RESUMO
Bioelectric signaling, intercellular communication facilitated by membrane potential and electrochemical coupling, is emerging as a key regulator of animal development. Gap junction (GJ) channels can mediate bioelectric signaling by creating a fast, direct pathway between cells for the movement of ions and other small molecules. In vertebrates, GJ channels are formed by a highly conserved transmembrane protein family called the connexins. The connexin gene family is large and complex, creating challenges in identifying specific connexins that create channels within developing and mature tissues. Using the embryonic zebrafish neuromuscular system as a model, we identify a connexin conserved across vertebrate lineages, gjd4, which encodes the Cx46.8 protein, that mediates bioelectric signaling required for slow muscle development and function. Through mutant analysis and in vivo imaging, we show that gjd4/Cx46.8 creates GJ channels specifically in developing slow muscle cells. Using genetics, pharmacology, and calcium imaging, we find that spinal-cord-generated neural activity is transmitted to developing slow muscle cells, and synchronized activity spreads via gjd4/Cx46.8 GJ channels. Finally, we show that bioelectrical signal propagation within the developing neuromuscular system is required for appropriate myofiber organization and that disruption leads to defects in behavior. Our work reveals a molecular basis for GJ communication among developing muscle cells and reveals how perturbations to bioelectric signaling in the neuromuscular system may contribute to developmental myopathies. Moreover, this work underscores a critical motif of signal propagation between organ systems and highlights the pivotal role of GJ communication in coordinating bioelectric signaling during development.
Assuntos
Conexinas , Junções Comunicantes , Transdução de Sinais , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Peixe-Zebra/embriologia , Junções Comunicantes/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Conexinas/metabolismo , Conexinas/genética , Desenvolvimento MuscularRESUMO
Animal development proceeds in the presence of intimate microbial associations, but the extent to which different host cells across the body respond to resident microbes remains to be fully explored. Using the vertebrate model organism, the larval zebrafish, we assessed transcriptional responses to the microbiota across the entire body at single-cell resolution. We find that cell types across the body, not limited to tissues at host-microbe interfaces, respond to the microbiota. Responses are cell-type-specific, but across many tissues the microbiota enhances cell proliferation, increases metabolism, and stimulates a diversity of cellular activities, revealing roles for the microbiota in promoting developmental plasticity. This work provides a resource for exploring transcriptional responses to the microbiota across all cell types of the vertebrate body and generating new hypotheses about the interactions between vertebrate hosts and their microbiota.
Assuntos
Microbiota , Peixe-Zebra , Animais , Larva , Proliferação de CélulasRESUMO
The receptor tyrosine kinase Ret plays a critical role in regulating enteric nervous system (ENS) development. Ret is important for proliferation, migration, and survival of enteric progenitor cells (EPCs). Ret also promotes neuronal fate, but its role during neuronal differentiation and in the adult ENS is less well understood. Inactivating RET mutations are associated with ENS diseases, e.g., Hirschsprung Disease, in which distal bowel lacks ENS cells. Zebrafish is an established model system for studying ENS development and modeling human ENS diseases. One advantage of the zebrafish model system is that their embryos are transparent, allowing visualization of developmental phenotypes in live animals. However, we lack tools to monitor Ret expression in live zebrafish. Here, we developed a new BAC transgenic line that expresses GFP under the ret promoter. We find that EPCs and the majority of ENS neurons express ret:GFP during ENS development. In the adult ENS, GFP+ neurons are equally present in females and males. In homozygous mutants of ret and sox10-another important ENS developmental regulator gene-GFP+ ENS cells are absent. In summary, we characterize a ret:GFP transgenic line as a new tool to visualize and study the Ret signaling pathway from early development through adulthood.
Assuntos
Sistema Nervoso Entérico , Peixe-Zebra , Animais , Masculino , Feminino , Humanos , Adulto , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Sistema Nervoso Entérico/metabolismo , Transdução de Sinais , Animais Geneticamente Modificados , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismoRESUMO
Host-associated microbiotas guide the trajectory of developmental programs, and altered microbiota composition is linked to neurodevelopmental conditions such as autism spectrum disorder. Recent work suggests that microbiotas modulate behavioral phenotypes associated with these disorders. We discovered that the zebrafish microbiota is required for normal social behavior and reveal a molecular pathway linking the microbiota, microglial remodeling of neural circuits, and social behavior in this experimentally tractable model vertebrate. Examining neuronal correlates of behavior, we found that the microbiota restrains neurite complexity and targeting of forebrain neurons required for normal social behavior and is necessary for localization of forebrain microglia, brain-resident phagocytes that remodel neuronal arbors. The microbiota also influences microglial molecular functions, including promoting expression of the complement signaling pathway and the synaptic remodeling factor c1q. Several distinct bacterial taxa are individually sufficient for normal microglial and neuronal phenotypes, suggesting that host neuroimmune development is sensitive to a feature common among many bacteria. Our results demonstrate that the microbiota influences zebrafish social behavior by stimulating microglial remodeling of forebrain circuits during early neurodevelopment and suggest pathways for new interventions in multiple neurodevelopmental disorders.
Assuntos
Transtorno do Espectro Autista , Microbiota , Animais , Microglia/metabolismo , Peixe-Zebra , Transtorno do Espectro Autista/metabolismo , Neurônios/fisiologia , Comportamento Social , ProsencéfaloRESUMO
BACKGROUND: An essential determinant of a neuron's functionality is its neurotransmitter phenotype. We previously identified a defined subpopulation of cholinergic neurons required for social orienting behavior in zebrafish. RESULTS: We transcriptionally profiled these neurons and discovered that they are capable of synthesizing both acetylcholine and GABA. We also established a constellation of transcription factors and neurotransmitter markers that can be used as a "transcriptomic fingerprint" to recognize a homologous neuronal population in another vertebrate. CONCLUSION: Our results suggest that this transcriptomic fingerprint and the cholinergic-GABAergic neuronal subtype that it defines are evolutionarily conserved.
Assuntos
Acetilcolina , Peixe-Zebra , Animais , Colinérgicos , Neurônios Colinérgicos , Neurotransmissores , Comportamento Social , Fatores de Transcrição , Peixe-Zebra/genética , Ácido gama-AminobutíricoRESUMO
The enteric nervous system (ENS) controls many aspects of intestinal homeostasis, including parameters that shape the habitat of microbial residents. Previously we showed that zebrafish lacking an ENS, due to deficiency of the sox10 gene, develop intestinal inflammation and bacterial dysbiosis, with an expansion of proinflammatory Vibrio strains. To understand the primary defects resulting in dysbiosis in sox10 mutants, we investigated how the ENS shapes the intestinal environment in the absence of microbiota and associated inflammatory responses. We found that intestinal transit, intestinal permeability, and luminal pH regulation are all aberrant in sox10 mutants, independent of microbially induced inflammation. Treatment with the proton pump inhibitor, omeprazole, corrected the more acidic luminal pH of sox10 mutants to wild type levels. Omeprazole treatment also prevented overabundance of Vibrio and ameliorated inflammation in sox10 mutant intestines. Treatment with the carbonic anhydrase inhibitor, acetazolamide, caused wild type luminal pH to become more acidic, and increased both Vibrio abundance and intestinal inflammation. We conclude that a primary function of the ENS is to regulate luminal pH, which plays a critical role in shaping the resident microbial community and regulating intestinal inflammation.
Assuntos
Sistema Nervoso Entérico/fisiologia , Intestinos/microbiologia , Fenobarbital/metabolismo , Fatores de Transcrição SOXE/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/fisiologia , Animais , Disbiose/microbiologia , Microbioma Gastrointestinal , Homeostase , Concentração de Íons de Hidrogênio , Inflamação , MutaçãoRESUMO
Intestinal tract development is a coordinated process involving signaling among the progenitors and developing cells from all three germ layers. Development of endoderm-derived intestinal epithelium has been shown to depend on epigenetic modifications, but whether that is also the case for intestinal tract cell types from other germ layers remains unclear. We found that functional loss of a DNA methylation machinery component, ubiquitin-like protein containing PHD and RING finger domains 1 (uhrf1), leads to reduced numbers of ectoderm-derived enteric neurons and severe disruption of mesoderm-derived intestinal smooth muscle. Genetic chimeras revealed that Uhrf1 functions both cell-autonomously in enteric neuron precursors and cell-non-autonomously in surrounding intestinal cells, consistent with what is known about signaling interactions between these cell types that promote one another's development. Uhrf1 recruits the DNA methyltransferase Dnmt1 to unmethylated DNA during replication. Dnmt1 is also expressed in enteric neurons and smooth muscle progenitors. dnmt1 mutants have fewer enteric neurons and disrupted intestinal smooth muscle compared to wildtypes. Because dnmt1;uhrf1 double mutants have a similar phenotype to dnmt1 and uhrf1 single mutants, Dnmt1 and Uhrf1 must function together during enteric neuron and intestinal muscle development. This work shows that genes controlling epigenetic modifications are important to coordinate intestinal tract development, provides the first demonstration that these genes influence development of the ENS, and advances uhrf1 and dnmt1 as potential new Hirschsprung disease candidates.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1/fisiologia , Sistema Nervoso Entérico/embriologia , Epigênese Genética , Intestinos/embriologia , Transativadores/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Quimera , DNA (Citosina-5-)-Metiltransferase 1/genética , Células-Tronco Embrionárias/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Intestinos/citologia , Intestinos/inervação , Masculino , Músculo Liso/embriologia , Mutação , Neurônios , Transativadores/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Fishes of the genus Danio exhibit diverse pigment patterns that serve as useful models for understanding the genes and cell behaviors underlying the evolution of adult form. Among these species, zebrafish D. rerio exhibit several dark stripes of melanophores with sparse iridophores that alternate with light interstripes of dense iridophores and xanthophores. By contrast, the closely related species D. nigrofasciatus has an attenuated pattern with fewer melanophores, stripes and interstripes. Here we demonstrate species differences in iridophore development that presage the fully formed patterns. Using genetic and transgenic approaches we identify the secreted peptide Endothelin-3 (Edn3)-a known melanogenic factor of tetrapods-as contributing to reduced iridophore proliferation and fewer stripes and interstripes in D. nigrofasciatus. We further show the locus encoding this factor is expressed at lower levels in D. nigrofasciatus owing to cis-regulatory differences between species. Finally, we show that functions of two paralogous loci encoding Edn3 have been partitioned between skin and non-skin iridophores. Our findings reveal genetic and cellular mechanisms contributing to pattern differences between these species and suggest a model for evolutionary changes in Edn3 requirements for pigment patterning and its diversification across vertebrates.
Assuntos
Cromatóforos/fisiologia , Endotelina-3/metabolismo , Pigmentação/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Proliferação de Células , Embrião não Mamífero , Endotelina-3/genética , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Modelos Animais , Fenótipo , Transdução de Sinais/genética , Pele/citologia , Especificidade da Espécie , Proteínas de Peixe-Zebra/genéticaRESUMO
Deficits in social engagement are diagnostic of multiple neurodevelopmental disorders, including autism and schizophrenia [1]. Genetically tractable animal models like zebrafish (Danio rerio) could provide valuable insight into developmental factors underlying these social impairments, but this approach is predicated on the ability to accurately and reliably quantify subtle behavioral changes. Similarly, characterizing local molecular and morphological phenotypes requires knowledge of the neuroanatomical correlates of social behavior. We leveraged behavioral and genetic tools in zebrafish to both refine our understanding of social behavior and identify brain regions important for driving it. We characterized visual social interactions between pairs of adult zebrafish and discovered that they perform a stereotyped orienting behavior that reflects social attention [2]. Furthermore, in pairs of fish, the orienting behavior of one individual is the primary factor driving the same behavior in the other individual. We used manual and genetic lesions to investigate the forebrain contribution to this behavior and identified a population of neurons in the ventral telencephalon whose ablation suppresses social interactions, while sparing other locomotor and visual behaviors. These neurons are cholinergic and express the gene encoding the transcription factor Lhx8a, which is required for development of cholinergic neurons in the mouse forebrain [3]. The neuronal population identified in zebrafish lies in a region homologous to mammalian forebrain regions implicated in social behavior such as the lateral septum [4]. Our data suggest that an evolutionarily conserved population of neurons controls social orienting in zebrafish.
Assuntos
Neurônios/fisiologia , Orientação Espacial/fisiologia , Comportamento Social , Telencéfalo/fisiologia , Peixe-Zebra/fisiologia , Animais , Feminino , MasculinoRESUMO
Resident microbes promote many aspects of host development, although the mechanisms by which microbiota influence host tissues remain unclear. We showed previously that the microbiota is required for allocation of appropriate numbers of secretory cells in the zebrafish intestinal epithelium. Because Notch signaling is crucial for secretory fate determination, we conducted epistasis experiments to establish whether the microbiota modulates host Notch signaling. We also investigated whether innate immune signaling transduces microbiota cues via the Myd88 adaptor protein. We provide the first evidence that microbiota-induced, Myd88-dependent signaling inhibits host Notch signaling in the intestinal epithelium, thereby promoting secretory cell fate determination. These results connect microbiota activity via innate immune signaling to the Notch pathway, which also plays crucial roles in intestinal homeostasis throughout life and when impaired can result in chronic inflammation and cancer.
Assuntos
Mucosa Intestinal/metabolismo , Microbiota , Fator 88 de Diferenciação Mieloide/metabolismo , Receptores Notch/metabolismo , Animais , Mucosa Intestinal/microbiologia , Mucosa Intestinal/fisiologia , Transdução de Sinais/fisiologia , Peixe-Zebra/metabolismoRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0159277.].
RESUMO
Sustaining a balanced intestinal microbial community is critical for maintaining intestinal health and preventing chronic inflammation. The gut is a highly dynamic environment, subject to periodic waves of peristaltic activity. We hypothesized that this dynamic environment is a prerequisite for a balanced microbial community and that the enteric nervous system (ENS), a chief regulator of physiological processes within the gut, profoundly influences gut microbiota composition. We found that zebrafish lacking an ENS due to a mutation in the Hirschsprung disease gene, sox10, develop microbiota-dependent inflammation that is transmissible between hosts. Profiling microbial communities across a spectrum of inflammatory phenotypes revealed that increased levels of inflammation were linked to an overabundance of pro-inflammatory bacterial lineages and a lack of anti-inflammatory bacterial lineages. Moreover, either administering a representative anti-inflammatory strain or restoring ENS function corrected the pathology. Thus, we demonstrate that the ENS modulates gut microbiota community membership to maintain intestinal health.
Assuntos
Sistema Nervoso Entérico/fisiologia , Microbioma Gastrointestinal , Intestinos/microbiologia , Animais , Bactérias/crescimento & desenvolvimento , Bactérias/isolamento & purificação , Contagem de Células , Contagem de Colônia Microbiana , Disbiose/genética , Disbiose/microbiologia , Disbiose/patologia , Sistema Nervoso Entérico/citologia , Regulação da Expressão Gênica , Inflamação/genética , Inflamação/patologia , Intestinos/patologia , Contagem de Leucócitos , Modelos Biológicos , Mutação/genética , Neutrófilos/metabolismo , Filogenia , Fatores de Transcrição SOXE/metabolismo , Transplante de Células-Tronco , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
Recombination-based cloning is a quick and efficient way to generate expression vectors. Recent advancements have provided powerful recombinant DNA methods for molecular manipulations. Here, we describe a novel collection of three-fragment MultiSite Gateway cloning system-compatible vectors providing expanded molecular tools for vertebrate research. The components of this toolkit encompass a broad range of uses such as fluorescent imaging, dual gene expression, RNA interference, tandem affinity purification, chemically-inducible dimerization and lentiviral production. We demonstrate examples highlighting the utility of this toolkit for producing multi-component vertebrate expression vectors with diverse primary research applications. The vectors presented here are compatible with other Gateway toolkits and collections, facilitating the rapid generation of a broad range of innovative DNA constructs for biological research.
Assuntos
Cromatografia de Afinidade/métodos , Clonagem Molecular/métodos , DNA Recombinante/genética , Expressão Gênica , Vetores Genéticos , Interferência de RNA , Recombinação Genética , Animais , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Hipocampo , Humanos , Ratos , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismoRESUMO
BACKGROUND: To understand the basis of nervous system development, we must learn how multipotent progenitors generate diverse neuronal and glial lineages. We addressed this issue in the zebrafish enteric nervous system (ENS), a complex neuronal and glial network that regulates essential intestinal functions. Little is currently known about how ENS progenitor subpopulations generate enteric neuronal and glial diversity. RESULTS: We identified temporally and spatially dependent progenitor subpopulations based on coexpression of three genes essential for normal ENS development: phox2bb, sox10, and ret. Our data suggest that combinatorial expression of these genes delineates three major ENS progenitor subpopulations, (1) phox2bb + /ret- /sox10-, (2) phox2bb + /ret + /sox10-, and (3) phox2bb + /ret + /sox10+, that reflect temporal progression of progenitor maturation during migration. We also found that differentiating zebrafish neurons maintain phox2bb and ret expression, and lose sox10 expression. CONCLUSIONS: Our data show that zebrafish enteric progenitors constitute a heterogeneous population at both early and late stages of ENS development and suggest that marker gene expression is indicative of a progenitor's fate. We propose that a progenitor's expression profile reveals its developmental state: "younger" wave front progenitors express all three genes, whereas more mature progenitors behind the wave front selectively lose sox10 and/or ret expression, which may indicate developmental restriction. Developmental Dynamics 245:1081-1096, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Sistema Nervoso Entérico/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Sistema Nervoso Entérico/citologia , Sistema Nervoso Entérico/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Crista Neural/citologia , Crista Neural/enzimologia , Crista Neural/metabolismo , RNA Mensageiro/genética , Fatores de Transcrição SOXE/genética , Fatores de Transcrição SOXE/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
The gut microbiota is a complex consortium of microorganisms with the ability to influence important aspects of host health and development. Harnessing this "microbial organ" for biomedical applications requires clarifying the degree to which host and bacterial factors act alone or in combination to govern the stability of specific lineages. To address this issue, we combined bacteriological manipulation and light sheet fluorescence microscopy to monitor the dynamics of a defined two-species microbiota within a vertebrate gut. We observed that the interplay between each population and the gut environment produces distinct spatiotemporal patterns. As a consequence, one species dominates while the other experiences sudden drops in abundance that are well fit by a stochastic mathematical model. Modeling revealed that direct bacterial competition could only partially explain the observed phenomena, suggesting that a host factor is also important in shaping the community. We hypothesized the host determinant to be gut motility, and tested this mechanism by measuring colonization in hosts with enteric nervous system dysfunction due to a mutation in the ret locus, which in humans is associated with the intestinal motility disorder known as Hirschsprung disease. In mutant hosts we found reduced gut motility and, confirming our hypothesis, robust coexistence of both bacterial species. This study provides evidence that host-mediated spatial structuring and stochastic perturbation of communities can drive bacterial population dynamics within the gut, and it reveals a new facet of the intestinal host-microbe interface by demonstrating the capacity of the enteric nervous system to influence the microbiota. Ultimately, these findings suggest that therapeutic strategies targeting the intestinal ecosystem should consider the dynamic physical nature of the gut environment.
Assuntos
Microbioma Gastrointestinal/fisiologia , Motilidade Gastrointestinal/fisiologia , Trato Gastrointestinal/microbiologia , Microbiota/fisiologia , Aeromonas veronii/fisiologia , Animais , Antibiose/fisiologia , Larva/genética , Larva/microbiologia , Larva/fisiologia , Microscopia de Fluorescência , Mutação , Dinâmica Populacional , Especificidade da Espécie , Vibrio cholerae/fisiologia , Peixe-ZebraRESUMO
A central problem in development is how fates of closely related cells are segregated. Lineally related motoneurons (MNs) and interneurons (INs) express many genes in common yet acquire distinct fates. For example, in mouse and chick Lhx3 plays a pivotal role in the development of both cell classes. Here, we utilize the ability to recognize individual zebrafish neurons to examine the roles of Lhx3 and its paralog Lhx4 in the development of MNs and ventral INs. We show that Lhx3 and Lhx4 are expressed by post-mitotic axial MNs derived from the MN progenitor (pMN) domain, p2 domain progenitors and by several types of INs derived from pMN and p2 domains. In the absence of Lhx3 and Lhx4, early-developing primary MNs (PMNs) adopt a hybrid fate, with morphological and molecular features of both PMNs and pMN-derived Kolmer-Agduhr' (KA') INs. In addition, we show that Lhx3 and Lhx4 distinguish the fates of two pMN-derived INs. Finally, we demonstrate that Lhx3 and Lhx4 are necessary for the formation of late-developing V2a and V2b INs. In conjunction with our previous work, these data reveal that distinct transcription factor families are deployed in post-mitotic MNs to unequivocally assign MN fate and suppress the development of alternative pMN-derived IN fates.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Interneurônios/fisiologia , Proteínas com Homeodomínio LIM/fisiologia , Neurônios Motores/fisiologia , Fatores de Transcrição/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Axônios/fisiologia , Linhagem da Célula , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/química , Neurônios/metabolismo , Oligonucleotídeos/química , Fenótipo , Estrutura Terciária de Proteína , Transdução de Sinais , Medula Espinal/embriologia , Peixe-Zebra/embriologiaRESUMO
Pigment patterns are useful for elucidating fundamental mechanisms of pattern formation and how these mechanisms evolve. In zebrafish, several pigment cell classes interact to generate stripes, yet the developmental requirements and origins of these cells remain poorly understood. Using zebrafish and a related species, we identified roles for thyroid hormone (TH) in pigment cell development and patterning, and in postembryonic development more generally. We show that adult pigment cells arise from distinct lineages having distinct requirements for TH and that differential TH dependence can evolve within lineages. Our findings demonstrate critical functions for TH in determining pigment pattern phenotype and highlight the potential for evolutionary diversification at the intersection of developmental and endocrine mechanisms.