RESUMO
Transglutaminase 2 (TG2) is a multifunctional protein widely distributed in various tissues and involved in many physiological and pathological processes. However, its actual role in biological processes is often controversial as TG2 shows different effects in these processes depending on its localization, cell type, or experimental conditions. We characterized the enzymatic and functional properties of TG2 proteins expressed in Danio rerio (zebrafish) to provide the basis for using this established animal model as a reliable tool to characterize TG2 functions in vivo. We confirmed the existence of three genes orthologous to human TG2 (zTGs2) in the zebrafish genome and their expression and function during embryonic development. We produced and purified the zTGs2s as recombinant proteins and showed that, like the human enzyme, zTGs2 catalyzes a Ca2+ dependent transamidation reaction that can be inhibited with TG2-specific inhibitors. In a cell model of human fibroblasts, we also demonstrated that zTGs2 can mediate RGD-independent cell adhesion in the extracellular environment. Finally, we transfected and selected zTGs2-overexpressing HEK293 cells and demonstrated that intracellular zTGs2 plays a very comparable protective/damaging role in the apoptotic process, as hTG2. Overall, our results suggest that zTGs2 proteins behave very similarly to the human ortholog and pave the way for future in vivo studies of TG2 functions in zebrafish.
Assuntos
Proteína 2 Glutamina gama-Glutamiltransferase , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Humanos , Apoptose/genética , Catálise , Adesão Celular , Fibroblastos , Expressão Gênica , Células HEK293 , Filogenia , Conformação Proteica , Proteína 2 Glutamina gama-Glutamiltransferase/química , Proteína 2 Glutamina gama-Glutamiltransferase/classificação , Proteína 2 Glutamina gama-Glutamiltransferase/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/classificação , Proteínas de Peixe-Zebra/genéticaRESUMO
Aminoacyl tRNA synthetases (ARSs) are a group of proteins, acting as transporters to transfer and attach the appropriate amino acids onto their cognate tRNAs for translation. So far, 18 out of 20 cytoplasmic ARSs are reported to be connected to different neuropathy disorders with multi-organ defects that are often accompanied with developmental delays. Thus, it is important to understand functions and impacts of ARSs at the whole organism level. Here, we systematically analyzed the spatiotemporal expression of 14 ars and 2 aimp genes during development in zebrafish that have not be previously reported. Not only in the brain, their dynamic expression patterns in several tissues such as in the muscles, liver and intestine suggest diverse roles in a wide range of development processes in addition to neuronal function, which is consistent with potential involvement in multiple syndrome diseases associated with ARS mutations. In particular, hinted by its robust expression pattern in the brain, we confirmed that aimp1 is required for the formation of cerebrovasculature by a loss-of-function approach. Overall, our systematic profiling data provides a useful basis for studying roles of ARSs during development and understanding their potential functions in the etiology of related diseases.
Assuntos
Aminoacil-tRNA Sintetases/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/genética , RNA de Transferência/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Aminoacil-tRNA Sintetases/classificação , Aminoacil-tRNA Sintetases/metabolismo , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Embrião não Mamífero , Perfilação da Expressão Gênica , Ontologia Genética , Humanos , Intestinos/crescimento & desenvolvimento , Intestinos/metabolismo , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Anotação de Sequência Molecular , Morfolinos/administração & dosagem , Morfolinos/genética , Morfolinos/metabolismo , Músculos/metabolismo , Proteínas do Tecido Nervoso/classificação , Proteínas do Tecido Nervoso/metabolismo , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/metabolismo , Transtornos do Neurodesenvolvimento/patologia , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/classificação , Proteínas de Peixe-Zebra/metabolismoRESUMO
GDF15 is frequently detected in patients suffering from various diseases, especially those associated with pro-inflammatory processes and/or metabolic disorders. Accordingly, sepsis, whose major complications are related to metabolic alterations and systemic inflammation, significantly increases the secretion of GDF15. Indeed, this cytokine could be considered a marker of sepsis severity. However, until the last several years, the involvement of GDF15 in these disorders had not been widely characterized. In mice, GDF15 was recently described as a pivotal inducer of sepsis tolerance by mediating metabolic alterations that reduce tissue damage. In this work we describe a zebrafish gdf15 gene. We found that gdf15 follows an expression pattern similar to that observed in mammals, being highly expressed in the liver and kidney and induced after pro-inflammatory stimuli. Moreover, larvae overexpressing gdf15 were more resistant to bacterial and viral challenges without affecting the pathogen load. Consequently, Gdf15 also protected zebrafish larvae against LPS-induced mortality. As in mice, zebrafish Gdf15 seems to induce sepsis tolerance by altering the metabolic parameters of the individuals.
Assuntos
Modelos Animais de Doenças , Fator 15 de Diferenciação de Crescimento/genética , Sepse/genética , Proteínas de Peixe-Zebra/genética , Aeromonas hydrophila/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Perfilação da Expressão Gênica/métodos , Fator 15 de Diferenciação de Crescimento/classificação , Fator 15 de Diferenciação de Crescimento/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Larva/genética , Larva/metabolismo , Larva/microbiologia , Lipopolissacarídeos , Camundongos , Filogenia , Sepse/induzido quimicamente , Sepse/microbiologia , Análise de Sobrevida , Proteínas de Peixe-Zebra/classificação , Proteínas de Peixe-Zebra/metabolismoRESUMO
Collagen VI (ColVI) is an extracellular matrix (ECM) protein involved in a range of physiological and pathological conditions. Zebrafish (Danio rerio) is a powerful model organism for studying vertebrate development and for in vivo analysis of tissue patterning. Here, we performed a thorough characterization of ColVI gene and protein expression in zebrafish during development and adult life. Bioinformatics analyses confirmed that zebrafish genome contains single genes encoding for α1(VI), α2(VI) and α3(VI) ColVI chains and duplicated genes encoding for α4(VI) chains. At 1 day post-fertilization (dpf) ColVI transcripts are expressed in myotomes, pectoral fin buds and developing epidermis, while from 2 dpf abundant transcript levels are present in myosepta, pectoral fins, axial vasculature, gut and craniofacial cartilage elements. Using newly generated polyclonal antibodies against zebrafish α1(VI) protein, we found that ColVI deposition in adult fish delineates distinct domains in the ECM of several organs, including cartilage, eye, skin, spleen and skeletal muscle. Altogether, these data provide the first detailed characterization of ColVI expression and ECM deposition in zebrafish, thus paving the way for further functional studies in this species.
Assuntos
Colágeno Tipo I/genética , Colágeno Tipo VI/genética , Regulação da Expressão Gênica no Desenvolvimento , Larva/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Colágeno Tipo I/classificação , Colágeno Tipo VI/classificação , Hibridização In Situ , Larva/crescimento & desenvolvimento , Músculo Esquelético/embriologia , Músculo Esquelético/crescimento & desenvolvimento , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/embriologia , Pele/crescimento & desenvolvimento , Análise Espaço-Temporal , Peixe-Zebra/embriologia , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/classificaçãoRESUMO
The zebrafish sensory posterior lateral line is an excellent model system to study collective cell migration and organogenesis. Shootin1 is a cytoplasmic protein involved in neuronal polarization and axon guidance. Previous studies have shown that shootin1 couples actin filament retrograde flow with extracellular adhesive substrates at the leading edge of axonal growth cones, thereby producing mechanical force for the migration and guidance of axonal growth cones. However, the functions of shootin in peripheral cells remain unknown. Here we identified two novel shootin family members, shootin2 and shootin3. In zebrafish, shootin1 and shootin3 are expressed in the posterior lateral line primordium (PLLP) and neuromasts during embryonic development. A shootin1 mutant displayed a reduced speed of PLLP migration, while shootin1;shootin3 double mutation inhibited cell proliferation in the PLLP. Furthermore, our results suggest that shootin1 and shootin3 positively regulate the number of neuromasts and the number of cells in deposited neuromasts. Our study demonstrates that shootins mediate collective cell migration of the posterior lateral line primordium and formation of neuromasts in zebrafish.
Assuntos
Proteínas de Transporte/metabolismo , Sistema da Linha Lateral/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Actinas/metabolismo , Animais , Proteínas de Transporte/classificação , Proteínas de Transporte/genética , Movimento Celular , Desenvolvimento Embrionário , Edição de Genes , Microscopia de Fluorescência , Neurônios/fisiologia , Organogênese , Filogenia , Ligação Proteica , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/classificação , Proteínas de Peixe-Zebra/genéticaRESUMO
Severe local acidosis causes tissue damage and pain, and is one of the hallmarks of many diseases including ischemia, cancer, and inflammation. However, the molecular mechanisms of the cellular response to acid are not fully understood. We performed an unbiased RNA interference screen and identified PAC (TMEM206) as being essential for the widely observed proton-activated Cl- (PAC) currents (I Cl,H). Overexpression of human PAC in PAC knockout cells generated I Cl,H with the same characteristics as the endogenous ones. Zebrafish PAC encodes a PAC channel with distinct properties. Knockout of mouse Pac abolished I Cl,H in neurons and attenuated brain damage after ischemic stroke. The wide expression of PAC suggests a broad role for this conserved Cl- channel family in physiological and pathological processes associated with acidic pH.
Assuntos
Canais de Cloreto/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Cálcio/metabolismo , Morte Celular , Canais de Cloreto/classificação , Canais de Cloreto/genética , Cloretos/metabolismo , Sequência Conservada , Evolução Molecular , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Hipóxia-Isquemia Encefálica/metabolismo , Hipóxia-Isquemia Encefálica/patologia , Proteínas de Membrana/classificação , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Neurônios/patologia , Filogenia , Interferência de RNA , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , Peixe-Zebra , Proteínas de Peixe-Zebra/classificação , Proteínas de Peixe-Zebra/genéticaRESUMO
Scrutiny of the zebrafish (Danio rerio) genomic database confirmed eight functional vitellogenin (vtg) genes, each with one or two transcript variants, and the encoded Vtg polypeptides were structurally and functionally characterized in detail by in silico and experimental analyses. There were five type I (vtgs1, 4, 5, 6, and 7), two type II (vtg2 and vtg8), and one type III (vtg3) vtg gene(s) encoding three major types of Vtg protein based on subdomain structure (Vtg-I, Vtg-II, and Vtg-III, respectively). Among various tissues of mature zebrafish, transcripts of the eight vtg genes were detected by RNA-Seq only in liver and intestine, with liver being the main site of vtg expression. All vtg transcripts except vtg8 were also detected in mature female liver by RT-qPCR. The relative abundances of Vtg proteins and their variants were quantified by LC-MS/MS in the liver of mature females and in eggs. The Vtgs were generally several fold more abundant in eggs, but profiles of abundance of the 19 different forms of Vtg evaluated were otherwise similar in liver and eggs, suggesting that yolk protein composition is determined largely by hepatic Vtg synthesis and secretion. Based on transcript and protein levels, Vtg-I is, by far, the dominant type of Vtg in zebrafish, followed by Vtg-II and then Vtg-III. When relative abundances of the different forms of Vtg were evaluated by LC-MS/MS in egg batches of good versus poor quality, no differences in the proportional abundance of individual forms of Vtg, or of different Vtg types, attributable to egg quality were observed.
Assuntos
Vitelogeninas/genética , Vitelogeninas/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Animais , Feminino , Expressão Gênica , Fígado/metabolismo , Masculino , Família Multigênica , Óvulo/metabolismo , Domínios Proteicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Distribuição Tecidual , Vitelogeninas/classificação , Proteínas de Peixe-Zebra/classificaçãoRESUMO
Tmem88a is a transmembrane protein that is thought to be a negative regulator of the Wnt signalling pathway. Several groups have used antisense morpholino oligonucleotides in an effort to characterise the role of tmem88a in zebrafish cardiovascular development, but they have not obtained consistent results. Here, we generate an 8 bp deletion in the coding region of the tmem88a locus using TALENs, and we have gone on to establish a viable homozygous tmem88aΔ8 mutant line. Although tmem88aΔ8 mutants have reduced expression of some key haematopoietic genes, differentiation of erythrocytes and neutrophils is unaffected, contradicting our previous study using antisense morpholino oligonucleotides. We find that expression of the tmem88a paralogue tmem88b is not significantly changed in tmem88aΔ8 mutants and injection of the tmem88a splice-blocking morpholino oligonucleotide into tmem88aΔ8 mutants recapitulates the reduction of erythrocytes observed in morphants using o-Dianisidine. This suggests that there is a partial, but inessential, requirement for tmem88a during haematopoiesis and that morpholino injection exacerbates this phenotype in tmem88a morpholino knockdown embryos.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes/métodos , Proteínas de Membrana/genética , Mutação , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Embrião não Mamífero/irrigação sanguínea , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Sistema Hematopoético/embriologia , Sistema Hematopoético/metabolismo , Hibridização In Situ , Proteínas de Membrana/classificação , Morfolinos/genética , Fenótipo , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/classificaçãoRESUMO
BACKGROUND: SLC22 protein family is a member of the SLC (Solute carriers) superfamily of polyspecific membrane transporters responsible for uptake of a wide range of organic anions and cations, including numerous endo- and xenobiotics. Due to the lack of knowledge on zebrafish Slc22 family, we performed initial characterization of these transporters using a detailed phylogenetic and conserved synteny analysis followed by the tissue specific expression profiling of slc22 transcripts. RESULTS: We identified 20 zebrafish slc22 genes which are organized in the same functional subgroups as human SLC22 members. Orthologies and syntenic relations between zebrafish and other vertebrates revealed consequences of the teleost-specific whole genome duplication as shown through one-to-many orthologies for certain zebrafish slc22 genes. Tissue expression profiles of slc22 transcripts were analyzed using qRT-PCR determinations in nine zebrafish tissues: liver, kidney, intestine, gills, brain, skeletal muscle, eye, heart, and gonads. Our analysis revealed high expression of oct1 in kidney, especially in females, followed by oat3 and oat2c in females, oat2e in males and orctl4 in females. oct1 was also dominant in male liver. oat2d showed the highest expression in intestine with less noticeable gender differences. All slc22 genes showed low expression in gills, and moderate expression in heart and skeletal muscle. Dominant genes in brain were oat1 in females and oct1 in males, while the highest gender differences were determined in gonads, with dominant expression of almost all slc22 genes in testes and the highest expression of oat2a. CONCLUSIONS: Our study offers the first insight into the orthology relationships, gene expression and potential role of Slc22 membrane transporters in zebrafish. Clear orthological relationships of zebrafish slc22 and other vertebrate slc22 genes were established. slc22 members are mostly highly conserved, suggesting their physiological and toxicological importance. One-to-many orthologies and differences in tissue expression patterns of zebrafish slc22 genes in comparison to human orthologs were observed. Our expression data point to partial similarity of zebrafish versus human Slc22 members, with possible compensatory roles of certain zebrafish transporters, whereas higher number of some orthologs implies potentially more diverse and specific roles of these proteins in zebrafish.
Assuntos
Proteínas de Transporte de Cátions Orgânicos/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Mapeamento Cromossômico , Feminino , Humanos , Masculino , Proteínas de Transporte de Cátions Orgânicos/classificação , Proteínas de Transporte de Cátions Orgânicos/genética , Filogenia , Ligação Proteica , RNA/isolamento & purificação , RNA/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Distribuição Tecidual , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Transcriptoma , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/classificação , Proteínas de Peixe-Zebra/genéticaRESUMO
Antigen processing and presentation genes found within the MHC are among the most highly polymorphic genes of vertebrate genomes, providing populations with diverse immune responses to a wide array of pathogens. Here, we describe transcriptome, exome, and whole-genome sequencing of clonal zebrafish, uncovering the most extensive diversity within the antigen processing and presentation genes of any species yet examined. Our CG2 clonal zebrafish assembly provides genomic context within a remarkably divergent haplotype of the core MHC region on chromosome 19 for six expressed genes not found in the zebrafish reference genome: mhc1uga, proteasome-ß 9b (psmb9b), psmb8f, and previously unknown genes psmb13b, tap2d, and tap2e We identify ancient lineages for Psmb13 within a proteasome branch previously thought to be monomorphic and provide evidence of substantial lineage diversity within each of three major trifurcations of catalytic-type proteasome subunits in vertebrates: Psmb5/Psmb8/Psmb11, Psmb6/Psmb9/Psmb12, and Psmb7/Psmb10/Psmb13. Strikingly, nearby tap2 and MHC class I genes also retain ancient sequence lineages, indicating that alternative lineages may have been preserved throughout the entire MHC pathway since early diversification of the adaptive immune system â¼500 Mya. Furthermore, polymorphisms within the three MHC pathway steps (antigen cleavage, transport, and presentation) are each predicted to alter peptide specificity. Lastly, comparative analysis shows that antigen processing gene diversity is far more extensive than previously realized (with ancient coelacanth psmb8 lineages, shark psmb13, and tap2t and psmb10 outside the teleost MHC), implying distinct immune functions and conserved roles in shaping MHC pathway evolution throughout vertebrates.
Assuntos
Evolução Biológica , Cisteína Endopeptidases/genética , Genoma , Haplótipos , Antígenos de Histocompatibilidade Classe I/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Apresentação de Antígeno , Clonagem de Organismos , Cisteína Endopeptidases/classificação , Cisteína Endopeptidases/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Antígenos de Histocompatibilidade Classe I/classificação , Antígenos de Histocompatibilidade Classe I/imunologia , Filogenia , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/imunologia , Isoformas de Proteínas/classificação , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Transcriptoma , Peixe-Zebra/classificação , Peixe-Zebra/imunologia , Proteínas de Peixe-Zebra/classificação , Proteínas de Peixe-Zebra/imunologiaRESUMO
Multidrug and toxin extrusion (MATE) proteins are involved in the extrusion of endogenous compounds and xenobiotics across the plasma membrane. They are conserved from bacteria to mammals, with different numbers of genes within groups. Here, we present the first data on identification and functional characterization of Mate proteins in zebrafish (Danio rerio). Phylogenetic analysis revealed six Mates in teleost fish, annotated as Mate3-8, which form a distinct cluster separated from the tetrapod MATEs/Mates. Synteny analysis showed that zebrafish mate genes are orthologous to human MATEs. Gene expression analysis revealed that all the mate transcripts were constitutively and differentially expressed during embryonic development, followed by pronounced and tissue-specific expression in adults. Functional analyses were performed using transport activity assays with model substrates after heterologous overexpression of five zebrafish Mates in HEK293T cells. The results showed that zebrafish Mates interact with both physiological and xenobiotic substances but also substantially differ with respect to the interacting compounds and interaction strength in comparison to mammalian MATEs/Mates. Taken together, our data clearly indicate a potentially important role for zebrafish Mate transporters in zebrafish embryos and adults and provide a basis for detailed functional characterizations of single zebrafish Mate transporters.
Assuntos
Proteínas de Transporte de Cátions Orgânicos/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Clonagem Molecular , Feminino , Expressão Gênica , Células HEK293 , Humanos , Cinética , Fígado/metabolismo , Masculino , Proteínas de Transporte de Cátions Orgânicos/classificação , Proteínas de Transporte de Cátions Orgânicos/genética , Filogenia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/classificação , Proteínas de Peixe-Zebra/genéticaRESUMO
Gene co-option, usually after gene duplication, in the evolution of development is found to contribute to vertebrate morphological innovations, including the endothelium-based vascular system. Recently, a zebrafish kank gene was found expressed in the vascular vessel primordium, suggesting KANK genes are a component of the developmental tool kit for the vertebrate vascular system. However, how the KANK gene family is involved in vascular vessel development during evolution remains largely unknown. First, we analyzed the molecular evolution of the KANK genes in metazoan, and found that KANK1, KANK2, KANK3 and KANK4 emerged in the lineage of vertebrate, consistent with the two rounds of vertebrate whole-genome duplications (WGD). Moreover, KANK genes were further duplicated in teleosts through the bony-fish specific WGD, while only kank1 and kank4 duplicates were retained in some of the examined fish species. We also found all zebrafish kank genes, except kank1b, are primarily expressed during embryonic vascular development. Compared to invertebrate KANK gene expression in the central nervous system, the vascular expression of zebrafish kank genes suggested KANK genes were co-opted for vertebrate vascular development. Given the cellular roles of KANK genes, our results suggest that this co-option may facilitate the evolutionary origin of vertebrate vascular vessels.
Assuntos
Evolução Molecular , Proteínas Supressoras de Tumor/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Teorema de Bayes , Vasos Sanguíneos/crescimento & desenvolvimento , Vasos Sanguíneos/metabolismo , Cromossomos/genética , Embrião não Mamífero/metabolismo , Duplicação Gênica , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Filogenia , Proteínas Supressoras de Tumor/classificação , Proteínas Supressoras de Tumor/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/classificação , Proteínas de Peixe-Zebra/metabolismoRESUMO
The precise positioning of organ progenitor cells constitutes an essential, yet poorly understood step during organogenesis. Using primordial germ cells that participate in gonad formation, we present the developmental mechanisms maintaining a motile progenitor cell population at the site where the organ develops. Employing high-resolution live-cell microscopy, we find that repulsive cues coupled with physical barriers confine the cells to the correct bilateral positions. This analysis revealed that cell polarity changes on interaction with the physical barrier and that the establishment of compact clusters involves increased cell-cell interaction time. Using particle-based simulations, we demonstrate the role of reflecting barriers, from which cells turn away on contact, and the importance of proper cell-cell adhesion level for maintaining the tight cell clusters and their correct positioning at the target region. The combination of these developmental and cellular mechanisms prevents organ fusion, controls organ positioning and is thus critical for its proper function.
Assuntos
Embrião não Mamífero/metabolismo , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Organogênese/genética , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Sistemas CRISPR-Cas/genética , Adesão Celular/genética , Movimento Celular/genética , Sistema Digestório/citologia , Sistema Digestório/embriologia , Sistema Digestório/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Perfilação da Expressão Gênica/métodos , Gônadas/citologia , Gônadas/embriologia , Gônadas/metabolismo , Metaloproteínas/classificação , Metaloproteínas/genética , Metaloproteínas/metabolismo , Microscopia de Fluorescência , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de Aminoácidos , Imagem com Lapso de Tempo , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/classificação , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The nucleolar protein 4-like (NOL4L) gene is present on chromosome 20 (20q11.21) in humans. Parts of this gene have been shown to fuse with RUNX1 and PAX5 in acute myeloid leukemia and acute lymphoblastic leukemia, respectively. The normal function of NOL4L in humans and other organisms is not well understood. The expression patterns and functions of NOL4L homologs during vertebrate development have not been reported. We sought to address these questions by studying the expression pattern of zebrafish nol4l during embryogenesis. Our data show that Znol4l mRNA is expressed in multiple organs in zebrafish embryos. The sites of expression include parts of the brain, spinal cord, pronephros, hematopoietic cells and gut.
Assuntos
Embrião não Mamífero/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Nucleares/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Embrião não Mamífero/embriologia , Humanos , Hibridização In Situ , Proteínas Nucleares/classificação , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/classificaçãoRESUMO
The BTB-POZ transcription factor Promyelocytic Leukemia Zinc Finger (PLZF, or ZBTB16) has been recently identified as a major factor regulating the induction of a subset of Interferon stimulated genes in human and mouse. We show that the two co-orthologues of PLZF found in zebrafish show distinct expression patterns, especially in larvae. Although zbtb16a/plzfa and zbtb16b/plzfb are not modulated by IFN produced during viral infection, their over-expression increases the level of the early type I IFN response, at a critical phase in the race between the virus and the host response. The effect of Plzfb on IFN induction was also detectable after cell infection by different non-enveloped RNA viruses, but not after infection by the rhabdovirus SVCV. Our findings indicate that plzf implication in the regulation of type I IFN responses is conserved across vertebrates, but at multiple levels of the pathway and through different mechanisms.
Assuntos
Interferon Tipo I/imunologia , Fatores de Transcrição Kruppel-Like/metabolismo , Infecções por Vírus de RNA/imunologia , Vírus de RNA/imunologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/imunologia , Animais , Humanos , Imunidade Inata , Interferon Tipo I/metabolismo , Fatores de Transcrição Kruppel-Like/classificação , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Filogenia , Poli I-C/imunologia , Proteína com Dedos de Zinco da Leucemia Promielocítica , RNA Viral/imunologia , Transcriptoma , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/classificação , Proteínas de Peixe-Zebra/genéticaRESUMO
Bone morphogenetic proteins (BMPs) are multifunctional growth factors that play crucial roles during embryonic development and cell fate determination. Nuclear transduction of BMP signals requires the receptor type Smad proteins, Smad1, Smad5, and Smad9. However, how these Smad proteins cooperate in vivo to regulate various developmental processes is largely unknown. In zebrafish, it was widely believed that the maternally expressed smad5 is essential for dorso-ventral (DV) patterning, and the zygotically transcribed smad1 is not required for normal DV axis establishment. In the present study, we have identified zygotically expressed smad9, which cooperates with smad1 downstream of smad5, to mediate zebrafish early DV patterning in a functional redundant manner. Although knockdown of smad1 or smad9 alone does not lead to visible dorsalization, double knockdown strongly dorsalizes zebrafish embryos, which cannot be efficiently rescued by smad5 overexpression, whereas the dorsalization induced by smad5 knockdown can be fully rescued by overexpression of smad1 or smad9. We have further revealed that the transcription initiations of smad1 and smad9 are repressed by each other, that they are direct transcriptional targets of Smad5, and that smad9, like smad1, is required for myelopoiesis. In conclusion, our study uncovers that smad1 and smad9 act redundantly to each other downstream of smad5 to mediate ventral specification and to regulate embryonic myelopoiesis.
Assuntos
Padronização Corporal/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Mielopoese/genética , Proteína Smad1/metabolismo , Proteína Smad5/metabolismo , Proteína Smad8/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Proteínas Morfogenéticas Ósseas/genética , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Dados de Sequência Molecular , Filogenia , Proteína Smad1/classificação , Proteína Smad1/genética , Proteína Smad5/classificação , Proteína Smad5/genética , Proteína Smad8/classificação , Proteína Smad8/genética , Iniciação da Transcrição Genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/classificação , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: The transient receptor potential melastatin (TRPM) gene family belongs to the superfamily of nonselective TRP ion channels. TRP channels are cellular sensors, detecting a multitude of inputs, including temperature, light, chemical, and mechanical stimuli. Recent studies revealed diverse roles during development, linking TRP channels to differentiation, proliferation, cell motility, cell death, and survival. A detailed description of this gene family in the zebrafish is still missing. RESULTS: Phylogenetic analysis revealed 11 trpm genes in the zebrafish genome. The zebrafish orthologs of mammalian TRPM1 and TRPM4 are duplicated and quadruplicated, respectively, and TRPM8, a cold sensitive channel has been lost in zebrafish. Whole-mount in situ hybridization experiments revealed dynamic expression pattern of trpm genes in the developing embryo and early larva. Transcripts were mainly found in neural cell clusters, but also in tissues involved in ion homeostasis. CONCLUSIONS: Our results suggest a role of TRPM channels in sensory information processing, including vision, olfaction, taste, and mechanosensation. An involvement in developmental processes is likely, as some trpm genes were found to be expressed in differentiating cells. Our data now provide a basis for functional analyses of this gene family of ion channels in the vertebrate model organism Danio rerio.
Assuntos
Filogenia , Canais de Cátion TRPM/classificação , Proteínas de Peixe-Zebra/classificação , Animais , Hibridização In Situ , Pronefro/metabolismo , Células Receptoras Sensoriais/metabolismo , Canais de Cátion TRPM/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
In mammals, IFIT (Interferon [IFN]-induced proteins with Tetratricopeptide Repeat [TPR] motifs) family genes are involved in many cellular and viral processes, which are tightly related to mammalian IFN response. However, little is known about non-mammalian IFIT genes. In the present study, IFIT genes are identified in the genome databases from the jawed vertebrates including the cartilaginous elephant shark but not from non-vertebrates such as lancelet, sea squirt and acorn worm, suggesting that IFIT gene family originates from a vertebrate ancestor about 450 million years ago. IFIT family genes show conserved gene structure and gene arrangements. Phylogenetic analyses reveal that this gene family has expanded through lineage-specific and species-specific gene duplication. Interestingly, IFN gene family seem to share a common ancestor and a similar evolutionary mechanism; the function link of IFIT genes to IFN response is present early since the origin of both gene families, as evidenced by the finding that zebrafish IFIT genes are upregulated by fish IFNs, poly(I:C) and two transcription factors IRF3/IRF7, likely via the IFN-stimulated response elements (ISRE) within the promoters of vertebrate IFIT family genes. These coevolution features creates functional association of both family genes to fulfill a common biological process, which is likely selected by viral infection during evolution of vertebrates. Our results are helpful for understanding of evolution of vertebrate IFN system.
Assuntos
Evolução Molecular , Interferons/genética , Família Multigênica/genética , Vertebrados/genética , Sequência de Aminoácidos , Animais , Células Cultivadas , Mapeamento Cromossômico , Proteínas de Peixes/genética , Peixes/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 7 de Interferon/genética , Interferons/farmacologia , Dados de Sequência Molecular , Filogenia , Poli I-C/farmacologia , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas/classificação , Isoformas de Proteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Sintenia , Vertebrados/classificação , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/classificação , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: Chondroitin/dermatan sulfate (CS/DS) proteoglycans present in the extracellular matrix have important structural and regulatory functions. RESULTS: Six human genes have previously been shown to catalyze CS/DS polymerization. Here we show that one of these genes, chpf, is represented by two copies in the zebrafish genome, chpfa and chpfb, while the other five human CS/DS glycosyltransferases csgalnact1, csgalnact2, chpf2, chsy1, and chsy3 all have single zebrafish orthologues. The putative zebrafish CS/DS glycosyltransferases are spatially and temporally expressed. Interestingly, overlapping expression of multiple glycosyltransferases coincides with high CS/DS deposition. Finally, whereas the relative levels of the related polysaccharide HS reach steady-state at around 2 days post fertilization, there is a continued relative increase of the CS amounts per larvae during the first 6 days of development, matching the increased cartilage formation. CONCLUSIONS: There are 7 CS/DS glycosyltransferases in zebrafish, which, based on homology, can be divided into the CSGALNACT, CHSY, and CHPF families. The overlap between intense CS/DS production and the expression of multiple CS/DS glycosyltransferases suggests that efficient CS/DS biosynthesis requires a combination of several glycosyltransferases.
Assuntos
Sulfatos de Condroitina/metabolismo , Dermatan Sulfato/metabolismo , Glicosiltransferases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Condroitina , Glicosiltransferases/classificação , Glicosiltransferases/genética , Filogenia , Peixe-Zebra , Proteínas de Peixe-Zebra/classificação , Proteínas de Peixe-Zebra/genéticaRESUMO
Female zebrafish have a prolific reproductive capacity, suggesting that a germline stem cell (GSC) population drives oocyte production. However, a zebrafish female GSC population has yet to be identified. Adult stem cells are defined by their ability to both self-renew and differentiate, and by their localization to a stem cell niche. We show here that mitotic and early meiotic germ cells are present in the adult ovary and that the zebrafish homolog of the conserved vertebrate GSC marker, nanos2, is expressed in a subset of pre-meiotic oogonia in the adult gonad. We propose that these nanos2(+) cells are GSCs. Importantly, we find that mitotic, nanos2(+), and early meiotic germ cells localize to the germinal zone, thus identifying this region as the probable ovarian GSC niche in zebrafish. nanos3, which encodes a conserved RNA-binding protein, is known to be required for the continued production of oocytes in the zebrafish. Although mammalian homologs of nanos3 are expressed in early spermatogonia, no study has defined the role of nanos3 in the regulation of vertebrate GSCs. Here we demonstrate that nanos3 function is required for the maintenance of GSCs, but not for their specification, and propose that nanos2 and nanos3 are partially redundant in this role.