/patologia , Células Ganglionares da Retina/ultraestrutura , Sinapses/patologia , Sinapses/ultraestrutura , Peixe-Zebra/crescimento & desenvolvimento
15.
Dev Dyn ; 239(8): 2298-306, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20658694

RESUMO

The Dlx genes encode a family of transcription factors important for the development of the vertebrate forebrain. These genes have very similar expression domains during the development of the telencephalon in mice and play a role in gamma-aminobutyric acid (GABAergic) interneuron differentiation. We have used triple fluorescent in situ hybridization to study the relative expression domains of the dlx and gad1 genes in the zebrafish telencephalon and diencephalon. We also generated transgenic zebrafish with regulatory elements from the zebrafish dlx1a/2a locus. The zebrafish dlx regulatory elements recapitulated dlx expression in the forebrain and mimicked the relationship between the expression of the dlx genes and gad1. Finally, we show that a putative enhancer located downstream of dlx2b can also activate reporter gene expression in a tissue-specific manner similar to endogenous dlx2b expression. Our results indicate the dlx genes are regulated by an evolutionarily conserved genetic pathway and may play a role in GABAergic interneuron differentiation in the zebrafish forebrain.


Assuntos
Glutamato Descarboxilase/análise , Proteínas de Homeodomínio/análise , Prosencéfalo/metabolismo , Fatores de Transcrição/análise , Animais , Diencéfalo/química , Hibridização in Situ Fluorescente , Interneurônios/citologia , Organismos Geneticamente Modificados , Prosencéfalo/crescimento & desenvolvimento , Telencéfalo/química , Peixe-Zebra , Ácido gama-Aminobutírico
16.
BMC Evol Biol ; 10: 157, 2010 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-20504318

RESUMO

BACKGROUND: The phylogenetic position of the elephant shark (Callorhinchus milii ) is particularly relevant to study the evolution of genes and gene regulation in vertebrates. Here we examine the evolution of Dlx homeobox gene regulation during vertebrate embryonic development with a particular focus on the forebrain. We first identified the elephant shark sequence orthologous to the URE2 cis -regulatory element of the mouse Dlx1/Dlx2 locus (herein named CmURE2). We then conducted a comparative study of the sequence and enhancer activity of CmURE2 with that of orthologous regulatory sequences from zebrafish and mouse. RESULTS: The CmURE2 sequence shows a high percentage of identity with its mouse and zebrafish counterparts but is overall more similar to mouse URE2 (MmURE2) than to zebrafish URE2 (DrURE2). In transgenic zebrafish and mouse embryos, CmURE2 displayed enhancer activity in the forebrain that overlapped with that of DrURE2 and MmURE2. However, we detected notable differences in the activity of the three sequences in the diencephalon. Outside of the forebrain, CmURE2 shows enhancer activity in areas such as the pharyngeal arches and dorsal root ganglia where its' counterparts are also active. CONCLUSIONS: Our transgenic assays show that part of the URE2 enhancer activity is conserved throughout jawed vertebrates but also that new characteristics have evolved in the different groups. Our study demonstrates that the elephant shark is a useful outgroup to study the evolution of regulatory mechanisms in vertebrates and to address how changes in the sequence of cis -regulatory elements translate into changes in their regulatory activity.


Assuntos
Elementos Facilitadores Genéticos , Evolução Molecular , Prosencéfalo/embriologia , Tubarões/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Sequência Conservada/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox , Camundongos , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de DNA , Tubarões/embriologia , Peixe-Zebra/embriologia
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