3,043 research outputs found

    The Tetraodon nigroviridis reference transcriptome: Developmental transition, length retention and microsynteny of long non-coding RNAs in a compact vertebrate genome

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    Pufferfish such as fugu and tetraodon carry the smallest genomes among all vertebrates and are ideal for studying genome evolution. However, comparative genomics using these species is hindered by the poor annotation of their genomes. We performed RNA sequencing during key stages of maternal to zygotic transition of Tetraodon nigroviridis and report its first developmental transcriptome. We assembled 61,033 transcripts (23,837 loci) representing 80% of the annotated gene models and 3816 novel coding transcripts from 2667 loci. We demonstrate the similarities of gene expression profiles between pufferfish and zebrafish during maternal to zygotic transition and annotated 1120 long non-coding RNAs (lncRNAs) many of which differentially expressed during development. The promoters for 60% of the assembled transcripts result validated by CAGE-seq. Despite the extreme compaction of the tetraodon genome and the dramatic loss of transposons, the length of lncRNA exons remain comparable to that of other vertebrates and a small set of lncRNAs appears enriched for transposable elements suggesting a selective pressure acting on lncRNAs length and composition. Finally, a set of lncRNAs are microsyntenic between teleost and vertebrates, which indicates potential regulatory interactions between lncRNAs and their flanking coding genes. Our work provides a fundamental molecular resource for vertebrate comparative genomics and embryogenesis studies

    The Molecular Mechanisms Of Sex Determination In Vertebrates

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    Many reptiles display temperature-dependent sex determination (TSD), in which the primary sex is determined by incubation temperatures rather than sex chromosomes. However, temperature is not the only factor that play critical roles in sex determination in the species with TSD. Previous studies in the snapping turtle, a species with TSD, showed that dihydrotestosterone (DHT) induces ovary development at temperatures that normally produce males or mixed sex ratios. In addition, the feminizing effect of DHT was found to be associated with increased expression of the ovary-determining gene Foxl2, suggesting a potential androgen-Foxl2 regulatory mechanism. This dissertation aims to clarify the molecular mechanisms underlying TSD in several aspects. First, determine the role of androgen in TSD; second, identify novel thermosensitive genes involved in TSD and lastly, reconstruct gene regulatory networks underlying sex determination. To test the hypothetical androgen-Foxl2 interaction, I cloned the proximal promoter (1.6 kb) and coding sequence for snapping turtle Foxl2 (tFoxl2) in frame with mCherry, a red fluorescent protein. The tFoxl2-mCherry fusion plasmid or mCherry plasmid were stably transfected into mouse KK1 granulosa cells. Although expression of tFoxl2-mCherry was not affected by androgen treatment in KK1 cells, androgen inhibited expression of the endogenous mouse Foxl2 gene, suggesting the androgen-Foxl2 interaction does exist but it differs between species. We also found tFoxl2-mCherry potentiated low dose DHT effects on aromatase expression, which has not been reported in any other studies. To identify novel sex-determining genes in TSD, I first de novo assembled and annotated the transcriptome of the snapping turtle using next-generation sequencing (NGS) and then performed RNA-seq analyses on the newly assembled reference transcriptome. With the differential gene expression analyses, I identified 293 thermosensitive genes. Among these genes, I find AEBP2, JARID2, and KDM6B of particular interest because these genes could influence expression of many other genes via epigenetic modifications. To further investigate the molecular mechanisms underlying sex determination, I reconstructed gene regulatory networks using an entropy based network reconstructing algorithm – ARACNE with public microarray experiments in mouse gonads. The subsequent hub gene analyses revealed the basic molecular pathways underlying gonadal development and the master regulator analyses identified 110 candidate sex-determining genes including both known sex-determining genes and novel candidate genes. My findings demonstrate that androgens can influence expression of key ovarian genes but further studies are needed to understand the androgen signaling in TSD. Furthermore, my study provides a first description of the snapping turtle transcriptome and the effects of temperature on transcriptome-wide patterns of gene expression during the TSP. In addition, hub genes and master regulators identified for mammalian gonad determination will guide the direction of future studies in the field of sex determination. However, additional studies are needed to validate the computational findings

    The immunome and embryo quality in sea bream and sea bass

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    Gilthead sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax) are teleosts belonging to Eupercaria and are the most important aquaculture fish species in the Mediterranean region. These two species are ranked second after the Atlantic salmon (Salmo salar) in production volume and value in the European Union (EU) aquaculture sector. Unpredictable fertilized egg/embryo quality and performance remain a bottleneck that threatens sustainability of sea bream and sea bass aquaculture, impeding the increased productivity of aquaculture that entirely depends on hatchery production. To address this issue, criteria and molecular markers linked to embryo quality that could be used to monitor and manage hatchery production were procured. Comparative molecular approaches using molecular biology, proteomics and transcriptomics were performed to analyze embryo performance and immunity in samples from several European commercial hatcheries. The core achievements were the: a) identification and characterization of lysozyme and complement 5 (C5) gene families and embryo and larval gene expression and enzyme activity from a diversity of hatcheries, b) characterization of the embryo proteome from three Mediterranean fish species [white sea bream (Diplodus sargus), meagre (Argyrosomus regius) and sea bream] 24h before hatch and at hatch and identification of common and species specific molecular patterns linked to biological function and putative quality-related proteins, c) comparative transcriptomics of good and poor quality sea bream embryos from several Mediterranean hatcheries. Qualitylinked transcripts and some elements of the regulatory epitranscriptome (non-coding RNA) were identified as well as the contribution of maternal proteins to embryos. Taken together, the results provide a comprehensive description of the molecular basis of sea bream and sea bass embryo development and reveal that immune-related molecules in fertilized eggs are low abundance. The development (quality)-related candidate markers identified will be of value for management of fish embryos in aquaculture hatcheries.O mar Mediterrâneo cobre apenas 0,7% da área oceânica mundial, mas é um dos principais reservatórios de biodiversidade marinha e costeira com cerca de 28% de espécies endémicas. A dourada (Sparus aurata) e o robalo (Dicentrarchus labrax), são duas espécies de peixe que pertencem à série Eupercaria e das mais comercializadas pela indústria de aquicultura nesta região. No sector da aquicultura da União Europeia (EU), estas duas espécies ocupam o segundo lugar no “ranking” da cadeia de valor, depois do salmão do Atlântico. Contudo, as suas características morfológicas e de crescimento são os parâmetros de qualidade relevantes considerados pela indústria. Esta abordagem deve-se à sua comercialização em formato de peixe inteiro, o que reduz o valor de mercado na cadeia de valor e também à ausência de critérios de qualidade para seleção de ovos e embriões, bem como, de marcadores moleculares de qualidade com maior grau de sensibilidade. Todos estes obstáculos, limitam substancialmente o desenvolvimento das indústrias associadas á comercialização destas duas espécies de peixe, impedindo a expansão da aquicultura e das “hatcheries” (maternidades incubadoras de ovos/embriões controlados artificialmente para fins comerciais). Para mitigar este problema, utilizou-se técnicas de biologia molecular e tecnologias ómicas e estabeleceu-se uma abordagem comparativa direcionada á descoberta de moléculas e vias metabólicas funcionais de importância crítica para o sistema imune dos peixes associada á “performance” de desenvolvimento de ovos e embriões. Esta tese está organizada em seis capítulos. Inicia-se com uma visão geral dos critérios morfológicos, físico-químicos e moleculares existentes para avaliar a qualidade de ovos e embriões para melhorar a gestão da aquicultura de peixes (Capítulo 1). Subsequentemente, caracterizou-se a família de genes do sistema imune: a) a das lisozimas em peixes teleósteos com enfase na sua caracterização molecular e funcional em dourada (Capítulo 2) e análises moleculares estruturais e evolutivas e b) a do complemento C5 (C5) em peixes especialmente em espécies da faamilia Cyprinidae (Capítulo 3). Os capítulos 4 e 5, integram abordagens de proteómica e transcriptómica em espécies de peixes mediterrânicos [pargo (Diplodus sargus), corvina (Argyrosomus regius) e dourada], focando os processos de desenvolvimento e de eclosão e na função da enzima “hatching enzyme” em dourada e robalo. Foi feita uma associação entre os capítulos e a análise integrada dos dados do transcritoma do embrião (Capítulo 5) revelou um padrão de expressão significativamente diferente (p-valor < 0,05) para o C5 (Capítulo 3) em diferentes lotes de embriões de dourada nas comparações entre graus de qualidade (Boa vrs Má) e entre estágios de desenvolvimento (Pré- eclosão vrs Eclosão). A variação do C5 em relação ao lote de embriões não foi afetada pela origem da “hatchery”, indicando que as prática de manejo ou os próprios reprodutores não influenciam a sua expressão. Os resultados sugerem que este gene e o seu produto proteico, são provavelmente importantes na proteção imunológica precoce e também em outras funções ainda não descritas na dourada ou em outras espécies de peixes. Também a integração dos resultados do proteoma (Capítulo 4) e do transcritoma (Capítulo 5) do embrião de dourada nos mesmos estágios de desenvolvimento, identificou um grupo de proteínas que se especula serem de origem materna. No último capítulo, sumarizou-se os resultados e são apresentadas perspetivas baseadas nos avanços e desafios atuais e propostas para o desenvolvimento de uma ferramenta integrada de monitorização da qualidade dos embriões e uma base biológica do desenvolvimento de ovos e embriões de peixes (Capítulo 6). Neste projeto foram: 1) identificadas duas importantes famílias de genes associadas à imunidade inata em peixes, a das lisozimas e a do C5. Caracterizou-se pela primeira vez a função das lisozimas através da sua expressão e atividade enzimática em embriões e em diferentes estágios larvares de uma diversidade de reprodutores de dourada. Estudou-se a função do C5 em peixes, através da construção de redes génicas, modelação por homologia e “docking” molecular entre o C5 e o seu receptor (C5R/CD88); 2) mapeou-se e caracterizou-se o proteoma do embrião de três espécies de peixes mediterrânicos (sargo, corvina e dourada) em duas fases do seu desenvolvimento (24h antes da eclosão e na eclosão) e identificou-se um grupo de proteínas potencialmente relacionadas com a imunidade e a qualidade dos embriões. Avaliou-se a função do gene para enzima “hatching enzyme”, com base na sua expressão em embriões de dourada e robalo nos estágios acima referidos; 3) mapeou-se e caracterizou-se o transcritoma de embriões de dourada com origem em diferentes “hatcheries” na região mediterrânica através de uma abordagem comparativa entre qualidade e estágios de desenvolvimento (qualidade- Boa vrs Má; estágios- Pré- eclosão vrs Eclosão) e identificou-se uma diversidade de transcritos, vias metabólicas e elementos do epitranscriptoma regulatório do RNA-não codificante. Foram identificados em comum 42 candidatos a marcadores de qualidade e enriquecidas duas vias metabólicas relacionadas com o sistema imunológico e associadas às “hatcheries”: a via de infeção por Salmonella (constituída por 7 genes relacionados com o sistema imune) e a via de sinalização MAPK (mitogen-activated protein kinase). Foram identificadas 543 proteínas que são expressas apenas no proteoma, sugerindo que podem ter origem materna e destas, 7 (diferencialmente expressas) estão potencialmente relacionadas com o sistema imune. Globalmente, os resultados forneceram um grupo de marcadores relacionados à imunidade e ao desenvolvimento (qualidade), com potencial de se traduzirem em critérios de qualidade de ovos e embriões para a indústria da aquicultura. Estes resultados, foram amplamente estudados para descrever a base molecular biológica entre os diferentes estágios de desenvolvimento de ovos e embriões de peixes e também, entre diferentes lotes de embriões de diferente qualidade. As ferramentas biológicas e critérios desenvolvidos neste trabalho, oferecem uma orientação para as “hatcheries” de peixes e a sua aplicação contribuirá para melhorar no futuro o sector da aquicultura.O apoio financeiro ao trabalho relatado na presente tese de doutoramento é reconhecido com gratidão, pois foi crucial para o progresso positivo e sucesso do trabalho científico. Uma diversidade de fontes de financiamento apoiou o trabalho desenvolvido nesta tese, quer diretamente através da compra de consumíveis/materiais (PerformFISH a European Union’s Horizon 2020 research and innovation grant, agreement Nº 727610), quer indiretamente através do financiamento do CCMAR de apoio a serviços e equipamentos utilizados durante a execução do trabalho (projetos da Fundação para a Ciência e Tecnologia (FCT) - UIDB/04326/2020, UIDP/04326/2020 e dos programas operacionais CRESC Algarve 2020 e COMPETE 2020 através do projeto EMBRC.PT ALG-01-0145-FEDER-022121)

    Transcriptional Regulation: a Genomic Overview

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    The availability of the Arabidopsis thaliana genome sequence allows a comprehensive analysis of transcriptional regulation in plants using novel genomic approaches and methodologies. Such a genomic view of transcription first necessitates the compilation of lists of elements. Transcription factors are the most numerous of the different types of proteins involved in transcription in eukaryotes, and the Arabidopsis genome codes for more than 1,500 of them, or approximately 6% of its total number of genes. A genome-wide comparison of transcription factors across the three eukaryotic kingdoms reveals the evolutionary generation of diversity in the components of the regulatory machinery of transcription. However, as illustrated by Arabidopsis, transcription in plants follows similar basic principles and logic to those in animals and fungi. A global view and understanding of transcription at a cellular and organismal level requires the characterization of the Arabidopsis transcriptome and promoterome, as well as of the interactome, the localizome, and the phenome of the proteins involved in transcription

    Effect of Larval Nutrition on Maternal mRNA Contribution to the Drosophila Egg.

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    Embryonic development begins under the control of maternal gene products, mRNAs and proteins that the mother deposits into the egg; the zygotic genome is activated some time later. Maternal control of early development is conserved across metazoans. Gene products contributed by mothers are critical to many early developmental processes, and set up trajectories for the rest of development. Maternal deposition of these factors is an often-overlooked aspect of parental investment. If the mother experiences challenging environmental conditions, such as poor nutrition, previous studies in Drosophila melanogaster have demonstrated a plastic response wherein these mothers may produce larger eggs to buffer the offspring against the same difficult environment. This additional investment can produce offspring that are more fit in the challenging environment. With this study, we ask whether D. melanogaster mothers who experience poor nutrition during their own development change their gene product contribution to the egg. We perform mRNA-Seq on eggs at a stage where all mRNAs are maternally derived, from mothers with different degrees of nutritional limitation. We find that nutritional limitation produces similar transcript changes at all degrees of limitation tested. Genes that have lower transcript abundance in nutritionally limited mothers are those involved in translation, which is likely one of the most energetically costly processes occurring in the early embryo. We find an increase in transcripts for transport and localization of macromolecules, and for the electron transport chain. The eggs produced by nutrition-limited mothers show a plastic response in mRNA deposition, which may better prepare the future embryo for development in a nutrition-limited environment

    Comparative Analysis of Small Non-Coding RNA and Messenger RNA Expression in Somatic Cell Nuclear Transfer and In Vitro-Fertilized Bovine Embryos During Early Development Through the Maternal-to-Embryonic Transition

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    Cloning animals using somatic cell nuclear transfer (scNT) was first successfully demonstrated with the birth of Dolly the sheep, but the process of cloning remains highly inefficient. By improving our understanding of the errors that may occur during cloned cattle embryo development, we could obtain a greater understanding of how specific molecular events contribute to successful development. The central dogma of biology refers to the process of DNA being transcribed into messenger RNA (mRNA) and the translation of mRNA into proteins, which ultimately carry out the functions encoded by genes. The epigenetic code is defined as the array of chemical modifications, or “marks”, to DNA molecules that do not change the genome sequence but do allow for control of gene expression. During early development, genome reprogramming involves the removal of epigenetic marks from the sperm and egg and re-establishment of marks for the embryonic genome that code for proper gene expression to support embryo development. The point during this process at which the embryo’s genes are turned on is known as embryonic genome activation (EGA). Small non-coding RNAs (sncRNAs), including microRNAs (miRNAs), may also contribute to the this process. For example, miRNA molecules do not code for proteins themselves, but rather bind to mRNAs and effectively block their translation into protein. We hypothesized that aberrant expression of sncRNAs in cloned embryos may lead to anomalous abundance of mRNA molecules, thus explaining poor development of cloned embryos. First, we used RNA sequencing to examine the total population of sncRNAs in cattle embryos produced by in vitro fertilization (IVF) and found a dramatic shift in populations at the EGA. Next, we collected both sncRNA and mRNA from scNT cattle embryos, and again performed sequencing of both RNA fractions. We found that few sncRNAs were abnormally expressed in scNT embryos, with all differences appearing after EGA at the morula developmental stage. However, notable differences in the populations of sncRNAs were evident when comparing embryos by developmental stage. For populations of mRNA, we observed dramatic differences when comparing scNT and IVF cattle embryos, with the highest number of changes occurring at the EGA (8-cell stage) and after (morula stage). While changes in specific miRNA molecules (miR-34a and miR-345) were negatively correlated with some of their predicted target mRNAs, this pattern was not widespread as would be expected if these sncRNAs are functionally binding to all of the predicted mRNA targets. Collectively, our observations suggest that other mechanisms leading to altered expression of mRNA in cloned embryos may be responsible for their relatively poor development
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