RESUMO
Embryonic development is remarkably robust, but temperature stress can degrade its ability to generate animals with invariant anatomy. Phenotypes associated with environmental stress suggest that some cell types are more sensitive to stress than others, but the basis of this sensitivity is unknown. Here, we characterize hundreds of individual zebrafish embryos under temperature stress using whole-animal single-cell RNA sequencing (RNA-seq) to identify cell types and molecular programs driving phenotypic variability. We find that temperature perturbs the normal proportions and gene expression programs of numerous cell types and also introduces asynchrony in developmental timing. The notochord is particularly sensitive to temperature, which we map to a specialized cell type: sheath cells. These cells accumulate misfolded protein at elevated temperature, leading to a cascading structural failure of the notochord and anatomic defects. Our study demonstrates that whole-animal single-cell RNA-seq can identify mechanisms for developmental robustness and pinpoint cell types that constitute key failure points.
Assuntos
Proteostase , Peixe-Zebra , Animais , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Temperatura , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Nuclear pore complexes (NPCs) are channels for nucleocytoplasmic transport of proteins and RNAs. However, it remains unclear whether composition, structure, and permeability of NPCs dynamically change during the cleavage period of vertebrate embryos and affect embryonic development. Here, we report that the comprehensive NPC maturity (CNM) controls the onset of zygotic genome activation (ZGA) during zebrafish early embryogenesis. We show that more nucleoporin proteins are recruited to and assembled into NPCs with development, resulting in progressive increase of NPCs in size and complexity. Maternal transcription factors (TFs) transport into nuclei more efficiently with increasing CNM. Deficiency or dysfunction of Nup133 or Ahctf1/Elys impairs NPC assembly, maternal TFs nuclear transport, and ZGA onset, while nup133 overexpression promotes these processes. Therefore, CNM may act as a molecular timer for ZGA by controlling nuclear transport of maternal TFs that reach nuclear concentration thresholds at a given time to initiate ZGA.
Assuntos
Poro Nuclear , Peixe-Zebra , Animais , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Peixe-Zebra/metabolismo , Zigoto/metabolismo , GenomaRESUMO
To track and control self-location, animals integrate their movements through space. Representations of self-location are observed in the mammalian hippocampal formation, but it is unknown if positional representations exist in more ancient brain regions, how they arise from integrated self-motion, and by what pathways they control locomotion. Here, in a head-fixed, fictive-swimming, virtual-reality preparation, we exposed larval zebrafish to a variety of involuntary displacements. They tracked these displacements and, many seconds later, moved toward their earlier location through corrective swimming ("positional homeostasis"). Whole-brain functional imaging revealed a network in the medulla that stores a memory of location and induces an error signal in the inferior olive to drive future corrective swimming. Optogenetically manipulating medullary integrator cells evoked displacement-memory behavior. Ablating them, or downstream olivary neurons, abolished displacement corrections. These results reveal a multiregional hindbrain circuit in vertebrates that integrates self-motion and stores self-location to control locomotor behavior.
Assuntos
Neurônios , Peixe-Zebra , Animais , Peixe-Zebra/fisiologia , Neurônios/fisiologia , Rombencéfalo/fisiologia , Encéfalo/fisiologia , Natação/fisiologia , Homeostase , MamíferosRESUMO
Necrosis of macrophages in the granuloma, the hallmark immunological structure of tuberculosis, is a major pathogenic event that increases host susceptibility. Through a zebrafish forward genetic screen, we identified the mTOR kinase, a master regulator of metabolism, as an early host resistance factor in tuberculosis. We found that mTOR complex 1 protects macrophages from mycobacterium-induced death by enabling infection-induced increases in mitochondrial energy metabolism fueled by glycolysis. These metabolic adaptations are required to prevent mitochondrial damage and death caused by the secreted mycobacterial virulence determinant ESAT-6. Thus, the host can effectively counter this early critical mycobacterial virulence mechanism simply by regulating energy metabolism, thereby allowing pathogen-specific immune mechanisms time to develop. Our findings may explain why Mycobacterium tuberculosis, albeit humanity's most lethal pathogen, is successful in only a minority of infected individuals.
Assuntos
Mycobacterium marinum , Mycobacterium tuberculosis , Tuberculose , Animais , Mycobacterium tuberculosis/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Peixe-ZebraRESUMO
The central pathogen-immune interface in tuberculosis is the granuloma, a complex host immune structure that dictates infection trajectory and physiology. Granuloma macrophages undergo a dramatic transition in which entire epithelial modules are induced and define granuloma architecture. In tuberculosis, relatively little is known about the host signals that trigger this transition. Using the zebrafish-Mycobacterium marinum model, we identify the basis of granuloma macrophage transformation. Single-cell RNA-sequencing analysis of zebrafish granulomas and analysis of Mycobacterium tuberculosis-infected macaques reveal that, even in the presence of robust type 1 immune responses, countervailing type 2 signals associate with macrophage epithelialization. We find that type 2 immune signaling, mediated via stat6, is absolutely required for epithelialization and granuloma formation. In mixed chimeras, stat6 acts cell autonomously within macrophages, where it is required for epithelioid transformation and incorporation into necrotic granulomas. These findings establish the signaling pathway that produces the hallmark structure of mycobacterial infection.
Assuntos
Granuloma/patologia , Imunidade/fisiologia , Infecções por Mycobacterium não Tuberculosas/patologia , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Caderinas/genética , Caderinas/metabolismo , Diferenciação Celular , Modelos Animais de Doenças , Células Epitelioides/citologia , Células Epitelioides/imunologia , Células Epitelioides/metabolismo , Granuloma/imunologia , Granuloma/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Interferon gama/metabolismo , Interleucina-12/metabolismo , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Infecções por Mycobacterium não Tuberculosas/imunologia , Mycobacterium marinum/isolamento & purificação , Mycobacterium marinum/fisiologia , Necrose , RNA Guia de Cinetoplastídeos/metabolismo , Receptores de Interleucina-4/antagonistas & inibidores , Receptores de Interleucina-4/genética , Receptores de Interleucina-4/metabolismo , Fator de Transcrição STAT6/antagonistas & inibidores , Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismoRESUMO
How tissues acquire complex shapes is a fundamental question in biology and regenerative medicine. Zebrafish semicircular canals form from invaginations in the otic epithelium (buds) that extend and fuse to form the hubs of each canal. We find that conventional actomyosin-driven behaviors are not required. Instead, local secretion of hyaluronan, made by the enzymes uridine 5'-diphosphate dehydrogenase (ugdh) and hyaluronan synthase 3 (has3), drives canal morphogenesis. Charged hyaluronate polymers osmotically swell with water and generate isotropic extracellular pressure to deform the overlying epithelium into buds. The mechanical anisotropy needed to shape buds into tubes is conferred by a polarized distribution of actomyosin and E-cadherin-rich membrane tethers, which we term cytocinches. Most work on tissue morphogenesis ascribes actomyosin contractility as the driving force, while the extracellular matrix shapes tissues through differential stiffness. Our work inverts this expectation. Hyaluronate pressure shaped by anisotropic tissue stiffness may be a widespread mechanism for powering morphological change in organogenesis and tissue engineering.
Assuntos
Espaço Extracelular/química , Ácido Hialurônico/farmacologia , Morfogênese , Especificidade de Órgãos , Pressão , Canais Semicirculares/citologia , Canais Semicirculares/embriologia , Actomiosina/metabolismo , Animais , Anisotropia , Comportamento Animal , Matriz Extracelular/metabolismo , Ácido Hialurônico/biossíntese , Modelos Biológicos , Morfogênese/efeitos dos fármacos , Especificidade de Órgãos/efeitos dos fármacos , Pressão Osmótica , Canais Semicirculares/diagnóstico por imagem , Comportamento Estereotipado , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/metabolismoRESUMO
Changes in appendage structure underlie key transitions in vertebrate evolution. Addition of skeletal elements along the proximal-distal axis facilitated critical transformations, including the fin-to-limb transition that permitted generation of diverse modes of locomotion. Here, we identify zebrafish mutants that form supernumerary long bones in their pectoral fins. These new bones integrate into musculature, form joints, and articulate with neighboring elements. This phenotype is caused by activating mutations in previously unrecognized regulators of appendage patterning, vav2 and waslb, that function in a common pathway. This pathway is required for appendage development across vertebrates, and loss of Wasl in mice causes defects similar to those seen in murine Hox mutants. Concordantly, formation of supernumerary bones requires Hox11 function, and mutations in the vav2/wasl pathway drive enhanced expression of hoxa11b, indicating developmental homology with the forearm. Our findings reveal a latent, limb-like pattern ability in fins that is activated by simple genetic perturbation.
Assuntos
Osso e Ossos/embriologia , Extremidades/embriologia , Peixe-Zebra/embriologia , Actinas/metabolismo , Nadadeiras de Animais/embriologia , Animais , Sequência de Bases , Padronização Corporal , Sistemas CRISPR-Cas/genética , Linhagem da Célula , Epistasia Genética , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Genes Reporter , Células HeLa , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Mutação/genética , Fenótipo , Filogenia , Transdução de Sinais/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Goal-directed behavior requires the interaction of multiple brain regions. How these regions and their interactions with brain-wide activity drive action selection is less understood. We have investigated this question by combining whole-brain volumetric calcium imaging using light-field microscopy and an operant-conditioning task in larval zebrafish. We find global, recurring dynamics of brain states to exhibit pre-motor bifurcations toward mutually exclusive decision outcomes. These dynamics arise from a distributed network displaying trial-by-trial functional connectivity changes, especially between cerebellum and habenula, which correlate with decision outcome. Within this network the cerebellum shows particularly strong and predictive pre-motor activity (>10 s before movement initiation), mainly within the granule cells. Turn directions are determined by the difference neuroactivity between the ipsilateral and contralateral hemispheres, while the rate of bi-hemispheric population ramping quantitatively predicts decision time on the trial-by-trial level. Our results highlight a cognitive role of the cerebellum and its importance in motor planning.
Assuntos
Cerebelo/fisiologia , Tomada de Decisões/fisiologia , Tempo de Reação/fisiologia , Peixe-Zebra/fisiologia , Animais , Comportamento Animal/fisiologia , Mapeamento Encefálico/métodos , Cérebro/fisiologia , Cognição/fisiologia , Condicionamento Operante/fisiologia , Objetivos , Habenula/fisiologia , Temperatura Alta , Larva/fisiologia , Atividade Motora/fisiologia , Movimento , Neurônios/fisiologia , Desempenho Psicomotor/fisiologia , Rombencéfalo/fisiologiaRESUMO
During neural tube closure and spinal cord development, many cells die in both the central and peripheral nervous systems (CNS and PNS, respectively). However, myeloid-derived professional phagocytes have not yet colonized the trunk region during early neurogenesis. How apoptotic cells are removed from this region during these stages remains largely unknown. Using live imaging in zebrafish, we demonstrate that neural crest cells (NCCs) respond rapidly to dying cells and phagocytose cellular debris around the neural tube. Additionally, NCCs have the ability to enter the CNS through motor exit point transition zones and clear debris in the spinal cord. Surprisingly, NCCs phagocytosis mechanistically resembles macrophage phagocytosis and their recruitment toward cellular debris is mediated by interleukin-1ß. Taken together, our results reveal a role for NCCs in phagocytosis of debris in the developing nervous system before the presence of professional phagocytes.
Assuntos
Movimento Celular/fisiologia , Crista Neural/fisiologia , Neurogênese/fisiologia , Sistema Nervoso Periférico/crescimento & desenvolvimento , Fagocitose/fisiologia , Medula Espinal/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados , Apoptose/fisiologia , Diferenciação Celular/fisiologia , Interleucina-1beta/metabolismo , Fagócitos/fisiologia , Fagossomos/fisiologia , Peixe-Zebra/embriologiaRESUMO
Cell fate specification by lateral inhibition typically involves contact signaling through the Delta-Notch signaling pathway. However, whether this is the only signaling mode mediating lateral inhibition remains unclear. Here we show that in zebrafish oogenesis, a group of cells within the granulosa cell layer at the oocyte animal pole acquire elevated levels of the transcriptional coactivator TAZ in their nuclei. One of these cells, the future micropyle precursor cell (MPC), accumulates increasingly high levels of nuclear TAZ and grows faster than its surrounding cells, mechanically compressing those cells, which ultimately lose TAZ from their nuclei. Strikingly, relieving neighbor-cell compression by MPC ablation or aspiration restores nuclear TAZ accumulation in neighboring cells, eventually leading to MPC re-specification from these cells. Conversely, MPC specification is defective in taz-/- follicles. These findings uncover a novel mode of lateral inhibition in cell fate specification based on mechanical signals controlling TAZ activity.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Oogênese/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Comunicação Celular/fisiologia , Diferenciação Celular/fisiologia , Linhagem da Célula , Núcleo Celular/metabolismo , Feminino , Células da Granulosa/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Oócitos/metabolismo , Oócitos/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Ativação Transcricional/fisiologia , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidoresRESUMO
Segregation of maternal determinants within the oocyte constitutes the first step in embryo patterning. In zebrafish oocytes, extensive ooplasmic streaming leads to the segregation of ooplasm from yolk granules along the animal-vegetal axis of the oocyte. Here, we show that this process does not rely on cortical actin reorganization, as previously thought, but instead on a cell-cycle-dependent bulk actin polymerization wave traveling from the animal to the vegetal pole of the oocyte. This wave functions in segregation by both pulling ooplasm animally and pushing yolk granules vegetally. Using biophysical experimentation and theory, we show that ooplasm pulling is mediated by bulk actin network flows exerting friction forces on the ooplasm, while yolk granule pushing is achieved by a mechanism closely resembling actin comet formation on yolk granules. Our study defines a novel role of cell-cycle-controlled bulk actin polymerization waves in oocyte polarization via ooplasmic segregation.
Assuntos
Actinas/metabolismo , Ciclo Celular/fisiologia , Oócitos/metabolismo , Actinas/fisiologia , Animais , Polaridade Celular/fisiologia , Citoplasma/metabolismo , Gema de Ovo/fisiologia , Polimerização , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo , ZigotoRESUMO
Cell-cell junctions respond to mechanical forces by changing their organization and function. To gain insight into the mechanochemical basis underlying junction mechanosensitivity, we analyzed tight junction (TJ) formation between the enveloping cell layer (EVL) and the yolk syncytial layer (YSL) in the gastrulating zebrafish embryo. We found that the accumulation of Zonula Occludens-1 (ZO-1) at TJs closely scales with tension of the adjacent actomyosin network, revealing that these junctions are mechanosensitive. Actomyosin tension triggers ZO-1 junctional accumulation by driving retrograde actomyosin flow within the YSL, which transports non-junctional ZO-1 clusters toward the TJ. Non-junctional ZO-1 clusters form by phase separation, and direct actin binding of ZO-1 is required for stable incorporation of retrogradely flowing ZO-1 clusters into TJs. If the formation and/or junctional incorporation of ZO-1 clusters is impaired, then TJs lose their mechanosensitivity, and consequently, EVL-YSL movement is delayed. Thus, phase separation and flow of non-junctional ZO-1 confer mechanosensitivity to TJs.
Assuntos
Desenvolvimento Embrionário/genética , Mecanotransdução Celular/genética , Junções Íntimas/genética , Proteína da Zônula de Oclusão-1/genética , Citoesqueleto de Actina/genética , Actomiosina/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Embrião não Mamífero/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Fosfoproteínas/genética , Ligação Proteica , Junções Íntimas/fisiologia , Saco Vitelino/crescimento & desenvolvimento , Saco Vitelino/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Genomic studies have identified hundreds of candidate genes near loci associated with risk for schizophrenia. To define candidates and their functions, we mutated zebrafish orthologs of 132 human schizophrenia-associated genes. We created a phenotype atlas consisting of whole-brain activity maps, brain structural differences, and profiles of behavioral abnormalities. Phenotypes were diverse but specific, including altered forebrain development and decreased prepulse inhibition. Exploration of these datasets identified promising candidates in more than 10 gene-rich regions, including the magnesium transporter cnnm2 and the translational repressor gigyf2, and revealed shared anatomical sites of activity differences, including the pallium, hypothalamus, and tectum. Single-cell RNA sequencing uncovered an essential role for the understudied transcription factor znf536 in the development of forebrain neurons implicated in social behavior and stress. This phenotypic landscape of schizophrenia-associated genes prioritizes more than 30 candidates for further study and provides hypotheses to bridge the divide between genetic association and biological mechanism.
Assuntos
Esquizofrenia/genética , Esquizofrenia/fisiopatologia , Animais , Encéfalo , Córtex Cerebral , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Peixe-Zebra/genéticaRESUMO
Tyrosine phosphorylation regulates multi-layered signaling networks with broad implications in (patho)physiology, but high-throughput methods for functional annotation of phosphotyrosine sites are lacking. To decipher phosphotyrosine signaling directly in tissue samples, we developed a mass-spectrometry-based interaction proteomics approach. We measured the in vivo EGF-dependent signaling network in lung tissue quantifying >1,000 phosphotyrosine sites. To assign function to all EGF-regulated sites, we determined their recruited protein signaling complexes in lung tissue by interaction proteomics. We demonstrated how mutations near tyrosine residues introduce molecular switches that rewire cancer signaling networks, and we revealed oncogenic properties of such a lung cancer EGFR mutant. To demonstrate the scalability of the approach, we performed >1,000 phosphopeptide pulldowns and analyzed them by rapid mass spectrometric analysis, revealing tissue-specific differences in interactors. Our approach is a general strategy for functional annotation of phosphorylation sites in tissues, enabling in-depth mechanistic insights into oncogenic rewiring of signaling networks.
Assuntos
Carcinogênese/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Fosfotirosina/metabolismo , Células A549 , Animais , Humanos , Espectrometria de Massas/métodos , Mutação , Fosfoproteínas/metabolismo , Fosforilação , Proteômica , Ratos , Ratos Sprague-Dawley , Peixe-ZebraRESUMO
Genome-wide studies have identified genetic variants linked to neurologic diseases. Environmental factors also play important roles, but no methods are available for their comprehensive investigation. We developed an approach that combines genomic data, screens in a novel zebrafish model, computational modeling, perturbation studies, and multiple sclerosis (MS) patient samples to evaluate the effects of environmental exposure on CNS inflammation. We found that the herbicide linuron amplifies astrocyte pro-inflammatory activities by activating signaling via sigma receptor 1, inositol-requiring enzyme-1α (IRE1α), and X-box binding protein 1 (XBP1). Indeed, astrocyte-specific shRNA- and CRISPR/Cas9-driven gene inactivation combined with RNA-seq, ATAC-seq, ChIP-seq, and study of patient samples suggest that IRE1α-XBP1 signaling promotes CNS inflammation in experimental autoimmune encephalomyelitis (EAE) and, potentially, MS. In summary, these studies define environmental mechanisms that control astrocyte pathogenic activities and establish a multidisciplinary approach for the systematic investigation of the effects of environmental exposure in neurologic disorders.
Assuntos
Astrócitos/metabolismo , Sistema Nervoso Central/metabolismo , Animais , Sistema Nervoso Central/imunologia , Biologia Computacional/métodos , Encefalomielite Autoimune Experimental/imunologia , Endorribonucleases/metabolismo , Meio Ambiente , Exposição Ambiental/efeitos adversos , Genoma , Genômica , Humanos , Inflamação/metabolismo , Linurona/efeitos adversos , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptores sigma/efeitos dos fármacos , Receptores sigma/metabolismo , Transdução de Sinais , Proteína 1 de Ligação a X-Box/metabolismo , Peixe-ZebraRESUMO
Xenograft cell transplantation into immunodeficient mice has become the gold standard for assessing pre-clinical efficacy of cancer drugs, yet direct visualization of single-cell phenotypes is difficult. Here, we report an optically-clear prkdc-/-, il2rga-/- zebrafish that lacks adaptive and natural killer immune cells, can engraft a wide array of human cancers at 37°C, and permits the dynamic visualization of single engrafted cells. For example, photoconversion cell-lineage tracing identified migratory and proliferative cell states in human rhabdomyosarcoma, a pediatric cancer of muscle. Additional experiments identified the preclinical efficacy of combination olaparib PARP inhibitor and temozolomide DNA-damaging agent as an effective therapy for rhabdomyosarcoma and visualized therapeutic responses using a four-color FUCCI cell-cycle fluorescent reporter. These experiments identified that combination treatment arrested rhabdomyosarcoma cells in the G2 cell cycle prior to induction of apoptosis. Finally, patient-derived xenografts could be engrafted into our model, opening new avenues for developing personalized therapeutic approaches in the future.
Assuntos
Animais Geneticamente Modificados/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Musculares , Rabdomiossarcoma , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/imunologia , Feminino , Xenoenxertos , Humanos , Células K562 , Masculino , Neoplasias Musculares/tratamento farmacológico , Neoplasias Musculares/imunologia , Neoplasias Musculares/metabolismo , Neoplasias Musculares/patologia , Transplante de Neoplasias , Ftalazinas/farmacologia , Piperazinas/farmacologia , Rabdomiossarcoma/tratamento farmacológico , Rabdomiossarcoma/imunologia , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma/patologia , Temozolomida/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra/genética , Peixe-Zebra/imunologiaRESUMO
When a behavior repeatedly fails to achieve its goal, animals often give up and become passive, which can be strategic for preserving energy or regrouping between attempts. It is unknown how the brain identifies behavioral failures and mediates this behavioral-state switch. In larval zebrafish swimming in virtual reality, visual feedback can be withheld so that swim attempts fail to trigger expected visual flow. After tens of seconds of such motor futility, animals became passive for similar durations. Whole-brain calcium imaging revealed noradrenergic neurons that responded specifically to failed swim attempts and radial astrocytes whose calcium levels accumulated with increasing numbers of failed attempts. Using cell ablation and optogenetic or chemogenetic activation, we found that noradrenergic neurons progressively activated brainstem radial astrocytes, which then suppressed swimming. Thus, radial astrocytes perform a computation critical for behavior: they accumulate evidence that current actions are ineffective and consequently drive changes in behavioral states. VIDEO ABSTRACT.
Assuntos
Astrócitos/metabolismo , Comportamento Animal/fisiologia , Larva/fisiologia , Peixe-Zebra/fisiologia , Neurônios Adrenérgicos/metabolismo , Animais , Animais Geneticamente Modificados/fisiologia , Astrócitos/citologia , Encéfalo/diagnóstico por imagem , Encéfalo/fisiologia , Mapeamento Encefálico , Cálcio/metabolismo , Comunicação Celular/fisiologia , Retroalimentação Sensorial/fisiologia , Neurônios GABAérgicos/metabolismo , Potenciais da Membrana/fisiologia , Optogenética , Natação/fisiologiaRESUMO
Animal survival requires a functioning nervous system to develop during embryogenesis. Newborn neurons must assemble into circuits producing activity patterns capable of instructing behaviors. Elucidating how this process is coordinated requires new methods that follow maturation and activity of all cells across a developing circuit. We present an imaging method for comprehensively tracking neuron lineages, movements, molecular identities, and activity in the entire developing zebrafish spinal cord, from neurogenesis until the emergence of patterned activity instructing the earliest spontaneous motor behavior. We found that motoneurons are active first and form local patterned ensembles with neighboring neurons. These ensembles merge, synchronize globally after reaching a threshold size, and finally recruit commissural interneurons to orchestrate the left-right alternating patterns important for locomotion in vertebrates. Individual neurons undergo functional maturation stereotypically based on their birth time and anatomical origin. Our study provides a general strategy for reconstructing how functioning circuits emerge during embryogenesis. VIDEO ABSTRACT.
RESUMO
Much of current molecular and cell biology research relies on the ability to purify cell types by fluorescence-activated cell sorting (FACS). FACS typically relies on the ability to label cell types of interest with antibodies or fluorescent transgenic constructs. However, antibody availability is often limited, and genetic manipulation is labor intensive or impossible in the case of primary human tissue. To date, no systematic method exists to enrich for cell types without a priori knowledge of cell-type markers. Here, we propose GateID, a computational method that combines single-cell transcriptomics with FACS index sorting to purify cell types of choice using only native cellular properties such as cell size, granularity, and mitochondrial content. We validate GateID by purifying various cell types from zebrafish kidney marrow and the human pancreas to high purity without resorting to specific antibodies or transgenes.
Assuntos
Separação Celular/métodos , Citometria de Fluxo/métodos , Software , Transcriptoma , Animais , Humanos , Rim/citologia , Pâncreas/citologia , Análise de Célula Única , Peixe-Zebra/anatomia & histologiaRESUMO
The fate and function of epigenetic marks during the germline-to-embryo transition is a key issue in developmental biology, with relevance to stem cell programming and transgenerational inheritance. In zebrafish, DNA methylation patterns are programmed in transcriptionally quiescent cleavage embryos; paternally inherited patterns are maintained, whereas maternal patterns are reprogrammed to match the paternal. Here, we provide the mechanism by demonstrating that "Placeholder" nucleosomes, containing histone H2A variant H2A.Z(FV) and H3K4me1, virtually occupy all regions lacking DNA methylation in both sperm and cleavage embryos and reside at promoters encoding housekeeping and early embryonic transcription factors. Upon genome-wide transcriptional onset, genes with Placeholder become either active (H3K4me3) or silent (H3K4me3/K27me3). Notably, perturbations causing Placeholder loss confer DNA methylation accumulation, whereas acquisition/expansion of Placeholder confers DNA hypomethylation and improper gene activation. Thus, during transcriptionally quiescent gametic and embryonic stages, an H2A.Z(FV)/H3K4me1-containing Placeholder nucleosome deters DNA methylation, poising parental genes for either gene-specific activation or facultative repression.