Effects of adrenergic agents on the expression of zebrafish (Danio rerio) vitellogenin Ao1

Teleost vitellogenins (VTGs) are large multidomain apolipoproteins, traditionally considered to be estrogen-responsive precursors of the major egg yolk proteins, expressed and synthesized mainly in hepatic tissue. The inducibility of VTGs has made them one of the most frequently used in vivo and in vitro biomarkers of exposure to estrogen-active substances. A significant level of zebrafish vtgAo1, a major estrogen responsive form, has been unexpectedly found in heart tissue in our present studies. Our studies on zebrafish cardiomyopathy, caused by adrenergic agonist treatment, suggest a similar protective function of the cardiac expressed vtgAo1. We hypothesize that its function is to unload surplus intracellular lipids in cardiomyocytes for "reverse triglyceride transportation" similar to that found in lipid transport proteins in mammals. Our results also demonstrated that zebrafish vtgAo1 mRNA expression in heart can be suppressed by both (x-adrenergic agonist, phenylephrine (PE) and beta-adrenergic agonist, isoproterenol (ISO). Furthermore, the strong stimulation of zebrafish vtgAo1 expression in plasma induced by the beta-adrenergic antagonist, MOXIsylyl, was detected by Enzyme-Linked ImmunoSorbent Assay (ELISA). Such stimulation cannot be suppressed by taMOXIfen, an antagonist to estrogen receptors. Thus, Our present data indicate that the production of teleost VTG in vivo can be regulated not only by estrogenic agents, but by adrenergic signals as well. (c) 2009 Elsevier Inc. All rights reserved.

Identification of differential transcript profiles between mutual crossbred embryos of zebrafish (Danio rerio) and Chinese rare minnow (Gobiocypris rarus) by cDNA-AFLP

The crosstalk between naive nucleus and maternal factors deposited in egg cytoplasm before zygotic genome activation is crucial for early development. In this study, we utilized two laboratory fishes, zebrafish (Danio rerio) and Chinese rare minnow and Chinese rare minnow (Gobiocypris rarus), to obtain mutual crossbred embroys and examine the interaction between nucleus and egg cytoplasm from different species. Although these two types of crossbred embryos originated from common nuclei, various developmental capacities were gained due to different origins of the egg cytoplasm. Using cDNA amplified fragment length polymorphism (cDNA-AFLP), We Compared transcript profiles between the mutual crossbred embryos at two developmental stages (50%- and 90%-epiholy). Three thousand cDNA fragments were generated in four cDNA pools with 64 primer combinations. All differently displayed transcript-derived fragments (TDFs) were screened by (lot blot hybridization, and the selected sequences were further analyzed by semi-quantitative RT-PCR and quantitative real-time RT-PCR. Compared with ZR embryos, 12 genes were up-regulated and 12 were down-regulated in RZ embryos. The gene fragments were sequenced and subjected to BLASTN analysis. The sequences encoded various proteins which functioned at various levels of proliferation, growth, and development. One gene (ZR6), dramatically down-regulated in RZ embryos, was chosen for loss-of-function study; the knockdown of ZR6 gave rise to the phenotype resembling that of RZ embryos. (c) 2008 Elsevier Inc. All rights reserved.

RNAi suppression of zebrafish peptidoglycan recognition protein 6 (zfPGRP6) mediated differentially expressed genes involved in Toll-like receptor signaling pathway and caused increased susceptibility to Flavobacterium columnare

In Drosophila, Toll signaling cascade, which resembles the mammalian Toll-like receptor (TLR)/IL-1R signaling pathways and regulates the expression of anti-microbial peptide genes, mainly relies on peptidoglycan recognition proteins (PGRPs) for the detection of bacterial pathogens. To explore the effect of zebrafish peptidoglycan recognition protein 6 (zfPGRP6) on Toll-like receptor signaling pathway, RNA interference (siRNA) and real time quantitative PCR (RQ-PCR) methods were used to identify differentially expressed genes regulated by zfPGRP6. The target genes included TLR2, TLR3, TLR5, TLR7, TLR8, IL1R, Sterile-alpha and Armadillo motif containing protein (SARM), myeloid differentiation factor 88 (MyD88) and nuclear factor (NF)-kappa B2 (p100/p52). The results of RQ-PCR showed that RNAi-mediated Suppression of zfPGRP6 significantly down-regulated the expression of TLR2, TLR5, IL1R, SARM, MyD88 and p100/p52. The expression of beta-defensin-1 was also down-regulated in those embryos silenced by zfPGRP6. In challenge experiments to determine the anti-bacterial response to Gram-negative bacteria, RNAi knock-down of zfPGRP6 markedly increased susceptibility to Flavobacterium columnare. (C) 2008 Elsevier B.V. All rights reserved.

Characterization of DYRK2 (dual-specificity tyrosine-phosphorylation-regulated kinase 2) from Zebrafish (Dario rerio)

Proteins of the DYRK (dual-specificity tyrosine-phosphorylation-regulated kinase) family are characterized by the presence of a conserved kinase domain and N-terminal DH box. DYRK2 is involved in regulating key developmental and cellular processes, such as neurogenesis, cell proliferation, cytokinesis, and cellular differentiation. Herein, we report that the ortholog of DYRK2 found in zebrafish shares about 70% identity with that of human, mouse, and chick. RT-PCR showed that DYRK2 is expressed maternally and zygotically. In-situ hybridization results show that DYRK2 is expressed in somite cells that will develop into muscles. Our results provide preliminary evidence for investigating the in-vivo function of DYRK2 in zebrafish muscle development.

Characterization of proteotoxic stress in zebrafish

A fidelidade da síntese proteica é fundamental para a estabilidade do proteoma e para a homeostasia celular. Em condições fisiológicas normais as células têm uma taxa de erro basal associada e esta muitas vezes aumenta com o envelhecimento e doença. Problemas na síntese das proteínas estão associados a várias doenças humanas e aos processos de envelhecimento. De facto, a incorporação de erros nas proteínas devido a tRNAs carregados pelas aminoacil-tRNA sintetases com o amino ácido errado causa doenças neurodegenerativas em humanos e ratos. Ainda não é claro como é que estas doenças se desenvolvem e se são uma consequência directa da disrupção do proteoma ou se são o resultado da toxicidade produzida pela acúmulação de proteínas mal traduzidas ao nível do ribossoma. Para elucidar como é que as células eucarióticas lidam com proteínas aberrantes e agregados proteicos (stress proteotóxico) desenvolvemos uma estratégia para destabilizar o proteoma. Para isso estabelecemos um sistema de erros de tradução em embriões de peixe zebra que assenta em tRNAs mutantes capazes de incorporar erradamente serina nas proteínas. As proteínas produzidas neste sistema despoletam as vias de resposta ao stress, nomeadamente a via da ubiquitina-proteassoma (UPP – “ubiquitin protesome pathway”) e a via do retículo endoplasmático (UPR – “unfolded protein response”). O stress proteotóxico gerado pelos erros de tradução altera a expressão génica e perfis de expressão de miRNAs, o desenvolvimento embrionário e viabilidade, aumenta a produção de espécies reactivas de oxigénio (ROS), leva ainda à acumulação de agregados proteicos e à disfunção mitocondrial. As malformações embrionárias e fenótipos de viabilidade que observámos foram revertidos por antioxidantes, o que sugere que os ROS desempenham papéis importantes nos fenótipos degenerativos celulares induzidos pela produção de proteínas aberrantes e agregação proteica. Estabelecemos ainda uma linha de peixe zebra transgénica para o estudo do stress proteotóxico. Este trabalho mostra que a destabilização do proteoma em embriões de peixe zebra com tRNAs mutantes é uma boa metodologia para estudar a biologia do stress proteotóxico visto que permite a agregação controlada do proteoma, mimetizando os processos de agregação de proteínas que ocorrem naturalmente durante o envelhecimento e em doenças conformacionais humanas.

Insights into molecular and cellular events involved in normal and pathological vertebral development and fusion using zebrafish as model organism

The vertebral column and its units, the vertebrae, are fundamental features, characteristic of all vertebrates. Developmental segregation of the vertebral bodies as articulated units is an intrinsic requirement to guarantee the proper function of the spine. Whenever these units become fused either during development or postsegmentation, movement is affected in a more or less severe manner, depending on the number of vertebrae affected. Nevertheless, fusion may occur as part of regular development and as a physiological requirement, like in the tetrapod sacrum or in fish posterior vertebrae forming the urostyle. In order to meet the main objective of this PhD project, which aimed to better understand the molecular and cellular events underlying vertebral fusion under physiological and pathological conditions, a detailed characterization of the vertebral fusion occurring in zebrafish caudal fin region was conducted. This showed that fusion in the caudal fin region comprised 5 vertebral bodies, from which, only fusion between [PU1++U1] and ural2 [U2+] was still traceable during development. This involved bone deposition around the notochord sheath while fusion within the remaining vertebral bodies occur at the level of the notochord sheath, as during the early establishment of the vertebral bodies. A comparison approach between the caudal fin vertebrae and the remaining vertebral column showed conserved features such as the presence of mineralization related proteins as Osteocalcin were identified throughout the vertebral column, independently on the mineralization patterns. This unexpected presence of Osteocalcin in notochord sheath, here identified as Oc1, suggested that this gene, opposing to Oc2, generally associated with bone formation and mature osteoblast activity, is potentially associated with early mineralization events including chordacentrum formation. Nevertheless, major differences between caudal fin region and anterior vertebral bodies considering arch histology and mineralization patterns, led us to use RA as an inductive factor for vertebral fusion, allowing a direct comparison of equivalent structures under normal and fusion events. This fusion phenotype was associated with notochord sheath ectopic mineralization instead of ectopic perichordal bone formation related with increased osteoblast activity, as suggested in previous reports. Additionally, alterations in ECM content, cell adhesion and blood coagulation were discussed as potentially related with the fusion phenotype. Finally, Matrix gla protein, upregulated upon RA treatment and shown to be associated with chordacentrum mineralization sites in regular development, was further described considering its potential function in vertebral formation and pathological fusion. Therefore with this work we propose zebrafish caudal fin vertebral fusion as a potential model to study both congenital and postsegmentation fusion and we present candidate factors and genes that may be further explored in order to clarify whether we can prevent vertebral fusion.

Molecular structure and functional analysis of runx3 in zebrafish

Tese de doutoramento, Ciências Biomédicas, Universidade do Algarve, Departamento de Ciências Biomédicas e Medicina, 2014

Effects of vitamin K deficiency and its mechanisms in vertebrate's early development: zebrafish as a model

Disertação de mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015

Conservation, duplication and divergence of the zebrafish stat5 genes

There are seven mammalian signal transducer and activator of transcription (Stat) proteins that act downstream of cytokine and growth factor receptors to mediate rapid changes in gene expression. The mammalian Stat5a and Stat5b genes show high sequence identity and lie adjacent in a head-to-head configuration next to the Stat3 gene, apparently the result of a relatively recent mammal-specific gene duplication event. We have identified and characterized two stat5 homologues that are expressed in zebrafish, named stat5.1 and stat5.2. The stat5.1 gene shows a high level of conservation with the single stat5 gene found in other teleosts and lies next to the stat3 gene, in the same relative orientation as the mammalian Stat5b gene. In contrast, the stat5.2 gene lies on a different chromosome to stat5.1 and stat3, and has diverged from the stat5 genes of other teleosts, with no apparent orthologue. Together, these data suggest that the ancestral Stat5 gene has undergone two independent gene duplication events to generate a stat5.2 paralogue in zebrafish and a Stat5a paralogue in mammals.

Characterization of the zebrafish matrix metalloproteinase 9 gene and its developmental expression pattern

Members of the matrix metalloproteinase (MMP) family are important for the remodeling of the extracellular matrix in a number of biological processes including a variety of immune responses. Two members of the family, MMP2 and MMP9, are highly expressed in specific myeloid cell populations in which they play a role in the innate immune response. To further expand the repertoire of molecular reagents available to study zebrafish myeloid cell development, the matrix metalloproteinase 9 (mmp9) gene from this organism has been identified and characterized. The encoded protein is 680 amino acids with high homology to known MMP9 proteins, particularly those of other teleost fish. Maternal transcripts of mmp9 are deposited in the oocyte and dispersed throughout the early embryo. These are replaced by specific zygotic transcripts in the notochord from 12 h post fertilization (hpf) and also transiently in the anterior mesoderm from 14 to 16 h post fertilization. From 24 h post fertilization, mmp9 expression was detected in a population of circulating white blood cells that are distinct from macrophages, and which migrate to the site of trauma, and so likely represent zebrafish heterophils. In the adult, mmp9 expression was most prominent in the splenic cords, a site occupied by mature myeloid cells in other species. These results suggest that mmp9 will serve as a useful marker of mature myeloid cells in the zebrafish.

Tissue specific roles for the Ribosome Biogenesis Factor Wdr43 in zebrafish development

During vertebrate craniofacial development, neural crest cells (NCCs) contribute to most of the craniofacial pharyngeal skeleton. Defects in NCC specification, migration and differentiation resulting in malformations in the craniofacial complex are associated with human craniofacial disorders including Treacher-Collins Syndrome, caused by mutations in TCOF1. It has been hypothesized that perturbed ribosome biogenesis and resulting p53 mediated neuroepithelial apoptosis results in NCC hypoplasia in mouse Tcof1 mutants. However, the underlying mechanisms linking ribosome biogenesis and NCC development remain poorly understood. Here we report a new zebrafish mutant, fantome (fan), which harbors a point mutation and predicted premature stop codon in zebrafish wdr43, the ortholog to yeast UTP5. Although wdr43 mRNA is widely expressed during early zebrafish development, and its deficiency triggers early neural, eye, heart and pharyngeal arch defects, later defects appear fairly restricted to NCC derived craniofacial cartilages. Here we show that the C-terminus of Wdr43, which is absent in fan mutant protein, is both necessary and sufficient to mediate its nucleolar localization and protein interactions in metazoans. We demonstrate that Wdr43 functions in ribosome biogenesis, and that defects observed in fan mutants are mediated by a p53 dependent pathway. Finally, we show that proper localization of a variety of nucleolar proteins, including TCOF1, is dependent on that of WDR43. Together, our findings provide new insight into roles for Wdr43 in development, ribosome biogenesis, and also ribosomopathy-induced craniofacial phenotypes including Treacher-Collins Syndrome.

Suppressor of cytokine signaling 4 and 5 in zebrafish development

This research provides insight into the evolution and function of zebrafish cell signaling proteins, Suppressor of Cytokine Signaling 4a, 4b and 5a. These signaling proteins were found to play essential roles during early and late stages of embryonic development, with specific roles in sensory processing and immune cell development.

Muscle-specific growth hormone receptor (GHR) overexpression induces hyperplasia but not hypertrophy in transgenic zebrafish

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Spatio-temporal distribution of fatty acid-binding protein 6 (fabp6) gene transcripts in the developing and adult zebrafish (Danio rerio)

We have determined the structure of the fatty acid-binding protein 6 (fabp6) gene and the tissue-specific distribution of its transcripts in embryos, larvae and adult zebrafish (Danio rerio). Like most members of the vertebrate FABP multigene family, the zebrafish fabp6 gene contains four exons separated by three introns. The coding region of the gene and expressed sequence tags code for a polypeptide of 131 amino acids (14 kDa, pI 6.59). The putative zebrafish Fabp6 protein shared greatest sequence identity with human FABP6 (55.3%) compared to other orthologous mammalian FABPs and paralogous zebrafish Fabps. Phylogenetic analysis showed that the zebrafish Fabp6 formed a distinct clade with the mammalian FABP6s. The zebrafish fabp6 gene was assigned to linkage group (chromosome) 21 by radiation hybrid mapping. Conserved gene synteny was evident between the zebrafish fabp6 gene on chromosome 21 and the FABP6/Fabp6 genes on human chromosome 5, rat chromosome 10 and mouse chromosome 11. Zebrafish fabp6 transcripts were first detected in the distal region of the intestine of embryos at 72 h postfertilization. This spatial distribution remained constant to 7-day-old larvae, the last stage assayed during larval development. In adult zebrafish, fabp6 transcripts were detected by RT-PCR in RNA extracted from liver, heart, intestine, ovary and kidney (most likely adrenal tissue), but not in RNA from skin, brain, gill, eye or muscle. In situ hybridization of a fabp6 riboprobe to adult zebrafish sections revealed intense hybridization signals in the adrenal homolog of the kidney and the distal region of the intestine, and to a lesser extent in ovary and liver, a transcript distribution that is similar, but not identical, to that seen for the mammalian FABP6/Fabp6 gene. © 2008 The Authors.

Analysis of protein expression in zebrafish during gonad differentiation by targeted proteomics

The molecular mechanisms governing sex determination and differentiation in the zebrafish (Danio rerio) are not fully understood. To gain more insights into the function of specific genes in these complex processes, the expression of multiple candidates needs to be assessed, preferably on the protein level. Here, we developed a targeted proteomics method based on selected reaction monitoring (SRM) to study the candidate sex-related proteins in zebrafish which were selected based on a global proteomics analysis of adult gonads and representational difference analysis of male and female DNA, as well as on published information on zebrafish and other vertebrates. We employed the developed SRM protocols to acquire time-resolved protein expression profiles during the gonad differentiation period in vas::EGFP transgenic zebrafish. Evidence on protein expression was obtained for the first time for several candidate genes previously studied only on the mRNA level or suggested by bioinformatic predictions. Tuba1b (tubulin alpha 1b), initially included in the study as one of the potential housekeeping proteins, was found to be preferentially expressed in the adult testis with nearly absent expression in the ovary. The revealed changes in protein expression patterns associated with gonad differentiation suggest that several of the examined proteins, especially Ilf2 and Ilf3 (interleukin enhancer-binding factors 2 and 3), Raldh3 (retinaldehyde dehydrogenase type 3), Zgc:195027 (low density lipoprotein-related receptor protein 3) and Sept5a (septin 5a), may play a specific role in the sexual differentiation in zebrafish.