RESUMEN
Using a transgenic zebrafish line harboring a heat-inducible dominant-interference pou5f3 gene (en-pou5f3), we reported that this PouV gene is involved in isthmus development at the midbrain-hindbrain boundary (MHB), which patterns the midbrain and cerebellum. Importantly, the functions of pou5f3 reportedly differ before and after the end of gastrulation. In the present study, we examined in detail the effects of en-pou5f3 induction on isthmus development during embryogenesis. When en-pou5f3 was induced around the end of gastrulation (bud stage), the isthmus was abrogated or deformed by the end of somitogenesis (24 hours post-fertilization). At this stage, the expression of MHB markers -- such as pax2a, fgf8a, wnt1, and gbx2 -- was absent in embryos lacking the isthmus structure, whereas it was present, although severely distorted, in embryos with a deformed isthmus. We further found that, after en-pou5f3 induction at late gastrulation, pax2a, fgf8a, and wnt1 were immediately and irreversibly downregulated, whereas the expression of en2a and gbx2 was reduced only weakly and slowly. Induction of en-pou5f3 at early somite stages also immediately downregulated MHB genes, particularly pax2a, but their expression was restored later. Overall, the data suggested that pou5f3 directly upregulates at least pax2a and possibly fgf8a and wnt1, which function in parallel in establishing the MHB, and that the role of pou5f3 dynamically changes around the end of gastrulation. We next examined the transcriptional regulation of pax2a using both in vitro and in vivo reporter analyses; the results showed that two upstream 1.0-kb regions with sequences conserved among vertebrates specifically drove transcription at the MHB. These reporter analyses confirmed that development of the isthmic organizer is regulated by PouV through direct regulation of pax2/pax2a in vertebrate embryos.
RESUMEN
The vertebrate telencephalic lobes consist of the pallium (dorsal) and subpallium (ventral). The subpallium gives rise to the basal ganglia, encompassing the pallidum and striatum. The development of this region is believed to depend on Foxg1/Foxg1a functions in both mice and zebrafish. This study aims to elucidate the genetic regulatory network controlled by foxg1a in subpallium development using zebrafish as a model. The expression gradient of foxg1a within the developing telencephalon was examined semi-quantitatively in initial investigations. Utilizing the CRISPR/Cas9 technique, we subsequently established a foxg1a mutant line and observed the resultant phenotypes. Morphological assessment revealed that foxg1a mutants exhibit a thin telencephalon together with a misshapen preoptic area (POA). Notably, accumulation of apoptotic cells was identified in this region. In mutants at 24 h postfertilization, the expression of pallium markers expanded ventrally, while that of subpallium markers was markedly suppressed. Concurrently, the expression of fgf8a, vax2, and six3b was shifted ventrally, causing anomalous expression in regions typical of POA formation in wild-type embryos. Consequently, the foxg1a mutation led to expansion of the pallium and disrupted the subpallium and POA. This highlights a pivotal role of foxg1a in directing the dorsoventral patterning of the telencephalon, particularly in subpallium differentiation, mirroring observations in mice. Additionally, reduced expression of neural progenitor maintenance genes was detected in mutants, suggesting the necessity of foxg1a in preserving neural progenitors. Collectively, these findings underscore evolutionarily conserved functions of foxg1 in the development of the subpallium in vertebrate embryos.
Asunto(s)
Factores de Transcripción Forkhead , Redes Reguladoras de Genes , Pez Cebra , Animales , Corteza Cerebral/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Telencéfalo/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Nuclear receptor subfamily 2 group F (Nr2f) proteins are essential for brain development in mice, but little is known about their precise roles and their evolutionary diversification. In the present study, the expression patterns of major nr2f genes (nr2f1a, nr2f1b, and nr2f2) during early brain development were investigated in zebrafish. Comparisons of their expression patterns revealed similar but temporally and spatially distinct patterns after early somite stages in the brain. Frameshift mutations in the three nr2f genes, achieved using the CRISPR/Cas9 method, resulted in a smaller telencephalon and smaller eyes in the nr2f1a mutants; milder forms of those defects were present in the nr2f1b and nr2f2 mutants. Acridine orange staining revealed enhanced cell death in the brain and/or eyes in all nr2f homozygous mutants. The expression of regional markers in the brain did not suggest global defects in brain regionalization; however, shha expression in the preoptic area and hypothalamus, as well as fgf8a expression in the anterior telencephalon, was disturbed in nr2f1a and nr2f1b mutants, potentially leading to a defective telencephalon. Specification of the retina and optic stalk was also significantly affected. The overexpression of nr2f1b by injection of mRNA disrupted the anterior brain at a high dose, and the expression of pax6a in the eyes and fgf8a in the telencephalon at a low dose. The results of these loss- and gain-of-function approaches showed that nr2f genes regulate the development of the telencephalon and eyes in zebrafish embryos.
Asunto(s)
Ojo , Miembro 2 del Grupo F de la Subfamilia 1 de Receptores Nucleares , Receptores Nucleares Huérfanos , Proteínas de Pez Cebra , Pez Cebra , Animales , Ratones , Encéfalo/metabolismo , Ojo/crecimiento & desarrollo , Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Telencéfalo/metabolismo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Receptores Nucleares Huérfanos/metabolismoRESUMEN
BACKGROUND: Elongation of the spinal cord is dependent on neural development from neuromesodermal progenitors in the tail bud. We previously showed the involvement of the Oct4-type gene, pou5f3, in this process in zebrafish mainly by dominant-interference gene induction, but, to compensate for the limitation of this transgene approach, mutant analysis was indispensable. pou5f3 involvement in the signaling pathways was another unsolved question. RESULTS: We examined the phenotypes of pou5f3 mutants and the effects of Pou5f3 activation by the tamoxifen-ERT2 system in the posterior neural tube, together confirming the involvement of pou5f3. The reporter assays using P19 cells implicated tail bud-related transcription factors in pou5f3 expression. Regulation of tail bud development by retinoic acid (RA) signaling was confirmed by treatment of embryos with RA and the synthesis inhibitor, and in vitro reporter assays further showed that RA signaling regulated pou5f3 expression. Importantly, the expression of the RA degradation enzyme gene, cyp26a1, was down-regulated in embryos with disrupted pou5f3 activity. CONCLUSIONS: The involvement of pou5f3 in spinal cord extension was supported by using mutants and the gain-of-function approach. Our findings further suggest that pou5f3 regulates the RA level, contributing to neurogenesis in the posterior neural tube.
Asunto(s)
Factores de Transcripción , Pez Cebra , Animales , Regulación del Desarrollo de la Expresión Génica , Ácido Retinoico 4-Hidroxilasa/genética , Ácido Retinoico 4-Hidroxilasa/metabolismo , Médula Espinal/metabolismo , Factores de Transcripción/metabolismo , Tretinoina/metabolismo , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
In the present study, we determined the developmental toxicity of endosulfan at an elevated ambient temperature using the zebrafish animal model. Zebrafish embryos of various developmental stages were exposed to endosulfan through E3 medium, raised under two selected temperature conditions (28.5 °C and an elevated temperature of 35 °C), and monitored under the microscope. Zebrafish embryos of very early developmental stages (cellular cleavage stages, such as the 64-cell stage) were highly sensitive to the elevated temperature as 37.5% died and 47.5% developed into amorphous type, while only 15.0% of embryos developed as normal embryos without malformation. Zebrafish embryos that were exposed concurrently to endosulfan and an elevated temperature showed stronger developmental defects (arrested epiboly progress, shortened body length, curved trunk) compared to the embryos exposed to either endosulfan or an elevated temperature. The brain structure of the embryos that concurrently were exposed to the elevated temperature and endosulfan was either incompletely developed or malformed. Furthermore, the stress-implicated genes hsp70, p16, and smp30 regulations were synergistically affected by endosulfan treatment under the elevated thermal condition. Overall, the elevated ambient temperature synergistically enhanced the developmental toxicity of endosulfan in zebrafish embryos.
Asunto(s)
Endosulfano , Pez Cebra , Animales , Endosulfano/toxicidad , Temperatura , Desarrollo Embrionario , Embrión no Mamífero/anomalíasRESUMEN
In vertebrate embryogenesis, elongation of the posterior body is driven by de novo production of the axial and paraxial mesoderm as well as the neural tube at the posterior end. This process is presumed to depend on the stem cell-like population in the tail bud region, but the details of the gene regulatory network involved are unknown. Previous studies suggested the involvement of pou5f3, an Oct4-type POU gene in zebrafish, in axial elongation. In the present study, we first found that pou5f3 is expressed mainly in the dorsal region of the tail bud immediately after gastrulation, and that this expression is restricted to the posterior-most region of the elongating neural tube during somitogenesis. This pou5f3 expression was complementary to the broad expression of sox3 in the neural tube, and formed a sharp boundary with specific expression of tbxta (orthologue of mammalian T/Brachyury) in the tail bud, implicating pou5f3 in the specification of tail bud-derived cells toward neural differentiation in the spinal cord. When pou5f3 was functionally impaired after gastrulation by induction of a dominant-interfering pou5f3 mutant gene (en-pou5f3), trunk and tail elongation were markedly disturbed at distinct positions along the axis depending on the stage. This finding showed involvement of pou5f3 in de novo generation of the body from the tail bud. Conditional functional abrogation also showed that pou5f3 downregulates mesoderm-forming genes but promotes neural development by activating neurogenesis genes around the tail bud. These results suggest that pou5f3 is involved in formation of the posterior spinal cord.
Asunto(s)
Proteínas de Pez Cebra , Pez Cebra , Animales , Desarrollo Embrionario , Mesodermo , Médula Espinal , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
The zebrafish is an excellent model animal that is amenable to forward genetics approaches. To uncover unknown developmental regulatory mechanisms in vertebrates, we conducted chemical mutagenesis screening and identified a novel mutation, kanazutsi (kzt). This mutation is recessive, and its homozygotes are embryonic lethal. Mutant embryos suffered from a variety of morphological defects, such as head flattening, pericardial edema, circulation defects, disrupted patterns of melanophore distribution, dwarf eyes, a defective jaw, and extensive apoptosis in the head, which indicates that the main affected tissues are derived from neural crest cells (NCCs). The expression of tissue-specific markers in kzt mutants showed that the early specification of NCCs was normal, but their later differentiation was severely affected. The mutation was mapped to chromosome 3 by linkage analyses, near cytoglobin 1 (cygb1), the product of which is a globin-family respiratory protein. cygb1 expression was activated during somitogenesis in somites and cranial NCCs in wild-type embryos but was significantly downregulated in mutant embryos, despite the normal primary structure of the gene product. The kzt mutation was phenocopied by cygb1 knockdown with low-dose morpholino oligos and was partially rescued by cygb1 overexpression. Both severe knockdown and null mutation of cygb1, established by the CRISPR/Cas9 technique, resulted in far more severe defects at early stages. Thus, it is highly likely that the downregulation of cygb1 is responsible for many, if not all, of the phenotypes of the kzt mutation. These results reveal a requirement for globin family proteins in vertebrate embryos, particularly in the differentiation and subsequent development of NCCs.
Asunto(s)
Citoglobina/genética , Regulación del Desarrollo de la Expresión Génica , Cresta Neural/citología , Cresta Neural/embriología , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Apoptosis/genética , Sistemas CRISPR-Cas , Diferenciación Celular/genética , Cromosomas/genética , Citoglobina/metabolismo , Desarrollo Embrionario/genética , Expresión Génica , Técnicas de Silenciamiento del Gen , Mutación , Cresta Neural/metabolismo , Fenotipo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
In the vertebrate body, a metameric structure is present along the anterior-posterior axis. Zebrafish tbx6-/- larvae, in which somite boundaries do not form during embryogenesis, were shown to exhibit abnormal skeletal morphology such as rib, neural arch and hemal arch. In this study, we investigated the role of somite patterning in the formation of anterior vertebrae and ribs in more detail. Using three-dimensional computed tomography scans, we found that anterior vertebrae including the Weberian apparatus were severely affected in tbx6-/- larvae. In addition, pleural ribs of tbx6 mutants exhibited severe defects in the initial ossification, extension of ossification, and formation of parapophyses. Two-colour staining revealed that bifurcation of ribs was caused by fusion or branching of ribs in tbx6-/- . The parapophyses in tbx6-/- juvenile fish showed irregular positioning to centra and abnormal attachment to ribs. Furthermore, we found that the ossification of the distal portion of ribs proceeded along myotome boundaries even in irregularly positioned myotome boundaries. These results provide evidence of the contribution of somite patterning to the formation of the Weberian apparatus and rib in zebrafish.
Asunto(s)
Tipificación del Cuerpo/genética , Costillas/embriología , Somitos/enzimología , Pez Cebra/embriología , Animales , Animales Modificados Genéticamente , Regulación del Desarrollo de la Expresión Génica , Costillas/diagnóstico por imagen , Somitos/diagnóstico por imagen , Proteínas de Dominio T Box/genética , Tomografía Computarizada por Rayos X , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
In early vertebrate embryos, the dorsal ectoderm is induced by the axial mesendoderm to form the neural plate, which is given competence to form neural cells by soxB1 genes. Subsequently, neurogenesis proceeds in proneural clusters that are generated by a gene network involving proneural genes and Notch signaling. However, what occurs between early neural induction and the later initiation of neurogenesis has not been fully revealed. In the present study, we demonstrated that during gastrulation, the expression of the Oct4-related PouV gene pou5f3 (also called pou2), which is widely observed at earlier stages, was rapidly localized to an array of isolated spotted domains, each of which coincided with individual proneural clusters. Two-color in situ hybridization confirmed that each pou5f3-expressing domain included a proneural cluster. Further analysis demonstrated that anterior pou5f3 domains straddled the boundaries between rhombomere 1 (r1) and r2, whereas posterior domains were included in r4. The effects of forced expression of an inducible negative dominant-interfering pou5f3 gene suggested that pou5f3 activated early proneural genes, such as neurog1 and ebf2, and also soxB1, but repressed the late proneural genes atoh1a and ascl1b. Furthermore, pou5f3 was considered to repress her4.1, a Notch-dependent Hairy/E(spl) gene involved in lateral inhibition in proneural clusters. These results suggest that pou5f3 promotes early neurogenesis in proneural clusters, but negatively regulates later neurogenesis. Suppression of pou5f3 also altered the expression of other her genes, including her3, her5, and her9, further supporting a role for pou5f3 in neurogenesis. In vitro reporter assays in P19â¯cells showed that pou5f3 was repressed by neurog1, but activated by Notch signaling. These findings together demonstrate the importance of the pou5f3-mediated gene regulatory network in neural development in vertebrate embryos.
Asunto(s)
Placa Neural/embriología , Neurogénesis , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Tipificación del Cuerpo , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Placa Neural/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factores de Transcripción SOXB1/genética , Proteínas de Pez Cebra/genéticaRESUMEN
The presumptive somite boundary in the presomitic mesoderm (PSM) is defined by the anterior border of the expression domain of Tbx6 protein. During somite segmentation, the expression domain of Tbx6 is regressed by Ripply-meditated degradation of Tbx6 protein. Although the expression of zebrafish tbx6 remains restricted to the PSM, the transcriptional regulation of tbx6 remains poorly understood. Here, we show that the expression of zebrafish tbx6 is maintained by transcriptional autoregulation. We find that a proximal-located cis-regulatory module, TR1, which contains two putative T-box sites, is required for somite segmentation in the intermediate body and for proper expression of segmentation genes. Embryos with deletion of TR1 exhibit significant reduction of tbx6 expression at the 12-somite stage, although its expression is initially observed. Additionally, Tbx6 is associated with TR1 and activates its own expression in the anterior PSM. Furthermore, the anterior expansion of tbx6 expression in ripply gene mutants is suppressed in a TR1-dependent manner. The results suggest that the autoregulatory loop of zebrafish tbx6 facilitates immediate removal of Tbx6 protein through termination of its own transcription at the anterior PSM.
Asunto(s)
Tipificación del Cuerpo/genética , Homeostasis/genética , Somitos/embriología , Proteínas de Dominio T Box/metabolismo , Transcripción Genética , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Animales , Sitios de Unión/genética , Embrión no Mamífero/metabolismo , Elementos de Facilitación Genéticos/genética , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Homocigoto , Dominios Proteicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Somitos/metabolismo , Proteínas de Dominio T Box/química , Proteínas de Dominio T Box/genética , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genéticaRESUMEN
One of the pivotal events in neural development is compartmentalization, wherein the neural tissue divides into domains and undergoes functional differentiation. For example, midbrain-hindbrain boundary (MHB) formation and subsequent isthmus development are key steps in cerebellar development. Although several regulatory mechanisms are known to underlie this event, little is known about cellular behaviors. In this study, to examine the cellular dynamics around the MHB region, we performed confocal time-lapse imaging in zebrafish embryos to track cell populations in the neural tube via 4D analysis. We used a transgenic line wherein enhanced green fluorescent protein (EGFP) expression is driven by the gastrulation brain homeobox 2 (gbx2) enhancer, which is involved in MHB maintenance. 4D time-lapse imaging of 5-20 h revealed a novel pattern in cell migration: a dynamic ventrocaudally directed migration from the MHB region toward the hindbrain. Furthermore, in the hindbrain region, these EGFP-positive cells altered their shapes and extended the axons. Immunohistochemical analysis and retrograde labeling showed that these cells in the hindbrain were in the process of neuronal differentiation, including reticulospinal neurons. These results revealed the dynamic and two-step behavior and possible fate of the cell population, which are linked to brain compartmentalization, leading to a deeper understanding of brain development and formation of neuronal circuits.
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Encéfalo/metabolismo , Movimiento Celular/fisiología , Proteínas de Homeodominio/metabolismo , Microscopía Confocal/métodos , Proteínas de Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Movimiento Celular/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Neurogénesis/fisiología , Neuroimagen/métodos , Imagen de Lapso de Tiempo , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
In the zebrafish segmentation clock, hairy/enhancer of split-related genes her1, her7, and hes6 encodes components of core oscillators. Since the expression of cyclic genes proceeds rapidly in the presomitic mesoderm (PSM), these hairy-related mRNAs are subject to strict post-transcriptional regulation. In this study, we demonstrate that inhibition of the CCR4-NOT deadenylase complex lengthens poly(A) tails of hairy-related mRNAs and increases the amount of these mRNAs, which is accompanied by defective somite segmentation. In transgenic embryos, we show that EGFP mRNAs with 3'UTRs of hairy-related genes exhibit turnover similar to endogenous mRNAs. Our results suggest that turnover rates of her1, her7, and hes6 mRNAs are differently regulated by the CCR4-NOT deadenylase complex possibly through their 3'UTRs in the zebrafish PSM.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Regulación del Desarrollo de la Expresión Génica , ARN Mensajero/genética , Somitos/metabolismo , Factores de Transcripción/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Relojes Biológicos , Tipificación del Cuerpo/genética , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Mesodermo/embriología , Mesodermo/metabolismo , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , Somitos/embriología , Factores de Transcripción/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
Voltage-sensitive dye (VSD) imaging enables fast, direct, and simultaneous detection of membrane potentials from a population of neurons forming neuronal circuits. This enables the detection of hyperpolarization together with depolarization, whose balance plays a pivotal role in the function of many brain regions. Among these is the cerebellum, which contains a significant number of inhibitory neurons. However, the mechanism underlying the functional development remains unclear. In this study, we used a model system ideal to study neurogenesis by applying VSD imaging to the cerebellum of zebrafish larvae to analyze the neuronal activity of the developing cerebellum, focusing on both excitation and inhibition. We performed in-vivo high-speed imaging of the entire cerebellum of the zebrafish, which was stained using Di-4-ANEPPS, a widely used VSD. To examine whether neuronal activity in the zebrafish cerebellum could be detected by this VSD, we applied electrical stimulation during VSD imaging, which showed that depolarization was detected widely in the cerebellum upon stimulation. These responses mostly disappeared following treatment with tetrodotoxin, indicating that Di-4-ANEPPS enabled optical measurement of neuronal activity in the developing cerebellum of zebrafish. Moreover, hyperpolarizing signals were also detected upon stimulation, but these were significantly reduced by treatment with picrotoxin, a GABAA receptor inhibitor, indicating that these responses represent inhibitory signals. This approach will enable a detailed analysis of the spatiotemporal dynamics of the excitation and inhibition in the cerebellum along its developmental stages, leading to a deeper understanding of the functional development of the cerebellum in vertebrates.
Asunto(s)
Cerebelo/citología , Cerebelo/crecimiento & desarrollo , Neuronas/fisiología , Imagen de Colorante Sensible al Voltaje/métodos , Animales , Animales Modificados Genéticamente , Proteína 3 Similar a ELAV/genética , Proteína 3 Similar a ELAV/metabolismo , Estimulación Eléctrica , Antagonistas del GABA/farmacología , Larva , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Neuronas/efectos de los fármacos , Picrotoxina/farmacología , Compuestos de Piridinio/metabolismo , Bloqueadores de los Canales de Sodio/farmacología , Tetrodotoxina/farmacología , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
Somites sequentially form with a regular interval by the segmentation from the anterior region of the presomitic mesoderm (PSM). The expression of several genes involved in the somite segmentation is switched off at the transition from the anterior PSM to somites. Zebrafish Ripply1, which down-regulates a T-box transcription factor Tbx6, is required for the suppression of segmentation gene expression. However, the functional roles of the Ripply-mediated suppression of segmentation gene expression at the anterior PSM remain elusive. In this study, we generated ripply1 mutants and examined genetic interaction between ripply1/2 and tbx6. Zebrafish ripply1-/- embryos failed to form the somite boundaries as was observed in knockdown embryos. We found that somite segmentation defects in ripply1 mutants were suppressed by heterozygous mutation of tbx6 or partial translational inhibition of tbx6 by antisense morpholino. We further showed that somite boundaries that were recovered in tbx6+/-; ripply1-/- embryos were dependent on the function of ripply2, indicating that relative gene dosage between ripply1/2 and tbx6 plays a critical role in the somite formation. Interestingly, the expression of segmentation genes such mesp as was still not fully suppressed at the anterior PSM of tbx6+/-; ripply1-/- embryos although the somite formation and rostral-caudal polarity of somites were properly established. Furthermore, impaired myogenesis was observed in the segmented somites in tbx6+/-; ripply1-/- embryos. These results revealed that partial suppression of the segmentation gene expression by Ripply is sufficient to establish the rostral-caudal polarity of somites, and that stronger suppression of the segmentation gene expression by Ripply is required for proper myogenesis in zebrafish embryos.
Asunto(s)
Tipificación del Cuerpo/genética , Desarrollo Embrionario/genética , Proteínas Nucleares/genética , Proteínas de Dominio T Box/genética , Proteínas de Pez Cebra/genética , Animales , Regulación del Desarrollo de la Expresión Génica , Mesodermo/crecimiento & desarrollo , Morfolinos/genética , Desarrollo de Músculos/genética , Somitos/crecimiento & desarrollo , Pez Cebra/genética , Pez Cebra/crecimiento & desarrolloRESUMEN
Optical measurement of membrane potentials enables fast, direct and simultaneous detection of membrane potentials from a population of neurons, providing a desirable approach for functional analysis of neuronal circuits. Here, we applied recently developed genetically encoded voltage indicators, ASAP1 (Accelerated Sensor of Action Potentials 1) and QuasAr2 (Quality superior to Arch 2), to zebrafish, an ideal model system for studying neurogenesis. To achieve this, we established transgenic lines which express the voltage sensors, and showed that ASAP1 is expressed in zebrafish neurons. To examine whether neuronal activity could be detected by ASAP1, we performed whole-cerebellum imaging, showing that depolarization was detected widely in the cerebellum and optic tectum upon electrical stimulation. Spontaneous activity in the spinal cord was also detected by ASAP1 imaging at single-cell resolution as well as at the neuronal population level. These responses mostly disappeared following treatment with tetrodotoxin, indicating that ASAP1 enabled optical measurement of neuronal activity in the zebrafish brain. Combining this method with other approaches, such as optogenetics and behavioural analysis may facilitate a deeper understanding of the functional organization of brain circuitry and its development.
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Potenciales de Acción , Red Nerviosa/fisiología , Optogenética , Imagen de Colorante Sensible al Voltaje , Pez Cebra/fisiología , Animales , Animales Modificados Genéticamente/genética , Cerebelo/citología , Cerebelo/fisiología , Expresión Génica , Potenciales de la Membrana , Red Nerviosa/citología , Optogenética/métodos , Médula Espinal/citología , Médula Espinal/fisiología , Imagen de Colorante Sensible al Voltaje/métodos , Pez Cebra/genéticaRESUMEN
The enteric nervous system (ENS) is the largest part of the peripheral nervous system in vertebrates. Toward the visualization of the development of the vertebrate ENS, we report our creation of a new transgenic line, Tg(chata:GGFF2) which has a 1.5-kb upstream region of the zebrafish choline acetyltransferase a (chata) gene followed by modified green fluorescent protein (gfp). During development, GFP + cells were detected in the gut by 60 h post-fertilization (hpf). In the gut of 6- and 12-days post-fertilization (dpf) larvae, an average of 92% of the GFP + cells were positive for the neuronal marker HuC/D, suggesting that GFP marks enteric neurons in this transgenic line. We also observed that 66% of the GFP + cells were choline acetyltransferase (ChAT)-immunopositive at 1.5 months. Thus, GFP is expressed at the larval stages at which ChAT protein expression is not yet detected by immunostaining. We studied the spatiotemporal pattern of neural differentiation in the ENS by live-imaging of this transgenic line. We observed that GFP + or gfpâ¯+ cells initially formed a pair of bilateral rows at 60 hpf or 53 hpf, respectively, in the migrating enteric neural crest cells. Most of the GFP + cells did not migrate, and most of the new GFP + cells were added to fill the space among the previously formed GFP + cells. GFP expression reached the anus by 72 hpf. New GFP + cells then also appeared in the dorsal and ventral sides of the initial GFP + rows, resulting in their distribution on the entire gut by 4 dpf. A small number of new GFP + cells were found to move among older GFP + cells just before the cells stopped migration, suggesting that the moving GFP + cells may represent neural precursor cells searching for a place for the final differentiation. Our data suggest that the Tg(chata:GGFF2) line could serve as a useful tool for studies of enteric neural differentiation and cell behavior.
Asunto(s)
Animales Modificados Genéticamente/crecimiento & desarrollo , Colina O-Acetiltransferasa/genética , Sistema Nervioso Entérico/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Pez Cebra/crecimiento & desarrollo , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/fisiología , Diferenciación Celular , Movimiento Celular , Sistema Nervioso Entérico/metabolismo , Proteínas Fluorescentes Verdes/genética , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Organogénesis , Pez Cebra/genética , Pez Cebra/fisiologíaRESUMEN
Zebrafish pou5f3 (previously named pou2), a close homologue of mouse Oct4, encodes a PouV-family transcription factor. pou5f3 has been implicated in diverse aspects of developmental regulation during embryogenesis. In the present study, we addressed the molecular function of Pou5f3 as a transcriptional regulator and the mechanism by which pou5f3 expression is transcriptionally regulated. We examined the influence of effector genes on the expression of the luciferase gene under the control of the upstream 2.1-kb regulatory DNA of pou5f3 (Luc-2.2) in HEK293T and P19 cells. We first confirmed that Pou5f3 functions as a transcriptional activator both in cultured cells and embryos, which confirmed autoregulation of pou5f3 in embryos. It was further shown that Luc-2.2 was activated synergistically by pou5f3 and sox3, which is similar to the co-operative activity of Oct4 and Sox2 in mice, although synergy between pou5f3 and sox2 was less obvious in this zebrafish system. The effects of pou5f3 deletion constructs on the regulation of Luc-2.2 expression revealed different roles for the three subregions of the N-terminal region in Pou5f3 in terms of its regulatory functions and co-operativity with Sox3. Electrophoretic mobility shift assays confirmed that Pou5f3 and Sox3 proteins specifically bind to adjacent sites in the 2.1-kb DNA and that there is an interaction between the two proteins. The synergy with sox3 was unique to pou5f3-the other POU factor genes examined did not show such synergy in Luc-2.2 regulation. Finally, functional interaction was observed between pou5f3 and sox3 in embryos in terms of the regulation of dorsoventral patterning and convergent extension movement. These findings together demonstrate co-operative functions of pou5f3 and sox3, which are frequently coexpressed in early embryos, in the regulation of early development.
Asunto(s)
Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Factor 3 de Transcripción de Unión a Octámeros/genética , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Embrión no Mamífero/citología , Células HEK293 , Humanos , Técnicas In Vitro , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factores de Transcripción/genética , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/metabolismoRESUMEN
During vertebrate brain development, the gastrulation brain homeobox 2 gene (gbx2) is expressed in the forebrain, but its precise roles are still unknown. In this study, we addressed this issue in zebrafish (Danio rerio) first by carefully examining gbx2 expression in the developing forebrain. We showed that gbx2 was expressed in the telencephalon during late somitogenesis, from 18h post-fertilization (hpf) to 24 hpf, and in the thalamic primordium after 26 hpf. In contrast, another gbx gene, gbx1, was expressed in the anterior-most ventral telencephalon after 36 hpf. Thus, the expression patterns of these two gbx genes did not overlap, arguing against their redundant function in the forebrain. Two-color fluorescence in situ hybridization (FISH) showed close relationships between the telencephalic expression of gbx2 and other forebrain-forming genes, suggesting that their interactions contribute to the regionalization of the telencephalon. FISH further revealed that gbx2 is expressed in the ventricular region of the telencephalon. By using transgenic fish in which gbx2 can be induced by heat shock, we found that gbx2 induction at 16 hpf repressed the expression of emx3, dlx2a, and six3b in the ventral telencephalon. Among secreted factor genes, bmp2b and wnt1 were repressed in the vicinity of the gbx2 domain in the telencephalon. The expression of forebrain-forming genes was examined in mutant embryos lacking gbx2, showing emx3 and dlx2a to be upregulated in the subpallium at 24 hpf. Taken together, these findings indicate that gbx2 contributes to the development of the subpallium through its repressive activities against other telencephalon-forming genes. We further showed that inhibiting FGF signaling and activating Wnt signaling repressed gbx2 and affected the regionalization of the telencephalon, supporting a functional link between gbx2, intracellular signaling, and telencephalon development.
Asunto(s)
Gastrulación/genética , Regulación del Desarrollo de la Expresión Génica/genética , Genes Homeobox/genética , Proteínas de Homeodominio/genética , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Animales , Tipificación del Cuerpo/genética , Neurogénesis/genética , Factores de Transcripción/genética , Pez Cebra/genéticaRESUMEN
It is well established that the gbx2 homeobox gene contributes to the positioning of the midbrain-hindbrain boundary (MHB) governing the development of adjacent brain regions in vertebrate embryos, but the specific aspects of the gene regulatory network regulated by gbx2 during brain development remain unclear. In the present study, we sought to comprehensively identify gbx2 target genes in zebrafish embryos by microarray analysis around the end of gastrulation, when the MHB is established, using transgenic embryos harboring heat-inducible gbx2. This analysis revealed that a large number of genes were either upregulated or downregulated following gbx2 induction, and the time course of induction differed depending on the genes. The differences in response to gbx2 were found by functional annotation analysis to be related to the functions and structures of the target genes. Among the significantly downregulated genes was her5, whose expression in the midbrain was precisely complementary to gbx2 expression around the MHB, suggesting that gbx2 expression in the anterior hindbrain restricts her5 expression to the midbrain. Because her5 represses neurogenesis, gbx2 may positively regulate neural development in its expression domain. Indeed, we showed further that gbx2 induction upregulated neural marker expression in the midbrain. Quantitative PCR analysis revealed that gbx2 upregulated the expression of the zebrafish proneural gene ebf2, whereas it repressed notch1a, which generally represses neurogenesis. Taken together, these results demonstrate that gbx2 not only functions to position the MHB but also regulates neurogenesis in the anterior hindbrain.
Asunto(s)
Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Neurogénesis/genética , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Encéfalo/embriología , Encéfalo/metabolismo , ADN/metabolismo , Células HEK293 , Proteínas de Homeodominio/genética , Humanos , Hibridación in Situ , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Transcripción Genética , Proteínas de Pez Cebra/genéticaRESUMEN
Chromatin immunoprecipitation (ChIP) against enhancer-associated marks with massive sequencing is a powerful approach to identify genome-wide distributions of putative enhancers. However, functional in vivo analysis is required to elucidate the activities of predicted enhancers. Using zebrafish embryos, we established a ChIP-Injection method that enables identification of functional enhancers based on their enhancer activities in embryos. Each reporter gene possessing the enhancer-associated genomic region enriched by ChIP was injected into zebrafish embryos to analyze the activity of putative enhancers. By using the ChIP-Injection, we identified 32 distinct putative enhancers that drove specific expression. Additionally, we generated transgenic lines that exhibit distributions of the EGFP signal as was observed in the screening. Furthermore, the expression pattern driven by the identified somite-specific enhancer resembled that of the gene acta2. The results indicate that ChIP-Injection provides an efficient approach for identification of active enhancers in a potentially wide variety of developmental tissues and stages.