RESUMO
Inherited retinal dystrophies (IRDs) are progressive diseases leading to vision loss. Mutation in the eyes shut homolog (EYS) gene is one of the most frequent causes of IRD. However, the mechanism of photoreceptor cell degeneration by mutant EYS has not been fully elucidated. Here, we generated retinal organoids from induced pluripotent stem cells (iPSCs) derived from patients with EYS-associated retinal dystrophy (EYS-RD). In photoreceptor cells of RD organoids, both EYS and G protein-coupled receptor kinase 7 (GRK7), one of the proteins handling phototoxicity, were not in the outer segment, where they are physiologically present. Furthermore, photoreceptor cells in RD organoids were vulnerable to light stimuli, and especially to blue light. Mislocalization of GRK7, which was also observed in eys-knockout zebrafish, was reversed by delivering control EYS into photoreceptor cells of RD organoids. These findings suggest that avoiding phototoxicity would be a potential therapeutic approach for EYS-RD.
Assuntos
Células-Tronco Pluripotentes Induzidas , Organoides , Distrofias Retinianas , Peixe-Zebra , Animais , Humanos , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Luz/efeitos adversos , Mutação , Organoides/metabolismo , Retina/metabolismo , Retina/patologia , Distrofias Retinianas/terapia , Distrofias Retinianas/genética , Distrofias Retinianas/metabolismoRESUMO
Spinal motor neurons (SMNs) are the primary target of degeneration in amyotrophic lateral sclerosis (ALS). Degenerating motor neurons accumulate cytoplasmic TAR DNA-binding protein 43 (TDP-43) aggregates in most ALS cases. This SMN pathology can occur without mutation in the coding sequence of the TDP-43-encoding gene, TARDBP. Whether and how wild-type TDP-43 drives pathological changes in SMNs in vivo remains largely unexplored. In this study, we develop a two-photon calcium imaging setup in which tactile-evoked neural responses of motor neurons in the brainstem and spinal cord can be monitored using the calcium indicator GCaMP. We devise a piezo-assisted tactile stimulator that reproducibly evokes a brainstem descending neuron upon tactile stimulation of the head. A direct comparison between caudal primary motor neurons (CaPs) with or without TDP-43 overexpression in contiguous spinal segments demonstrates that CaPs overexpressing TDP-43 display attenuated Ca2+ transients during fictive escape locomotion evoked by the tactile stimulation. These results show that excessive amounts of TDP-43 protein reduce the neuronal excitability of SMNs and potentially contribute to asymptomatic pathological lesions of SMNs and movement disorders in patients with ALS.
Assuntos
Esclerose Lateral Amiotrófica , Animais , Humanos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Peixe-Zebra/metabolismo , Cálcio/metabolismo , Proteostase , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Medula Espinal , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismoRESUMO
TAR DNA-binding protein 43 (TDP-43) is an evolutionarily conserved RNA/DNA-binding protein that is nuclear-enriched in healthy cells, but deposited in the cytoplasm as aggregates in affected neurons in certain neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). We have previously developed an optogenetic TDP-43 variant (opTDP-43h) whose oligomerization status can be modulated via the CRY2olig tag, which self-assembles upon absorption of blue light. Illumination of zebrafish spinal motor neurons expressing opTDP-43h with a blue light triggers its cytoplasmic mislocalization, eventually leading to cytoplasmic deposition of opTDP-43h aggregates. Intriguingly, a light illumination-dependent transient opTDP-43 mislocalization can halt motor axon outgrowth, even in the absence of cytoplasmic deposition of opTDP-43 aggregates. These observations point toward an oligomerization-dependent, but aggregation-independent, cytotoxic effect of TDP-43 that might contribute to pathogenesis of ALS. In the present review, we would like to overview the zebrafish ALS model based on the optogenetic TDP-43, and then discuss about the potential mechanisms of TDP-43 cytotoxicity that trigger and/or promote motor neuron degeneration in ALS.
Assuntos
Esclerose Lateral Amiotrófica , Animais , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Peixe-Zebra/metabolismo , Optogenética , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismoRESUMO
Abnormal protein aggregation and selective neuronal vulnerability are two major hallmarks of neurodegenerative diseases. Causal relationships between these features may be interrogated by controlling the phase transition of a disease-associated protein in a vulnerable cell type, although this experimental approach has been limited so far. Here, we describe a protocol to induce phase transition of the RNA/DNA-binding protein TDP-43 in spinal motor neurons of zebrafish larvae for modeling cytoplasmic aggregation of TDP-43 occurring in degenerating motor neurons in amyotrophic lateral sclerosis (ALS). We describe a bacterial artificial chromosome (BAC)-based genetic method to deliver an optogenetic TDP-43 variant selectively to spinal motor neurons of zebrafish. The high translucency of zebrafish larvae allows for the phase transition of the optogenetic TDP-43 in the spinal motor neurons by a simple external illumination using a light-emitting diode (LED) against unrestrained fish. We also present a basic workflow of live imaging of the zebrafish spinal motor neurons and image analysis with freely available Fiji/ImageJ software to characterize responses of the optogenetic TDP-43 to the light illumination. This protocol enables the characterization of TDP-43 phase transition and aggregate formation in an ALS-vulnerable cellular environment, which should facilitate an investigation of its cellular and behavioral consequences.
Assuntos
Esclerose Lateral Amiotrófica , Proteínas de Ligação a DNA , Proteínas de Peixe-Zebra , Peixe-Zebra , Esclerose Lateral Amiotrófica/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Larva/metabolismo , Neurônios Motores/metabolismo , Optogenética , Medula Espinal/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by progressive degeneration of motor neurons in the brain and spinal cord. Spinal motor neurons align along the spinal cord length within the vertebral column, and extend long axons to connect with skeletal muscles covering the body surface. Due to this anatomy, spinal motor neurons are among the most difficult cells to observe in vivo. Larval zebrafish have transparent bodies that allow non-invasive visualization of whole cells of single spinal motor neurons, from somas to the neuromuscular synapses. This unique feature, combined with its amenability to genome editing, pharmacology, and optogenetics, enables functional analyses of ALS-associated proteins in the spinal motor neurons in vivo with subcellular resolution. Here, we review the zebrafish skeletal neuromuscular system and the optical methods used to study it. We then introduce a recently developed optogenetic zebrafish ALS model that uses light illumination to control oligomerization, phase transition and aggregation of the ALS-associated DNA/RNA-binding protein called TDP-43. Finally, we will discuss how this disease-in-a-fish ALS model can help solve key questions about ALS pathogenesis and lead to new ALS therapeutics.
RESUMO
Transactive response DNA-binding protein 43 kDa (TDP-43) encoded by the TARDBP gene is an evolutionarily conserved heterogeneous nuclear ribonucleoprotein (hnRNP) that regulates multiple steps of RNA metabolism, and its cytoplasmic aggregation characterizes degenerating motor neurons in amyotrophic lateral sclerosis (ALS). In most ALS cases, cytoplasmic TDP-43 aggregation occurs in the absence of mutations in the coding sequence of TARDBP. Thus, a major challenge in ALS research is to understand the nature of pathological changes occurring in wild-type TDP-43 and to explore upstream events in intracellular and extracellular milieu that promote the pathological transition of TDP-43. Despite the inherent obstacles to analyzing TDP-43 dynamics in in vivo motor neurons due to their anatomical complexity and inaccessibility, recent studies using cellular and animal models have provided important mechanistic insights into potential links between TDP-43 and motor neuron vulnerability in ALS. This review is intended to provide an overview of the current literature on the function and regulation of TDP-43-containing RNP granules or membraneless organelles, as revealed by various models, and to discuss the potential mechanisms by which TDP-43 can cause selective vulnerability of motor neurons in ALS.
Assuntos
Esclerose Lateral Amiotrófica/complicações , Proteínas de Ligação a DNA/metabolismo , Doença dos Neurônios Motores/patologia , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Proteínas de Ligação a DNA/química , Humanos , Doença dos Neurônios Motores/etiologia , Doença dos Neurônios Motores/metabolismoRESUMO
Neurons in the inferior olivary nuclei (IO neurons) send climbing fibers to Purkinje cells to elicit functions of the cerebellum. IO neurons and Purkinje cells are derived from neural progenitors expressing the proneural gene ptf1a In this study, we found that the homeobox gene gsx2 was co-expressed with ptf1a in IO progenitors in zebrafish. Both gsx2 and ptf1a zebrafish mutants showed a strong reduction or loss of IO neurons. The expression of ptf1a was not affected in gsx2 mutants, and vice versa. In IO progenitors, the ptf1a mutation increased apoptosis whereas the gsx2 mutation did not, suggesting that ptf1a and gsx2 are regulated independently of each other and have distinct roles. The fibroblast growth factors (Fgf) 3 and 8a, and retinoic acid signals negatively and positively, respectively, regulated gsx2 expression and thereby the development of IO neurons. mafba and Hox genes are at least partly involved in the Fgf- and retinoic acid-dependent regulation of IO neuronal development. Our results indicate that gsx2 mediates the rostro-caudal positional signals to specify the identity of IO neurons from ptf1a-expressing neural progenitors.
Assuntos
Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Proteínas de Peixe-Zebra/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Neurogênese/genética , Neurogênese/fisiologia , Neurônios/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Cytoplasmic aggregation of TDP-43 characterizes degenerating neurons in most cases of amyotrophic lateral sclerosis (ALS). Here, we develop an optogenetic TDP-43 variant (opTDP-43), whose multimerization status can be modulated in vivo through external light illumination. Using the translucent zebrafish neuromuscular system, we demonstrate that short-term light stimulation reversibly induces cytoplasmic opTDP-43 mislocalization, but not aggregation, in the spinal motor neuron, leading to an axon outgrowth defect associated with myofiber denervation. In contrast, opTDP-43 forms pathological aggregates in the cytoplasm after longer-term illumination and seeds non-optogenetic TDP-43 aggregation. Furthermore, we find that an ALS-linked mutation in the intrinsically disordered region (IDR) exacerbates the light-dependent opTDP-43 toxicity on locomotor behavior. Together, our results propose that IDR-mediated TDP-43 oligomerization triggers both acute and long-term pathologies of motor neurons, which may be relevant to the pathogenesis and progression of ALS.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Medula Espinal/metabolismo , Medula Espinal/patologia , Esclerose Lateral Amiotrófica/genética , Animais , Animais Geneticamente Modificados , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Moleculares , Mutação , Optogenética , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Multimerização Proteica , Estabilidade Proteica , Regulação para Cima , Peixe-ZebraRESUMO
Cereblon (CRBN) is a primary target of thalidomide and mediates its multiple pharmacological activities, including teratogenic and antimyeloma activities. CRBN functions as a substrate receptor of the E3 ubiquitin ligase CRL4, whose substrate specificity is modulated by thalidomide and its analogs. Although a number of CRL4CRBN substrates have recently been identified, the substrate involved in thalidomide teratogenicity is unclear. Here we show that p63 isoforms are thalidomide-dependent CRL4CRBN neosubstrates that are responsible, at least in part, for its teratogenic effects. The p53 family member p63 is associated with multiple developmental processes. ∆Np63α is essential for limb development, while TAp63α is important for cochlea development and hearing. Using a zebrafish model, we demonstrate that thalidomide exerts its teratogenic effects on pectoral fins and otic vesicles by inducing the degradation of ∆Np63α and TAp63α, respectively. These results may contribute to the invention of new thalidomide analogs lacking teratogenic activity.
Assuntos
Proteínas de Membrana/metabolismo , Teratogênicos/toxicidade , Talidomida/toxicidade , Células HEK293 , Humanos , Especificidade por SubstratoRESUMO
Cranial motor nuclei in the brainstem innervate diverse types of head and neck muscles. Failure in establishing these neuromuscular connections causes congenital cranial dysinnervation disorders (CCDDs) characterized by abnormal craniofacial movements. However, mechanisms that link cranial motor nuclei to target muscles are poorly understood at the molecular level. Here, we report that protocadherin-mediated repulsion mediates neuromuscular connection in the ocular motor system in zebrafish. We identify pools of abducens motor neurons that are topographically arranged according to soma size and convergently innervate a single muscle. Disruptions of Duane retraction syndrome-associated transcription factors reveal that these neurons require Mafba/MAFB, but not Sall4/SALL4, for differentiation. Furthermore, genetic perturbations of Pcdh17/protocadherin-17 result in defective axon growth and soma clumping, thereby abolishing neuromuscular connectivity. Our results suggest that protocadherin-mediated repulsion forms the central topography and efferent projection pattern of the abducens nucleus following Mafba-dependent specification and imply potential involvement of protocadherins in CCDD etiology.
Assuntos
Núcleo do Nervo Abducente/fisiopatologia , Tronco Encefálico/metabolismo , Animais , Humanos , Peixe-ZebraRESUMO
BACKGROUND: The most frequent genetic cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) is the expansion of a GGGGCC hexanucleotide repeat in a non-coding region of the chromosome 9 open reading frame 72 (C9orf72) locus. The pathological hallmarks observed in C9orf72 repeat expansion carriers are the formation of RNA foci and deposition of dipeptide repeat (DPR) proteins derived from repeat associated non-ATG (RAN) translation. Currently, it is unclear whether formation of RNA foci, DPR translation products, or partial loss of C9orf72 predominantly drive neurotoxicity in vivo. By using a transgenic approach in zebrafish we address if the most frequently found DPR in human ALS/FTLD brain, the poly-Gly-Ala (poly-GA) protein, is toxic in vivo. METHOD: We generated several transgenic UAS responder lines that express either 80 repeats of GGGGCC alone, or together with a translation initiation ATG codon forcing the translation of GA80-GFP protein upon crossing to a Gal4 driver. The GGGGCC repeat and GA80 were fused to green fluorescent protein (GFP) lacking a start codon to monitor protein translation by GFP fluorescence. RESULTS: Zebrafish transgenic for the GGGGCC repeat lacking an ATG codon showed very mild toxicity in the absence of poly-GA. However, strong toxicity was induced upon ATG initiated expression of poly-GA, which was rescued by injection of an antisense morpholino interfering with start codon dependent poly-GA translation. This morpholino only interferes with GA80-GFP translation without affecting repeat transcription, indicating that the toxicity is derived from GA80-GFP. CONCLUSION: These novel transgenic C9orf72 associated repeat zebrafish models demonstrate poly-GA toxicity in zebrafish. Reduction of poly-GA protein rescues toxicity validating this therapeutic approach to treat C9orf72 repeat expansion carriers. These novel animal models provide a valuable tool for drug discovery to reduce DPR associated toxicity in ALS/FTLD patients with C9orf72 repeat expansions.
Assuntos
Esclerose Lateral Amiotrófica/genética , Degeneração Lobar Frontotemporal/genética , Fases de Leitura Aberta , Peptídeos/toxicidade , Esclerose Lateral Amiotrófica/patologia , Animais , Animais Geneticamente Modificados , Western Blotting , Repetições de Dinucleotídeos , Modelos Animais de Doenças , Degeneração Lobar Frontotemporal/patologia , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Reação em Cadeia da Polimerase , Polímeros , Peixe-ZebraRESUMO
Neuregulin1 (NRG1) plays diverse developmental roles and is likely involved in several neurological disorders including schizophrenia. The transmembrane NRG1 protein is proteolytically cleaved and released as a soluble ligand for ErbB receptors. Such post-translational processing, referred to as 'ectodomain shedding', is thought to be crucial for NRG1 function. However, little is known regarding the regulatory mechanism of NRG1 cleavage in vivo. Here, we developed a fluorescent probe, NRG1 Cleavage Indicating SenSOR (N-CISSOR), by fusing mCherry and GFP to the extracellular and intracellular domains of NRG1, respectively. N-CISSOR mimicked the subcellular localization and biochemical properties of NRG1 including cleavage dynamics and ErbB phosphorylation in cultured cells. mCherry/GFP ratio imaging of phorbol-12-myristate-13-acetate-stimulated N-CISSOR-expressing HEK293T cells enabled to monitor rapid ectodomain shedding of NRG1 at the subcellular level. Utilizing N-CISSOR in zebrafish embryos revealed preferential axonal NRG1 ectodomain shedding in developing motor neurons, demonstrating that NRG1 ectodomain shedding is spatially regulated at the subcellular level. Thus, N-CISSOR will be a valuable tool for elucidating the spatiotemporal regulation of NRG1 ectodomain shedding, both in vitro and in vivo.
Assuntos
Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/metabolismo , Neuregulina-1/metabolismo , Processamento de Proteína Pós-Traducional , Imagem com Lapso de Tempo/métodos , Animais , Animais Geneticamente Modificados , Sítios de Ligação/genética , Linhagem Celular , Embrião não Mamífero/metabolismo , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Camundongos , Microscopia de Fluorescência , Neuregulina-1/genética , Ésteres de Forbol/farmacologia , Proteólise/efeitos dos fármacos , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteína Vermelha FluorescenteRESUMO
Following their synthesis in the endoplasmic reticulum (ER), voltage-gated sodium channels (NaV) are transported to the membranes of excitable cells, where they often cluster, such as at the axon initial segment of neurons. Although the mechanisms by which NaV channels form and maintain clusters have been extensively examined, the processes that govern their transport and degradation have received less attention. Our entry into the study of these processes began with the isolation of a new allele of the zebrafish mutant alligator, which we found to be caused by mutations in the gene encoding really interesting new gene (RING) finger protein 121 (RNF121), an E3-ubiquitin ligase present in the ER and cis-Golgi compartments. Here we demonstrate that RNF121 facilitates two opposing fates of NaV channels: (i) ubiquitin-mediated proteasome degradation and (ii) membrane localization when coexpressed with auxiliary NaVß subunits. Collectively, these results indicate that RNF121 participates in the quality control of NaV channels during their synthesis and subsequent transport to the membrane.
Assuntos
Proteólise , Domínios RING Finger , Ubiquitina-Proteína Ligases/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Sequência de Bases , Membrana Celular/genética , Membrana Celular/metabolismo , Dados de Sequência Molecular , Mutação , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte Proteico/fisiologia , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Canais de Sódio Disparados por Voltagem/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
The cerebellum is involved in some forms of motor coordination and motor learning. Here we isolated transgenic (Tg) zebrafish lines that express a modified version of Gal4-VP16 (GFF) in the cerebellar neural circuits: granule, Purkinje, or eurydendroid cells, Bergmann glia, or the neurons in the inferior olive nuclei (IO) which send climbing fibers to Purkinje cells, with the transposon Tol2 system. By combining GFF lines with Tg lines carrying a reporter gene located downstream of Gal4 binding sequences (upstream activating sequence: UAS), we investigated the anatomy and developmental processes of the cerebellar neural circuitry. Combining an IO-specific Gal4 line with a UAS reporter line expressing the photoconvertible fluorescent protein Kaede demonstrated the contralateral projections of climbing fibers. Combining a granule cell-specific Gal4 line with a UAS reporter line expressing wheat germ agglutinin (WGA) confirmed direct and/or indirect connections of granule cells with Purkinje cells, eurydendroid cells, and IO neurons in zebrafish. Time-lapse analysis of a granule cell-specific Gal4 line revealed initial random movements and ventral migration of granule cell nuclei. Transgenesis of a reporter gene with another transposon Tol1 system visualized neuronal structure at a single cell resolution. Our findings indicate the usefulness of these zebrafish Gal4 Tg lines for studying the development and function of cerebellar neural circuits.
Assuntos
Cerebelo/embriologia , Proteínas de Ligação a DNA/genética , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Movimento Celular , Cerebelo/fisiologia , Elementos de DNA Transponíveis , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Técnicas Genéticas , Proteínas de Fluorescência Verde/metabolismo , Vias Neurais , Neurônios/fisiologia , Células de Purkinje/citologia , Sinapses , Transgenes , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
Blood vessels and neurons grow often side by side. However, the molecular and cellular mechanisms underlying their parallel development remain unclear. Here, we report that a subpopulation of secondary motoneurons extends axons ventrally outside of the neural tubes and rostrocaudally as a fascicle beneath the dorsal aorta (DA) in zebrafish. We tried to clarify the mechanism by which these motoneuron axons grow beneath the DA and found that Vegfc in the DA and Vegfr3 in the motoneurons were essential for the axon growth. Forced expression of either Vegfc in arteries or Vegfr3 in motoneurons resulted in enhanced axon growth of motoneurons over the DA. Both vegfr3 morphants and vegfc morphants lost the alignment of motoneuron axons with DA. In addition, forced expression of two mutant forms of Vegfr3 in motoneurons, potentially trapping endogenous Vegfc, resulted in failure of growth of motoneuron axons beneath the DA. Finally, a vegfr3 mutant fish lacked the motoneuron axons beneath the DA. Collectively, Vegfc from the preformed DA guides the axon growth of secondary motoneurons.
Assuntos
Aorta/citologia , Aorta/metabolismo , Axônios/metabolismo , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fator C de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Correct organ size must involve a balance between promotion and inhibition of cell proliferation. A mathematical model has been proposed in which an organ is assumed to produce its own growth activator as well as a growth inhibitor [1], but there is as yet no molecular evidence to support this model [2]. The mechanosensory organs of the fish lateral line system (neuromasts) are composed of a core of sensory hair cells surrounded by nonsensory support cells. Sensory cells are constantly replaced and are regenerated from surrounding nonsensory cells [3], while each organ retains the same size throughout life. Moreover, neuromasts also bud off new neuromasts, which stop growing when they reach the same size [4, 5]. Here, we show that the size of neuromasts is controlled by a balance between growth-promoting Wnt signaling activity in proliferation-competent cells and Wnt-inhibiting Dkk activity produced by differentiated sensory cells. This negative feedback loop from Dkk (secreted by differentiated cells) on Wnt-dependent cell proliferation (in surrounding cells) also acts during regeneration to achieve size constancy. This study establishes Wnt/Dkk as a novel mechanism to determine the final size of an organ.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/genética , Sistema da Linha Lateral/crescimento & desenvolvimento , Via de Sinalização Wnt , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/fisiologia , Diferenciação Celular , Proliferação de Células , Retroalimentação , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Sistema da Linha Lateral/anatomia & histologia , Tamanho do Órgão , Reação em Cadeia da Polimerase , Regeneração , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/metabolismoRESUMO
Bacterial artificial chromosome (BAC) transgenesis and gene/enhancer trapping are effective approaches for identification of genetically defined neuronal populations in the central nervous system (CNS). Here, we applied these techniques to zebrafish (Danio rerio) in order to obtain insights into the cellular architecture of the axial motor column in vertebrates. First, by using the BAC for the Mnx class homeodomain protein gene mnr2b/mnx2b, we established the mnGFF7 transgenic line expressing the Gal4FF transcriptional activator in a large part of the motor column. Single cell labeling of Gal4FF-expressing cells in the mnGFF7 line enabled a detailed investigation of the morphological characteristics of individual spinal motoneurons, as well as the overall organization of the motor column in a spinal segment. Secondly, from a large-scale gene trap screen, we identified transgenic lines that marked discrete subpopulations of spinal motoneurons with Gal4FF. Molecular characterization of these lines led to the identification of the ADAMTS3 gene, which encodes an evolutionarily conserved ADAMTS family of peptidases and is dynamically expressed in the ventral spinal cord. The transgenic fish established here, along with the identified gene, should facilitate an understanding of the cellular and molecular architecture of the spinal cord motor column and its connection to muscles in vertebrates.
Assuntos
Proteínas ADAM/biossíntese , Cromossomos Artificiais Bacterianos/metabolismo , Técnicas de Transferência de Genes , Neurônios Motores/metabolismo , Pró-Colágeno N-Endopeptidase/biossíntese , Medula Espinal/citologia , Medula Espinal/metabolismo , Proteínas ADAMTS , Animais , Animais Geneticamente Modificados , Cromossomos Artificiais Bacterianos/genética , Peixe-ZebraRESUMO
The vertebrate brain is anatomically and functionally asymmetric. The left and right cerebral hemispheres harbor neural stem cell niches at the ventricular-subventricular zone (V-SVZ) of the ventricular walls, where new neurons are continuously generated throughout life. However, any interhemispheric asymmetry of neural stem cell niches remains unclear. We performed gene-trap screens in adult zebrafish to identify genes that are differentially expressed in the two hemispheres and found that adult-born neurons expressing the neural zinc-finger protein Myt1 exist predominantly in the left V-SVZ. This lateralization could be reversed by left olfactory sensory deprivation-induced inactivation of Notch signaling. The olfactory behavioral preference for attractive amino acids was also impaired by sensory deprivation of the left olfactory system, but not of the right olfactory system. Our findings suggest that olfactory input generates interhemispheric differences in the fate of adult-born neurons in the zebrafish brain.