RESUMEN
Thousands of human disease-associated single nucleotide polymorphisms (SNPs) lie in the non-coding genome, but only a handful have been demonstrated to affect gene expression and human biology. We computationally identified risk-associated SNPs in deeply conserved non-exonic elements (CNEs) potentially contributing to 45 human diseases. We further demonstrated that human CNE1/rs17421627 associated with retinal vasculature defects showed transcriptional activity in the zebrafish retina, while introducing the risk-associated allele completely abolished CNE1 enhancer activity. Furthermore, deletion of CNE1 led to retinal vasculature defects and to a specific downregulation of microRNA-9, rather than MEF2C as predicted by the original genome-wide association studies. Consistent with these results, miR-9 depletion affects retinal vasculature formation, demonstrating MIR-9-2 as a critical gene underpinning the associated trait. Importantly, we validated that other CNEs act as transcriptional enhancers that can be disrupted by conserved non-coding SNPs. This study uncovers disease-associated non-coding mutations that are deeply conserved, providing a path for in vivo testing to reveal their cis-regulated genes and biological roles.
Asunto(s)
Elementos de Facilitación Genéticos/genética , MicroARNs/genética , Vasculitis Retiniana/genética , Alelos , Animales , Secuencia Conservada/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Estudio de Asociación del Genoma Completo , Humanos , Factores de Transcripción MEF2/genética , Mutación , Polimorfismo de Nucleótido Simple/genética , Retina/metabolismo , Retina/patología , Vasculitis Retiniana/patología , Pez Cebra/genéticaRESUMEN
In the developing brain, neurons expressing VEGF-A and blood vessels grow in close apposition, but many of the molecular pathways regulating neuronal VEGF-A and neurovascular system development remain to be deciphered. Here, we show that miR-9 links neurogenesis and angiogenesis through the formation of neurons expressing VEGF-A. We found that miR-9 directly targets the transcription factors TLX and ONECUTs to regulate VEGF-A expression. miR-9 inhibition leads to increased TLX and ONECUT expression, resulting in VEGF-A overexpression. This untimely increase of neuronal VEGF-A signal leads to the thickening of blood vessels at the expense of the normal formation of the neurovascular network in the brain and retina. Thus, this conserved transcriptional cascade is critical for proper brain development in vertebrates. Because of this dual role on neural stem cell proliferation and angiogenesis, miR-9 and its downstream targets are promising factors for cellular regenerative therapy following stroke and for brain tumor treatment.
Asunto(s)
Corteza Cerebral/metabolismo , MicroARNs/genética , Neovascularización Fisiológica/genética , Células-Madre Neurales/metabolismo , Neurogénesis/genética , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Secuencia de Bases , Sitios de Unión , Diferenciación Celular , Proliferación Celular , Corteza Cerebral/crecimiento & desarrollo , Embrión no Mamífero , Feto , Regulación del Desarrollo de la Expresión Génica , Factor Nuclear 6 del Hepatocito/genética , Factor Nuclear 6 del Hepatocito/metabolismo , Humanos , MicroARNs/metabolismo , Morfogénesis/genética , Células-Madre Neurales/citología , Neuronas/metabolismo , Neuronas/patología , Receptores Nucleares Huérfanos , Cultivo Primario de Células , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Retina/crecimiento & desarrollo , Retina/metabolismo , Transducción de Señal , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Pez CebraRESUMEN
Using genome-wide transcriptional profiling and whole-mount expression analyses of zebrafish larvae, we have identified hyaluronan synthase 3 (has3) as an upregulated gene during caudal fin regeneration. has3 expression is induced in the wound epithelium within hours after tail amputation, and its onset and maintenance requires fibroblast growth factor, phosphoinositide 3-kinase, and transforming growth factor-ß signaling. Inhibition of hyaluronic acid (HA) synthesis by the small molecule 4-methylumbelliferone (4-MU) impairs tail regeneration in zebrafish larvae by preventing injury-induced cell proliferation. In addition, 4-MU reduces the expression of genes associated with wound epithelium and blastema function. Treatment with glycogen synthase kinase 3 inhibitors rescues 4-MU-induced defects in cell proliferation and tail regeneration, while restoring a subset of wound epithelium and blastema markers. Our findings demonstrate a role for HA biosynthesis in zebrafish tail regeneration and delineate its epistatic relationships with other regenerative processes.
Asunto(s)
Aletas de Animales/fisiología , Glucuronosiltransferasa/fisiología , Ácido Hialurónico/fisiología , Regeneración/genética , Proteínas de Pez Cebra/fisiología , Pez Cebra/fisiología , Animales , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Epistasis Genética , Regulación de la Expresión Génica/efectos de los fármacos , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Hialuronano Sintasas , Ácido Hialurónico/biosíntesis , Himecromona/farmacología , Regeneración/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Cicatrización de Heridas/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
RFamide neuropeptide VF (NPVF) is expressed by neurons in the hypothalamus and has been implicated in nociception, but the circuit mechanisms remain unexplored. Here, we studied the structural and functional connections from NPVF neurons to downstream targets in the context of nociception, using novel transgenic lines, optogenetics, and calcium imaging in behaving larval zebrafish. We found a specific projection from NPVF neurons to serotonergic neurons in the ventral raphe nucleus (vRN). We showed NPVF neurons and vRN are suppressed and excited by noxious stimuli, respectively. We combined optogenetics with calcium imaging and pharmacology to demonstrate that stimulation of NPVF cells suppresses neuronal activity in vRN. During noxious stimuli, serotonergic neurons activation was due to a suppression of an inhibitory NPVF-ventral raphe peptidergic projection. This study reveals a novel NPVF-vRN functional circuit modulated by noxious stimuli in vertebrates.
Asunto(s)
Hipotálamo/metabolismo , Neuropéptidos/metabolismo , Nocicepción , Núcleos del Rafe/metabolismo , Pez Cebra/metabolismo , Secuencia de Aminoácidos , Animales , Neuronas/metabolismo , Neuropéptidos/química , Serotonina/metabolismoRESUMEN
The habenulae are highly conserved nuclei in the dorsal diencephalon that connect the forebrain to the midbrain and hindbrain. These nuclei have been implicated in a broad variety of behaviours in humans, primates, rodents and zebrafish. Despite this, the molecular mechanisms that control the genesis and differentiation of neural progenitors in the habenulae remain relatively unknown. We have previously shown that, in zebrafish, the timing of habenular neurogenesis is left-right asymmetric and that in the absence of Nodal signalling this asymmetry is lost. Here, we show that habenular neurogenesis requires the homeobox transcription factor Pax6a and the redundant action of two proneural bHLH factors, Neurog1 and Neurod4. We present evidence that Hedgehog signalling is required for the expression of pax6a, which is in turn necessary for the expression of neurog1 and neurod4. Finally, we demonstrate by pharmacological inhibition that Hedgehog signalling is required continuously during habenular neurogenesis and by cell transplantation experiments that pathway activation is required cell autonomously. Our data sheds light on the mechanism underlying habenular development that may provide insights into how Nodal signalling imposes asymmetry on the timing of habenular neurogenesis.
Asunto(s)
Habénula/embriología , Proteínas Hedgehog/fisiología , Neurogénesis , Factor de Transcripción PAX6/fisiología , Transducción de Señal , Proteínas de Pez Cebra/fisiología , Pez Cebra/embriología , Animales , Animales Modificados Genéticamente , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Tipificación del Cuerpo , Regulación del Desarrollo de la Expresión Génica , Genotipo , Heterocigoto , Mutación , Proteínas del Tejido Nervioso/fisiología , Neuronas/metabolismo , Reacción en Cadena de la Polimerasa , Factores de Transcripción/metabolismoRESUMEN
A variety of signaling pathways have been shown to regulate specification of neuronal subtype identity. However, the mechanisms by which future neurons simultaneously process information from multiple pathways to establish their identity remain poorly understood. The zebrafish pineal gland offers a simple system with which to address questions concerning the integration of signaling pathways during neural specification as it contains only two types of neurons - photoreceptors and projection neurons. We have previously shown that Notch signaling inhibits the projection neuron fate. Here, we show that BMP signaling is both necessary and sufficient to promote the photoreceptor fate. We also demonstrate that crosstalk between BMP and Notch signaling is required for the inhibition of a projection neuron fate in future photoreceptors. In this case, BMP signaling is required as a competence factor for the efficient activation of Notch targets. Our results indicate that both the induction of a photoreceptor fate and the interaction with Notch relies on a canonical BMP/Smad5 pathway. However, the activation of Notch-dependent transcription does not require a canonical Smad5-DNA interaction. Our results provide new insights into how multiple signaling influences are integrated during cell fate specification in the vertebrate CNS.