RESUMO
Escaping from danger is one of the most fundamental survival behaviors for animals. Most freshwater fishes display olfactory alarm reactions in which an injured fish releases putative alarm substances from the skin to notify its shoaling company about the presence of danger. Here, we identified two small compounds in zebrafish skin extract, designated as ostariopterin and daniol sulfate. Ostariopterin is a pterin derivative commonly produced in many freshwater fishes belonging to the Ostariophysi superorder. Daniol sulfate is a novel sulfated bile alcohol specifically present in the Danio species, including zebrafish. Ostariopterin and daniol sulfate activate distinct glomeruli in the olfactory bulb. Zebrafish display robust alarm reactions, composed of darting, freezing, and bottom dwelling, only when they are concomitantly stimulated with ostariopterin and daniol sulfate. These results demonstrate that the fish alarm reaction is driven through a coincidence detection mechanism of the two compounds along the olfactory neural circuitry.
Assuntos
Cyprinidae , Perciformes , Animais , Peixe-Zebra/fisiologia , Olfato , Bulbo Olfatório , SulfatosRESUMO
Escape responses to threatening stimuli are vital for survival in all animal species. Larval zebrafish display fast escape responses when exposed to tactile, acoustic, and visual stimuli. However, their behavioral responses to chemosensory stimuli remain unknown. In this study, we found that carbon dioxide (CO2) induced a slow avoidance response, which was distinct from the touch-evoked fast escape response. We identified the gonadotropin-releasing hormone 3-expressing terminal nerve as the CO2 sensor in the nose. Wide-field calcium imaging revealed downstream CO2-activated ensembles of neurons along three distinct neural pathways, olfactory, trigeminal, and habenulo-interpeduncular, further reaching the reticulospinal neurons in the hindbrain. Ablation of the nose, terminal nerve, or trigeminal ganglion resulted in a dramatic decrease in CO2-evoked avoidance responses. These findings demonstrate that the terminal nerve-trigeminal system plays a pivotal role in triggering a slow chemosensory avoidance behavior in the larval zebrafish.
Assuntos
Aprendizagem da Esquiva/fisiologia , Dióxido de Carbono/farmacologia , Hormônio Liberador de Gonadotropina/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ácido Pirrolidonocarboxílico/análogos & derivados , Animais , Animais Geneticamente Modificados , Aprendizagem da Esquiva/efeitos dos fármacos , Larva , Vias Neurais/efeitos dos fármacos , Vias Neurais/metabolismo , Ácido Pirrolidonocarboxílico/metabolismo , Gânglio Trigeminal/efeitos dos fármacos , Gânglio Trigeminal/metabolismo , Peixe-ZebraRESUMO
Nucleotides released from food sources into environmental water are supposed to act as feeding cues for many fish species. However, it remains unknown how fish can sensitively detect those nucleotides. Here we discover a novel olfactory mechanism for ATP sensing in zebrafish. Upon entering into the nostril, ATP is efficiently converted into adenosine through enzymatic reactions of two ecto-nucleotidases expressed in the olfactory epithelium. Adenosine subsequently activates a small population of olfactory sensory neurons expressing a novel adenosine receptor A2c that is unique to fishes and amphibians. The information is then transmitted to a single glomerulus in the olfactory bulb and further to four regions in higher olfactory centers. These results provide conclusive evidence for a sophisticated enzyme-linked receptor mechanism underlying detection of ATP as a food-derived attractive odorant linking to foraging behavior that is crucial and common to aquatic lower vertebrates.
Assuntos
Trifosfato de Adenosina/metabolismo , Neurônios Receptores Olfatórios/fisiologia , Olfato/fisiologia , Peixe-Zebra/fisiologia , Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Comportamento Animal , Cálcio/metabolismo , Nariz/fisiologia , Mucosa Olfatória/citologia , Mucosa Olfatória/fisiologia , Neurônios Receptores Olfatórios/citologia , Filogenia , Receptores Purinérgicos P1/metabolismoRESUMO
Pheromones play vital roles for survival and reproduction in various organisms. In many fishes, prostaglandin F2α acts not only as a female reproductive hormone, facilitating ovulation and spawning, but also as a sex pheromone inducing male reproductive behaviors. Here, we unravel the molecular and neural circuit mechanisms underlying the pheromonal action of prostaglandin F2α in zebrafish. Prostaglandin F2α specifically activates two olfactory receptors with different sensitivities and expression in distinct populations of ciliated olfactory sensory neurons. Pheromone information is then transmitted to two ventromedial glomeruli in the olfactory bulb and further to four regions in higher olfactory centers. Mutant male zebrafish deficient in the high-affinity receptor exhibit loss of attractive response to prostaglandin F2α and impairment of courtship behaviors toward female fish. These findings demonstrate the functional significance and activation of selective neural circuitry for the sex pheromone prostaglandin F2α and its cognate olfactory receptor in fish reproductive behavior.
Assuntos
Bulbo Olfatório/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Receptores Odorantes/metabolismo , Receptores de Prostaglandina/metabolismo , Olfato/fisiologia , Animais , Corte , Dinoprosta/metabolismo , Bulbo Olfatório/efeitos dos fármacos , Feromônios/metabolismo , Reprodução/fisiologia , Comportamento Sexual Animal/fisiologia , Peixe-ZebraRESUMO
Anticipation of danger at first elicits panic in animals, but later it helps them to avoid the real threat adaptively. In zebrafish, as fish experience more and more danger, neurons in the ventral habenula (vHb) showed tonic increase in the activity to the presented cue and activated serotonergic neurons in the median raphe (MR). This neuronal activity could represent the expectation of a dangerous outcome and be used for comparison with a real outcome when the fish is learning how to escape from a dangerous to a safer environment. Indeed, inhibiting synaptic transmission from vHb to MR impaired adaptive avoidance learning, while panic behavior induced by classical fear conditioning remained intact. Furthermore, artificially triggering this negative outcome expectation signal by optogenetic stimulation of vHb neurons evoked place avoidance behavior. Thus, vHb-MR circuit is essential for representing the level of expected danger and behavioral programming to adaptively avoid potential hazard.
Assuntos
Aprendizagem da Esquiva/fisiologia , Habenula/fisiologia , Vias Neurais/fisiologia , Núcleos da Rafe/fisiologia , Neurônios Serotoninérgicos/fisiologia , 5,7-Di-Hidroxitriptamina/metabolismo , Potenciais de Ação/fisiologia , Adaptação Psicológica/fisiologia , Animais , Animais Geneticamente Modificados , Condicionamento Clássico/fisiologia , Sinais (Psicologia) , Medo/fisiologia , Habenula/citologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Neurotransmissores/metabolismo , Núcleos da Rafe/citologia , Serotonina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/genética , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Olfactory glomeruli are innervated with great precision by the axons of different olfactory sensory neuron types and act as functional units in odor information processing. Approximately 140 glomeruli are present in each olfactory bulb of adult zebrafish; these units consist of either highly stereotypic large glomeruli or smaller anatomically indistinguishable glomeruli. In the present study, we investigated developmental differences among these types of glomeruli. We observed that 10 large and individually identifiable glomeruli already developed before hatching, at 72 h after fertilization, in configurations that resembled their mature organization. However, the cross-sectional area of these glomeruli increased throughout larval development, and they eventually comprised the largest units in postlarval olfactory bulbs. In contrast, small and anatomically indistinguishable glomeruli formed only after hatching, apparently by segregating from five larger precursors that were identifiable during embryonic development. The differentiation of these small glomeruli proceeded with conspicuous variation in number and arrangement, both among larvae and between olfactory bulbs of the same individuals. To determine factors that might contribute to this variability, we investigated the effects of olfactory enrichment on the development of amino acid-responsive lateral glomeruli, which include both large and small units. Larvae reared in an amino acid-enriched environment had normal large lateral glomeruli, but the small lateral glomeruli were more numerous and displayed reduced cross-sectional areas compared with glomeruli in control animals. Our results suggest that large and small glomeruli mature via distinct developmental processes that may be differentially influenced by sensory experience.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Rede Nervosa/fisiologia , Bulbo Olfatório , Neurônios Receptores Olfatórios/fisiologia , Aminoácidos/farmacologia , Animais , Animais Geneticamente Modificados , Embrião não Mamífero , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hemocianinas/metabolismo , Larva , Proteínas de Membrana/metabolismo , Microscopia Confocal , Rede Nervosa/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Vias Neurais/embriologia , Vias Neurais/crescimento & desenvolvimento , Bulbo Olfatório/citologia , Bulbo Olfatório/embriologia , Bulbo Olfatório/crescimento & desenvolvimento , Neurônios Receptores Olfatórios/efeitos dos fármacos , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
In fish, amino acids are food-related important olfactory cues to elicit an attractive response. However, the neural circuit underlying this olfactory behavior is not fully elucidated. In the present study, we applied the Tol2 transposon-mediated gene trap method to dissect the zebrafish olfactory system genetically. Four zebrafish lines (SAGFF27A, SAGFF91B, SAGFF179A, and SAGFF228C) were established in which the modified transcription activator Gal4FF was expressed in distinct subsets of olfactory sensory neurons (OSNs). The OSNs in individual lines projected axons to partially overlapping but mostly different glomeruli in the olfactory bulb (OB). In SAGFF27A, Gal4FF was expressed predominantly in microvillous OSNs innervating the lateral glomerular cluster that corresponded to the amino acid-responsive region in the OB. To clarify the olfactory neural pathway mediating the feeding behavior, we genetically expressed tetanus neurotoxin in the Gal4FF lines to block synaptic transmission in distinct populations of glomeruli and examined their behavioral response to amino acids. The attractive response to amino acids was abolished only in SAGFF27A fish carrying the tetanus neurotoxin transgene. These findings clearly demonstrate the functional significance of the microvillous OSNs innervating the lateral glomerular cluster in the amino acid-mediated feeding behavior of zebrafish. Thus, the integrated approach combining genetic, neuroanatomical, and behavioral methods enables us to elucidate the neural circuit mechanism underlying various olfactory behaviors in adult zebrafish.
Assuntos
Aminoácidos/metabolismo , Elementos de DNA Transponíveis , Condutos Olfatórios , Peixe-Zebra/fisiologia , Animais , Imuno-Histoquímica , Peixe-Zebra/genéticaRESUMO
Cholesterol regulates Hedgehog (Hh) signaling during early vertebrate development. Smith-Lemli-Opitz syndrome (SLOS) is caused by defects in 7-dehydrocholesterol reductase (DHCR7), an enzyme catalyzing the final step of cholesterol biosynthesis. Many developmental malformations attributed to SLOS occur in tissues and organs where Hh signaling is required for development, but the precise role of DHCR7 deficiency in this disease remains murky. We report that DHCR7 and Sonic Hedgehog (Shh) are co-expressed during midline development in Xenopus embryos. DHCR7 has previously been implicated to function as a positive regulator of Hh signaling that acts to regulate the cholesterol adduction of Hh ligand or to affect Hh signaling in the responding cell. We present gain- and loss-of-function analyses suggesting that DHCR7 functions as a negative regulator of Hh signaling at the level or downstream of Smoothened (Smo) and affects intracellular Hh signaling. Our analysis also raises the possibility that the human condition SLOS is caused not only by disruption of the enzymatic role of DHCR7 as a reductase in cholesterol biosynthesis, but may also involve defects in DHCR7 resulting in derepression of Shh signaling.
Assuntos
Colesterol/biossíntese , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Padronização Corporal , Estruturas Embrionárias/anatomia & histologia , Estruturas Embrionárias/fisiologia , Epistasia Genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog , Humanos , Hibridização In Situ , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Síndrome de Smith-Lemli-Opitz/enzimologia , Síndrome de Smith-Lemli-Opitz/genética , Receptor Smoothened , Transativadores/genética , Proteínas de Xenopus/genética , Xenopus laevis/anatomia & histologia , Xenopus laevis/metabolismoRESUMO
Development is controlled by a complex series of events requiring sequential gene activation. Understanding the logic of gene networks during development is necessary for a complete understanding of how genes contribute to phenotype. Pioneering work initiated in the sea urchin and Drosophila has demonstrated that reasonable transcriptional regulatory network diagrams representing early development in multicellular animals can be generated through use of appropriate genomic, genetic, and biochemical tools. Establishment of similar regulatory network diagrams for vertebrate development is a necessary step. The amphibian Xenopus has long been used as a model for vertebrate early development and has contributed greatly to the elucidation of gene regulation. Because the best and most extensively studied transcriptional regulatory network in Xenopus is that underlying the formation and function of Spemann's organizer, we describe the current status of our understanding of this gene regulatory network and its relationship to mesodermal patterning. Seventy-four transcription factors currently known to be expressed in the mesoendoderm of Xenopus gastrula were characterized according to their modes of action, DNA binding consensus sequences, and target genes. Among them, nineteen transcription factors were characterized sufficiently in detail, allowing us to generate a gene regulatory network diagram. Additionally, we discuss recent amphibian work using a combined DNA microarray and bioinformatics approach that promises to accelerate regulatory network studies.
Assuntos
Xenopus/embriologia , Xenopus/genética , Animais , Sequência de Bases , DNA/genética , Endoderma/citologia , Regulação da Expressão Gênica no Desenvolvimento , Genes Reguladores , Genômica , Peptídeos e Proteínas de Sinalização Intercelular , Mesoderma/citologia , Modelos Genéticos , Análise de Sequência com Séries de Oligonucleotídeos , Filogenia , Regiões Promotoras Genéticas , Proteínas/genética , Transdução de Sinais , Transcrição Gênica , Ativação Transcricional , Proteínas de XenopusRESUMO
We have undertaken a large-scale microarray gene expression analysis using cDNAs corresponding to 21,000 Xenopus laevis ESTs. mRNAs from 37 samples, including embryos and adult organs, were profiled. Cluster analysis of embryos of different stages was carried out and revealed expected affinities between gastrulae and neurulae, as well as between advanced neurulae and tadpoles, while egg and feeding larvae were clearly separated. Cluster analysis of adult organs showed some unexpected tissue-relatedness, e.g. kidney is more related to endodermal than to mesodermal tissues and the brain is separated from other neuroectodermal derivatives. Cluster analysis of genes revealed major phases of co-ordinate gene expression between egg and adult stages. During the maternal-early embryonic phase, genes maintaining a rapidly dividing cell state are predominantly expressed (cell cycle regulators, chromatin proteins). Genes involved in protein biosynthesis are progressively induced from mid-embryogenesis onwards. The larval-adult phase is characterised by expression of genes involved in metabolism and terminal differentiation. Thirteen potential synexpression groups were identified, which encompass components of diverse molecular processes or supra-molecular structures, including chromatin, RNA processing and nucleolar function, cell cycle, respiratory chain/Krebs cycle, protein biosynthesis, endoplasmic reticulum, vesicle transport, synaptic vesicle, microtubule, intermediate filament, epithelial proteins and collagen. Data filtering identified genes with potential stage-, region- and organ-specific expression. The dataset was assembled in the iChip microarray database, , which allows user-defined queries. The study provides insights into the higher order of vertebrate gene expression, identifies synexpression groups and marker genes, and makes predictions for the biological role of numerous uncharacterized genes.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Análise de Sequência com Séries de Oligonucleotídeos , Xenopus laevis/genética , Animais , Clonagem Molecular , Análise por Conglomerados , Colágeno/metabolismo , DNA Complementar/metabolismo , Bases de Dados Genéticas , Bases de Dados de Proteínas , Desenvolvimento Embrionário , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica/métodos , Família Multigênica , RNA/metabolismo , Fatores de Tempo , Distribuição Tecidual , XenopusRESUMO
To isolate novel genes regulating neural induction, we used a DNA microarray approach. As neural induction is thought to occur by means of the inhibition of bone morphogenetic protein (BMP) signaling, BMP signaling was inhibited in ectodermal cells by overexpression of a dominant-negative receptor. RNAs were isolated from control animal cap explants and from dominant-negative BMP receptor expressing animal caps and subjected to a microarray experiment using newly generated high-density Xenopus DNA microarray chips representing over 17,000 unigenes. We have identified 77 genes that are induced in animal caps after inhibition of BMP signaling, and all of these genes were subjected to whole-mount in situ hybridization analysis. Thirty-two genes showed specific expression in neural tissues. Of the 32, 14 genes have never been linked to neural induction. Two genes that are highly induced by BMP inhibition are inhibitors of Wnt signaling, suggesting that a key step in neural induction is to produce Wnt antagonists to promote anterior neural plate development. Our current analysis also proves that a microarray approach is useful in identifying novel candidate factors involved in neural induction and patterning.
Assuntos
Proteínas Morfogenéticas Ósseas/biossíntese , Regulação da Expressão Gênica , Neurônios/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Padronização Corporal , Cromatina/metabolismo , Clonagem Molecular , Genes Dominantes , Hibridização In Situ , Crista Neural/metabolismo , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fatores de Tempo , Xenopus laevisRESUMO
Retinoid signaling is important for patterning the vertebrate hindbrain and midaxial regions. We recently showed that signaling through retinoic acid receptors (RARs) is essential for anteroposterior patterning along the entire body axis. To further investigate the mechanisms through which RARs act, we used microarray analysis to investigate the effects of modulating RAR activity on target gene expression. We identified 334 up-regulated genes (92% of which were validated), including known RA-responsive genes, known genes that have never been proposed as RA targets and many hypothetical and unidentified genes (n = 166). Sixty-seven validated down-regulated genes were identified, including known RA-responsive genes and anterior marker genes. The expression patterns of selected up-regulated genes (n = 45) were examined at neurula stages using whole-mount in situ hybridization. We found that most of these genes were expressed in the neural tube and many were expressed in anterior tissues such as neural crest, brain, eye anlagen, and cement gland. Some were expressed in tissues such as notochord, somites, pronephros, and blood islands, where retinoic acid (RA) plays established roles in organogenesis. Members of this set of newly identified RAR target genes are likely to play important roles in neural patterning and organogenesis under the control of RAR signaling pathways, and their further characterization will expand our understanding of RA signaling during development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Análise de Sequência com Séries de Oligonucleotídeos , Receptores do Ácido Retinoico/metabolismo , Xenopus/embriologia , Animais , Northern Blotting , Padronização Corporal , Análise por Conglomerados , Regulação para Baixo , Ácidos Graxos/metabolismo , Hibridização In Situ , Rim/embriologia , Crista Neural/embriologia , Crista Neural/metabolismo , Retinoides/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Esteroides/metabolismo , Fatores de Tempo , Tretinoína/metabolismo , Regulação para CimaRESUMO
Anteroposterior (AP) patterning of the developing CNS is crucial for both regional specification and the timing of neurogenesis. Several important factors are involved in AP patterning, including members of the WNT and FGF growth factor families, retinoic acid receptors, and HOX genes. We have examined the interactions between FGF and retinoic signaling pathways. Blockade of FGF signaling downregulates the expression of members of the RAR signaling pathway, RARalpha, RALDH2 and CYP26. Overexpression of a constitutively active RARalpha2 rescues the effects of FGF blockade on the expression of XCAD3 and HOXB9. This suggests that RARalpha2 is required as a downstream target of FGF signaling for the posterior expression of XCAD3 and HOXB9. Surprisingly, we found that posterior expression of FGFR1 and FGFR4 was dependent on the expression of RARalpha2. Anterior expression was also altered with FGFR1 expression being lost, whereas FGFR4 expression was expanded beyond its normal expression domain. RARalpha2 is required for the expression of XCAD3 and HOXB9, and for the ability of XCAD3 to induce HOXB9 expression. We conclude that RARalpha2 is required at multiple points in the posteriorization pathway, suggesting that correct AP neural patterning depends on a series of mutually interactive feedback loops among FGFs, RARs and HOX genes.
Assuntos
Vértebra Cervical Áxis/embriologia , Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Tretinoína/metabolismo , Xenopus/embriologia , Família Aldeído Desidrogenase 1 , Aldeído Oxidase , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Animais , Vértebra Cervical Áxis/metabolismo , Padronização Corporal/genética , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Embrião não Mamífero , Epistasia Genética , Proteínas Fetais/genética , Proteínas Fetais/metabolismo , Fator 8 de Crescimento de Fibroblasto , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Retinal Desidrogenase , Ácido Retinoico 4 Hidroxilase , Receptor alfa de Ácido Retinoico , Xenopus/genética , Xenopus/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMO
During amphibian gastrulation, the anterior endomesoderm is thought to move forward along the inner surface of the blastocoel roof toward the animal pole where it comes into physical contact with the anterior-most portion of the prospective head neuroectoderm (PHN), and it is also believed that this physical interaction occurs during the mid-gastrula stage. However, using Xenopus embryos we found that the interaction between the anterior endomesoderm and the PHN occurs as early as stage 10.25 and the blastocoel roof ectoderm at this stage contributed only to the epidermal tissue. We also found that once the interaction was established, these tissues continued to associate in register and ultimately became the head structures. From these findings, we propose a new model of Xenopus gastrulation. The anterior endomesoderm migrates only a short distance on the inner surface of the blastocoel roof during very early stages of gastrulation (by stage 10.25). Then, axial mesoderm formation occurs, beginning dorsally (anterior) and progressing ventrally (posterior) to complete gastrulation. This new view of Xenopus gastrulation makes it possible to directly compare vertebrate gastrulation movements.