RESUMO
Re-epithelialization of cutaneous wounds in adult mammals takes days to complete and relies on numerous signalling cues and multiple overlapping cellular processes that take place both within the epidermis and in other participating tissues. Re-epithelialization of partial- or full-thickness skin wounds of adult zebrafish, however, is extremely rapid and largely independent of the other processes of wound healing. Live imaging after treatment with transgene-encoded or chemical inhibitors reveals that re-epithelializing keratinocytes repopulate wounds by TGF-ß- and integrin-dependent lamellipodial crawling at the leading edges of the epidermal tongue. In addition, re-epithelialization requires long-range epithelial rearrangements, involving radial intercalations, flattening and directed elongation of cells - processes that are dependent on Rho kinase, JNK and, to some extent, planar cell polarity within the epidermis. These rearrangements lead to a massive recruitment of keratinocytes from the adjacent epidermis and make re-epithelialization independent of keratinocyte proliferation and the mitogenic effect of FGF signalling, which are only required after wound closure, allowing the epidermis outside the wound to re-establish its normal thickness. Together, these results demonstrate that the adult zebrafish is a valuable in vivo model for studying and visualizing the processes involved in cutaneous wound closure, facilitating the dissection of direct from indirect and motogenic from mitogenic effects of genes and molecules affecting wound re-epithelialization.
Assuntos
Envelhecimento/fisiologia , Embrião de Mamíferos/fisiologia , Mamíferos/embriologia , Reepitelização , Pele/patologia , Peixe-Zebra/fisiologia , Citoesqueleto de Actina/metabolismo , Animais , Movimento Celular , Proliferação de Células , Epiderme/patologia , Células Epiteliais/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Integrinas/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Queratinócitos/patologia , Morfogênese , Pseudópodes/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
Cilia are microtubule-based organelles that are present on most cells and are required for normal tissue development and function. Defective cilia cause complex syndromes with multiple organ manifestations termed ciliopathies. A crucial step during ciliogenesis in multiciliated cells (MCCs) is the association of future basal bodies with the apical plasma membrane, followed by their correct spacing and planar orientation. Here, we report a novel role for ELMO-DOCK1, which is a bipartite guanine nucleotide exchange factor complex for the small GTPase Rac1, and for the membrane-cytoskeletal linker Ezrin, in regulating centriole/basal body migration, docking and spacing. Downregulation of each component results in ciliopathy-related phenotypes in zebrafish and disrupted ciliogenesis in Xenopus epidermal MCCs. Subcellular analysis revealed a striking impairment of basal body docking and spacing, which is likely to account for the observed phenotypes. These results are substantiated by showing a genetic interaction between elmo1 and ezrin b. Finally, we provide biochemical evidence that the ELMO-DOCK1-Rac1 complex influences Ezrin phosphorylation and thereby probably serves as an important molecular switch. Collectively, we demonstrate that the ELMO-Ezrin complex orchestrates ciliary basal body migration, docking and positioning in vivo.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Corpos Basais/metabolismo , Cílios/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Axonema/metabolismo , Axonema/ultraestrutura , Membrana Celular/metabolismo , Cílios/ultraestrutura , Embrião não Mamífero/metabolismo , Embrião não Mamífero/ultraestrutura , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Modelos Biológicos , Fosforilação , Ligação Proteica , Xenopus laevis , Peixe-Zebra/embriologia , Proteínas rac de Ligação ao GTPRESUMO
Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein that is dynamically expressed in human and murine renal epithelia during development. The levels of EpCAM in the renal epithelium are upregulated both during regeneration after ischemia/reperfusion injury and in renal-derived carcinomas. The role of EpCAM in early kidney development, however, has remained unclear. The zebrafish pronephros shows a similar segmentation pattern to the mammalian metanephric nephron, and has recently emerged as a tractable model to study the regulatory programs governing early nephrogenesis. Since EpCAM shows persistent expression in the pronephros throughout early development, we developed a method to study the global changes in gene expression in specific pronephric segments of wild type and EpCAM-deficient zebrafish embryos. In epcam mutants, we found 379 differentially expressed genes. Gene ontology analysis revealed that EpCAM controls various developmental programs, including uretric bud development, morphogenesis of branching epithelium, regulation of cell differentiation and cilium morphogenesis.
Assuntos
Glicoproteínas de Membrana/metabolismo , Morfogênese/fisiologia , Pronefro/embriologia , Pronefro/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Pronefro/crescimento & desenvolvimento , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Morphogenesis of the semicircular canal ducts in the vertebrate inner ear is a dramatic example of epithelial remodelling in the embryo, and failure of normal canal development results in vestibular dysfunction. In zebrafish and Xenopus, semicircular canal ducts develop when projections of epithelium, driven by extracellular matrix production, push into the otic vesicle and fuse to form pillars. We show that in the zebrafish, extracellular matrix gene expression is high during projection outgrowth and then rapidly downregulated after fusion. Enzymatic disruption of hyaluronan in the projections leads to their collapse and a failure to form pillars: as a result, the ears swell. We have cloned a zebrafish mutant, lauscher (lau), identified by its swollen ear phenotype. The primary defect in the ear is abnormal projection outgrowth and a failure of fusion to form the semicircular canal pillars. Otic expression of extracellular matrix components is highly disrupted: several genes fail to become downregulated and remain expressed at abnormally high levels into late larval stages. The lau mutations disrupt gpr126, an adhesion class G protein-coupled receptor gene. Expression of gpr126 is similar to that of sox10, an ear and neural crest marker, and is partially dependent on sox10 activity. Fusion of canal projections and downregulation of otic versican expression in a hypomorphic lau allele can be restored by cAMP agonists. We propose that Gpr126 acts through a cAMP-mediated pathway to control the outgrowth and adhesion of canal projections in the zebrafish ear via the regulation of extracellular matrix gene expression.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Morfogênese/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Canais Semicirculares/embriologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , AMP Cíclico/metabolismo , Matriz Extracelular/metabolismo , Genótipo , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Hibridização In Situ , Repetições de Microssatélites/genética , Faloidina , Polimorfismo de Nucleotídeo Único/genética , Fatores de Transcrição SOXE/metabolismo , Canais Semicirculares/anormalidades , Análise de Sequência de DNA , Versicanas/metabolismoRESUMO
Recordings of the physiological history of cells provide insights into biological processes, yet obtaining such recordings is a challenge. To address this, we introduce a method to record transient cellular events for later analysis. We designed proteins that become labeled in the presence of both a specific cellular activity and a fluorescent substrate. The recording period is set by the presence of the substrate, whereas the cellular activity controls the degree of the labeling. The use of distinguishable substrates enabled the recording of successive periods of activity. We recorded protein-protein interactions, G protein-coupled receptor activation, and increases in intracellular calcium. Recordings of elevated calcium levels allowed selections of cells from heterogeneous populations for transcriptomic analysis and tracking of neuronal activities in flies and zebrafish.
Assuntos
Cálcio , Fenômenos Fisiológicos Celulares , Células , Coloração e Rotulagem , Animais , Corantes , Perfilação da Expressão Gênica , Peixe-Zebra , Células/química , Domínios e Motivos de Interação entre ProteínasRESUMO
NPHP4 mutations cause nephronophthisis, an autosomal recessive cystic kidney disease associated with renal fibrosis and kidney failure. The NPHP4 gene product nephrocystin-4 interacts with other nephrocystins, cytoskeletal and ciliary proteins; however, the molecular and cellular functions of nephrocystin-4 have remained elusive. Here we demonstrate that nephrocystin-4 is required for normal cloaca formation during zebrafish embryogenesis. Time-lapse imaging of the developing zebrafish pronephros revealed that tubular epithelial cells at the distal pronephros actively migrate between the yolk sac extension and the blood island towards the ventral fin fold to join the proctodeum and to form the cloaca. Nphp4-deficient pronephric duct cells failed to connect with their ectodermal counterparts, and instead formed a vesicle at the obstructed end of the pronephric duct. Nephrocystin-4 interacts with nephrocystin-1 and Par6. Depletion of zebrafish NPHP1 (nphp1) increased the incidence of cyst formation and randomization of the normal body axis, but did not augment cloaca malformation in nphp4-deficient zebrafish embryos. However, simultaneous depletion of zebrafish Par6 (pard6) aggravated cloaca formation defects in nphp4-depleted embryos, suggesting that nphp4 orchestrates directed cell migration and cloaca formation through interaction with the Par protein complex.
Assuntos
Cloaca/embriologia , Néfrons/embriologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Movimento Celular , Cílios/metabolismo , Cloaca/metabolismo , Cloaca/patologia , Clonagem Molecular , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Dados de Sequência Molecular , Néfrons/metabolismo , Néfrons/patologia , Fenótipo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genéticaRESUMO
Using forward genetics, we have identified the genes mutated in two classes of zebrafish fin mutants. The mutants of the first class are characterized by defects in embryonic fin morphogenesis, which are due to mutations in a Laminin subunit or an Integrin alpha receptor, respectively. The mutants of the second class display characteristic blistering underneath the basement membrane of the fin epidermis. Three of them are due to mutations in zebrafish orthologues of FRAS1, FREM1, or FREM2, large basement membrane protein encoding genes that are mutated in mouse bleb mutants and in human patients suffering from Fraser Syndrome, a rare congenital condition characterized by syndactyly and cryptophthalmos. Fin blistering in a fourth group of zebrafish mutants is caused by mutations in Hemicentin1 (Hmcn1), another large extracellular matrix protein the function of which in vertebrates was hitherto unknown. Our mutant and dose-dependent interaction data suggest a potential involvement of Hmcn1 in Fraser complex-dependent basement membrane anchorage. Furthermore, we present biochemical and genetic data suggesting a role for the proprotein convertase FurinA in zebrafish fin development and cell surface shedding of Fras1 and Frem2, thereby allowing proper localization of the proteins within the basement membrane of forming fins. Finally, we identify the extracellular matrix protein Fibrillin2 as an indispensable interaction partner of Hmcn1. Thus we have defined a series of zebrafish mutants modelling Fraser Syndrome and have identified several implicated novel genes that might help to further elucidate the mechanisms of basement membrane anchorage and of the disease's aetiology. In addition, the novel genes might prove helpful to unravel the molecular nature of thus far unresolved cases of the human disease.
Assuntos
Embrião não Mamífero/metabolismo , Proteínas da Matriz Extracelular/genética , Síndrome de Frasier/genética , Furina/genética , Mutação , Pró-Proteína Convertases/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas da Matriz Extracelular/metabolismo , Furina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Dados de Sequência Molecular , Pró-Proteína Convertases/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Mutations of the immunoglobulin superfamily proteins nephrin and Neph1 lead to congenital nephrotic syndrome in humans or mice. Neph proteins are three closely related molecules that are evolutionarily conserved and mediate cell recognition. Their importance for morphogenetic processes including the formation of the kidney filtration barrier in vertebrates and synaptogenesis in Caenorhabditis elegans has recently been uncovered. However, the individual morphogenetic function of mammalian Neph1-3 isoforms remained elusive. We demonstrate now that the Neph/nephrin family proteins can form cell-cell adhesion modules across species. Expression of all three mammalian Neph isoforms partially rescued mutant C. elegans lacking their Neph homolog syg-1 and restored synapse formation, suggesting a functional redundancy between the three isoforms. Strikingly, the rescue of defective synaptic connectivity was prevented by deletion of the highly conserved cytoplasmic PSD95/Dlg/ZO-1-binding motif of SYG-1/Neph proteins, indicating the critical role of this intracellular signaling motif for SYG-1/Neph-dependent morphogenetic events. To determine the significance of Neph isoform redundancy for vertebrate kidney development, we analyzed the expression pattern and the functional role of Neph proteins in zebrafish. In situ hybridizations identified zNeph1 and zNeph2 as glomerular proteins. Morpholino knockdown of either zNeph1 or zNeph2 resulted in loss of slit diaphragms and leakiness of the glomerular filtration barrier. This is the first report utilizing C. elegans to study mammalian Neph/nephrin protein function and to demonstrate a functional overlap of Neph1-3 proteins. Furthermore, we identify Neph2 as a novel critical regulator of glomerular function, indicating that both Neph1 and Neph2 are required for glomerular maintenance and development.
Assuntos
Imunoglobulinas/fisiologia , Rim/crescimento & desenvolvimento , Proteínas de Membrana/fisiologia , Neurônios/fisiologia , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/classificação , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiologia , Adesão Celular , Células HeLa , Humanos , Imunoglobulinas/classificação , Imunoglobulinas/genética , Proteínas de Membrana/classificação , Proteínas de Membrana/genética , Camundongos , Modelos Animais , Morfogênese/genética , Domínios PDZ , FilogeniaRESUMO
The aberrant expression of the transmembrane protein EpCAM is associated with tumor progression, affecting different cellular processes such as cell-cell adhesion, migration, proliferation, differentiation, signaling, and invasion. However, the in vivo function of EpCAM still remains elusive due to the lack of genetic loss-of-function studies. Here, we describe epcam (tacstd) null mutants in zebrafish. Maternal-zygotic mutants display compromised basal protrusive activity and epithelial morphogenesis in cells of the enveloping layer (EVL) during epiboly. In partial redundancy with E-cadherin (Ecad), EpCAM made by EVL cells is further required for cell-cell adhesion within the EVL and, possibly, for proper attachment of underlying deep cells to the inner surface of the EVL, thereby also affecting deep cell epiboly movements. During later development, EpCAM per se becomes indispensable for epithelial integrity within the periderm of the skin, secondarily leading to disrupted morphology of the underlying basal epidermis and moderate hyper-proliferation of skin cells. On the molecular level, EVL cells of epcam mutant embryos display reduced levels of membranous Ecad, accompanied by an enrichment of tight junction proteins and a basal extension of apical junction complexes (AJCs). Our data suggest that EpCAM acts as a partner of E-cadherin to control adhesiveness and integrity as well as plasticity and morphogenesis within simple epithelia. In addition, EpCAM is required for the interaction of the epithelia with underlying cell layers.
Assuntos
Antígenos de Neoplasias/fisiologia , Moléculas de Adesão Celular/fisiologia , Epitélio/crescimento & desenvolvimento , Glicoproteínas de Membrana/fisiologia , Morfogênese , Pele/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/fisiologia , Animais , Caderinas/fisiologia , Adesão Celular , Embrião não Mamífero , Molécula de Adesão da Célula Epitelial , Epitélio/embriologia , Pele/embriologia , Peixe-ZebraRESUMO
Kidney injury is a common complication of severe disease. Here, we report that injuries of the zebrafish embryonal kidney are rapidly repaired by a migratory response in 2-, but not in 1-day-old embryos. Gene expression profiles between these two developmental stages identify cxcl12a and myca as candidates involved in the repair process. Zebrafish embryos with cxcl12a, cxcr4b, or myca deficiency display repair abnormalities, confirming their role in response to injury. In mice with a kidney-specific knockout, Cxcl12 and Myc gene deletions suppress mitochondrial metabolism and glycolysis, and delay the recovery after ischemia/reperfusion injury. Probing these observations in zebrafish reveal that inhibition of glycolysis slows fast migrating cells and delays the repair after injury, but does not affect the slow cell movements during kidney development. Our findings demonstrate that Cxcl12 and Myc facilitate glycolysis to promote fast migratory responses during development and repair, and potentially also during tumor invasion and metastasis.
Assuntos
Quimiocina CXCL12/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Nefropatias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Movimento Celular , Metabolismo Energético , Deleção de Genes , Perfilação da Expressão Gênica , Glicólise , Homeostase , Rim/lesões , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Tretinoína/químicaRESUMO
In most animals, primordial germ cell (PGC) specification and development depend on maternally provided cytoplasmic determinants that constitute the so-called germ plasm. Little is known about the role of germ plasm in vertebrate germ cell development, and its molecular mode of action remains elusive. While PGC specification in mammals occurs via different mechanisms, several germ plasm components required for early PGC development in lower organisms are expressed in mammalian germ cells after their migration to the gonad and are involved in gametogenesis. Here we show that the RNA of dead end, encoding a novel putative RNA binding protein, is a component of the germ plasm in zebrafish and is specifically expressed in PGCs throughout embryogenesis; Dead End protein is localized to perinuclear germ granules within PGCs. Knockdown of dead end blocks confinement of PGCs to the deep blastoderm shortly after their specification and results in failure of PGCs to exhibit motile behavior and to actively migrate thereafter. PGCs subsequently die, while somatic development is not effected. We have identified dead end orthologs in other vertebrates including Xenopus, mouse, and chick, where they are expressed in germ plasm and germ-line cells, suggesting a role in germ-line development in these organisms as well.
Assuntos
Células Germinativas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Sequência de Bases , Movimento Celular , Sobrevivência Celular , Clonagem Molecular , DNA Complementar/genética , Células Germinativas/citologia , Hibridização In Situ , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos Antissenso/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Genetic access to small, reproducible sets of neurons is key to an understanding of the functional wiring of the brain. Here we report the generation of a new Gal4- and Cre-driver resource for zebrafish neurobiology. Candidate genes, including cell type-specific transcription factors, neurotransmitter-synthesizing enzymes and neuropeptides, were selected according to their expression patterns in small and unique subsets of neurons from diverse brain regions. BAC recombineering, followed by Tol2 transgenesis, was used to generate driver lines that label neuronal populations in patterns that, to a large but variable extent, recapitulate the endogenous gene expression. We used image registration to characterize, compare, and digitally superimpose the labeling patterns from our newly generated transgenic lines. This analysis revealed highly restricted and mutually exclusive tissue distributions, with striking resolution of layered brain regions such as the tectum or the rhombencephalon. We further show that a combination of Gal4 and Cre transgenes allows intersectional expression of a fluorescent reporter in regions where the expression of the two drivers overlaps. Taken together, our study offers new tools for functional studies of specific neural circuits in zebrafish.
Assuntos
Encéfalo/fisiologia , Cromossomos Artificiais Bacterianos , Marcação de Genes , Neurônios/fisiologia , Transgenes , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/metabolismoRESUMO
The importance of the aquaculture production is increasing with the declining global fish stocks, but early sexual maturation in several farmed species reduces muscle growth and quality, and escapees could have a negative impact on wild populations. A possible solution to these problems is the production of sterile fish by ablation of the embryonic primordial germ cells (PGCs), a technique developed in zebrafish. Cell-specific regulation of mRNA stability is crucial for proper specification of the germ cell lineage and commonly involves microRNA (miRNA)-mediated degradation of targeted mRNAs in somatic cells. This study reports on the functional roles of conserved motifs in the 3' untranslated region (UTR) of the miRNA target gene nanos3 identified in Atlantic cod, Atlantic salmon, and zebrafish. The 3'UTR of cod nanos3 was sufficient for targeting the expression of green fluorescent protein (GFP) to the presumptive PGCs in injected embryos of the three phylogenetically distant species. 3'UTR elements of importance for PGC-specific expression were further examined by fusing truncated 3'UTR variants of cod nanos3 to GFP followed by injections in zebrafish embryos. The expression patterns of the GFP constructs in PGCs and somatic cells suggested that the proximal U-rich region is responsible for the PGC-specific stabilization of the endogenous nanos3 mRNA. Morpholino-mediated downregulation of the RNA-binding protein Dead end (DnD), a PGC-specific inhibitor of miRNA action, abolished the fluorescence of the PGCs in cod and zebrafish embryos, suggesting a conserved DnD-dependent mechanism for germ cell survival and migration.
Assuntos
Aquicultura/métodos , Peixes/fisiologia , Células Germinativas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Esterilização Reprodutiva/veterinária , Regiões 3' não Traduzidas/genética , Animais , Peixes/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Ligação a RNA/genética , Especificidade da Espécie , Esterilização Reprodutiva/métodosRESUMO
Bardet-Biedl syndrome (BBS) and nephronophthisis (NPH) are hereditary autosomal recessive disorders, encoded by two families of diverse genes. BBS and NPH display several overlapping phenotypes including cystic kidney disease, retinitis pigmentosa, liver fibrosis, situs inversus and cerebellar defects. Since most of the BBS and NPH proteins localize to cilia and/or their appendages, BBS and NPH are considered ciliopathies. In this study, we characterized the function of the transcription factor Nphp7 in zebrafish, and addressed the molecular connection between BBS and NPH. The knockdown of zebrafish bbs1 and nphp7.2 caused similar phenotypic changes including convergent extension defects, curvature of the body axis, hydrocephalus, abnormal heart looping and cystic pronephros, all consistent with an altered ciliary function. Immunoprecipitation assays revealed a physical interaction between BBS1 and NPHP7, and the simultaneous knockdown of zbbs1 and znphp7.2 enhanced the cystic pronephros phenotype synergistically, suggesting a genetic interaction between zbbs1 and znphp7.2 in vivo. Deletion of zBbs1 or zNphp7.2 did not compromise cilia formation, but disrupted cilia motility. Although NPHP7 has been shown to act as transcriptional repressor, our studies suggest a crosstalk between BBS1 and NPHP7 in regulating normal function of the cilium.
Assuntos
Cílios/metabolismo , Cílios/fisiologia , Proteínas Nucleares/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Proteínas Nucleares/genética , Ligação Proteica , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Upon injury, the skin must quickly regenerate to regain its barrier function. In mammals, wound healing is rapid and scar free during embryogenesis, whereas in adults it involves multiple steps including blood clotting, inflammation, re-epithelialization, vascularization, and granulation tissue formation and maturation, resulting in a scar. We have established a rapid and robust method to introduce full-thickness wounds onto the flank of adult zebrafish, and show that apart from external fibrin clot formation, all steps of adult mammalian wound repair also exist in zebrafish. Wound re-epithelialization is extremely rapid and initiates with no apparent lag phase, subsequently followed by the immigration of inflammatory cells and the formation of granulation tissue, consisting of macrophages, fibroblasts, blood vessels, and collagen. The granulation tissue later regresses, resulting in minimal scar formation. Studies after chemical treatment or with transgenic fish further suggest that wound re-epithelialization occurs independently of inflammation and fibroblast growth factor signaling, whereas both are essential for fibroblast recruitment and granulation tissue formation. Together, these results demonstrate that major steps and principles of cutaneous wound healing are conserved among adult mammals and adult zebrafish, making zebrafish a valuable model for studying vertebrate skin repair.
Assuntos
Cicatriz/fisiopatologia , Modelos Animais de Doenças , Pele/lesões , Cicatrização/fisiologia , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Coagulação Sanguínea/fisiologia , Cicatriz/patologia , Dermatite/patologia , Dermatite/fisiopatologia , Células Epiteliais/fisiologia , Fatores de Crescimento de Fibroblastos/metabolismo , Tecido de Granulação/fisiologia , Neovascularização Fisiológica/fisiologia , Transdução de Sinais/fisiologia , Pele/embriologia , Pele/patologiaRESUMO
Dead end (dnd) is a vertebrate-specific component of the germ plasm and germ-cell granules that is crucial for germ-cell development in zebrafish and mouse. Dnd counteracts the inhibitory function of miRNAs, thereby facilitating the expression of proteins such as Nanos and Tdrd7 in the germ cells. Here, we show that cis-acting elements within dnd mRNA and the RNA recognition motive (RRM) of the protein are essential for targeting protein expression to the germ cells and to the perinuclear granules, respectively. We demonstrate that as it executes its function, Dnd translocates between the germ-cell nucleus and germ-cell granules. This phenomenon is not observed in proteins mutated in the RRM motif, correlating with loss of function of Dnd. Based on molecular modeling, we identify the putative RNA binding domain of Dnd as a canonical RRM and propose that this domain is important for protein subcellular localization and function.
Assuntos
MicroRNAs/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Regiões 3' não Traduzidas/metabolismo , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Transporte de RNA , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Deleção de Sequência , Homologia Estrutural de Proteína , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genéticaRESUMO
MicroRNAs (miRNAs) are inhibitors of gene expression capable of controlling processes in normal development and cancer. In mammals, miRNAs use a seed sequence of 6-8 nucleotides (nt) to associate with 3' untranslated regions (3'UTRs) of mRNAs and inhibit their expression. Intriguingly, occasionally not only the miRNA-targeting site but also sequences in its vicinity are highly conserved throughout evolution. We therefore hypothesized that conserved regions in mRNAs may serve as docking platforms for modulators of miRNA activity. Here we demonstrate that the expression of dead end 1 (Dnd1), an evolutionary conserved RNA-binding protein (RBP), counteracts the function of several miRNAs in human cells and in primordial germ cells of zebrafish by binding mRNAs and prohibiting miRNAs from associating with their target sites. These effects of Dnd1 are mediated through uridine-rich regions present in the miRNA-targeted mRNAs. Thus, our data unravel a novel role of Dnd1 in protecting certain mRNAs from miRNA-mediated repression.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Células Germinativas/metabolismo , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica , Proteínas de Peixe-Zebra/metabolismo , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Conexina 43/genética , Conexina 43/metabolismo , Sequência Conservada , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Humanos , Dados de Sequência Molecular , Mutação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ligação a RNA/genética , Sequências Reguladoras de Ácido Ribonucleico , Transfecção , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
The progenitors of the gametes, the primordial germ cells (PGCs) are typically specified early in the development in positions, which are distinct from the gonad. These cells then migrate toward the gonad where they differentiate into sperms and eggs. Here, we study the role of the germ cells in somatic development and particularly the role of the germ line in the sex differentiation in zebrafish. To this end, we ablated the germ cells using two independent methods and followed the development of the experimental fish. First, PGCs were ablated by knocking down the function of dead end, a gene important for the survival of this lineage. Second, a method to eliminate the PGCs using the toxin-antitoxin components of the parD bacterial genetic system was used. Specifically, we expressed a bacterial toxin Kid preferentially in the PGCs and at the same time protected somatic cells by uniformly expressing the specific antidote Kis. Our results demonstrate an unexpected role for the germ line in promoting female development because PGC-ablated fish invariably developed as males.
Assuntos
Óvulo/fisiologia , Processos de Determinação Sexual , Espermatozoides/fisiologia , Peixe-Zebra/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxina Diftérica/farmacologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Feminino , Masculino , Óvulo/efeitos dos fármacos , Espermatozoides/efeitos dos fármacosRESUMO
MicroRNAs (miRNAs) represent a family of small, regulatory, noncoding RNAs that are found in plants and animals. Here, we describe the miRNA profile of the zebrafish Danio rerio resolved in a developmental and cell-type-specific manner. The profiles were obtained from larger-scale sequencing of small RNA libraries prepared from developmentally staged zebrafish, and two adult fibroblast cell lines derived from the caudal fin (ZFL) and the liver epithelium (SJD). We identified a total of 154 distinct miRNAs expressed from 343 miRNA genes. Other experimental/computational sources support an additional 10 miRNAs encoded by 19 genes. The miRNAs can be classified into 87 distinct families. Cross-species comparison indicates that 81 families are conserved in mammals, 17 of which also have at least one member conserved in an invertebrate. Our analysis reveals that the zygotes are essentially devoid of miRNAs and that their expression begins during the blastula period with a zebrafish-specific family of miRNAs encoded by closely spaced multicopy genes. Computational predictions of zebrafish miRNA targets are provided that take into account the depth of evolutionary conservation. Besides miRNAs, we identified a prominent class of repeat-associated small interfering RNAs (rasiRNAs).