RNA-seq of female gonads isolated from juvenile eif4e1b mutant and wild-type zebrafish
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ABSTRACT:
ORGANISM(S): Danio rerio
PROVIDER: GSE241537 | GEO | 2023/10/03
REPOSITORIES: GEO
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Project description:Maternal mRNAs are essential for protein synthesis during oogenesis and early embryogenesis. To adapt translation to specific needs during development, maternal mRNAs are translationally repressed by shortening the polyA tails. While mRNA deadenylation is associated with decapping and degradation in somatic cells, maternal mRNAs with short polyA tails are stable. Here we report an essential role for the germline-specific paralog of the mRNA cap-binding factor eIF4E, known as eIF4E1b, in the storage and repression of maternal mRNAs with short polyA tails. eIF4E1b binds to the mRNA cap and is targeted to ribonucleoprotein complexes through its direct interaction with eIF4ENIF1/4E-T. In early embryos, eIF4E1b binds to a specific set of translationally repressed mRNAs with short or no polyA tails, such as histone mRNAs, which are translated later on during embryogenesis. Consistent with an important role in maternal mRNA dormancy, mutation of eIF4E1b in zebrafish impairs female germline development. Understanding the mechanism and function of eIF4E1B provides new insights into fundamental post-transcriptional regulatory principles governing early vertebrate development.
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Project description:Piwi proteins specify an animal-specific subclass of the Argonaute family, that in vertebrates are specifically expressed in germ cells. We demonstrate that zebrafish Piwi (Ziwi) is expressed in both the male and the female gonad, and is a component of a germline specifying structure, called nuage. Loss of Ziwi function results in a progressive loss of germ cells due to apoptosis during larval development. In animals that have only reduced Ziwi function, germ cells are maintained, but display abnormal levels of apoptosis in adults. In mammals, Piwi proteins have been found to associate with approximately 29-nucleotide long, testis specific RNA molecules, called piRNAs. Here we show that zebrafish piRNAs are present in both ovaries and testes. Furthermore, we show that piRNAs are modified at their 3’ end and that they are produced by a Dicer independent mechanism. Many ovarian piRNAs are derived from transposons, implicating a role for these molecules in the silencing of these repetitive elements in vertebrates. Keywords: high-throughput 454 sequencing sequencing of ~30 nt small RNAs from zebrafish ovaries and testes using 454 technology
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Project description:We have identified eukaryotic translation initiation factor 4E family member 1B (Eif4e1b) as a regulator of maternal RNA translation, whose maternal deletion leads to 2-cell arrest phenotype in mouse embryos. eIF4E1b protein binds mRNA selectively in oocytes and controls their translation in early embryos. We aim to profile the global changes in protein expression in metaphase II (MII) eggs and zygotes collected from control or Eif4e1b knockout female mice. An encapsulated proteomic-sample processing protocol is used to perform this low-input mass spectrometry to quantify proteomic changes (Kulak et al. 2014/PMID 24497582).
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Project description:Piwi proteins specify an animal-specific subclass of the Argonaute family, that in vertebrates are specifically expressed in germ cells. We demonstrate that zebrafish Piwi (Ziwi) is expressed in both the male and the female gonad, and is a component of a germline specifying structure, called nuage. Loss of Ziwi function results in a progressive loss of germ cells due to apoptosis during larval development. In animals that have only reduced Ziwi function, germ cells are maintained, but display abnormal levels of apoptosis in adults. In mammals, Piwi proteins have been found to associate with approximately 29-nucleotide long, testis specific RNA molecules, called piRNAs. Here we show that zebrafish piRNAs are present in both ovaries and testes. Furthermore, we show that piRNAs are modified at their 3’ end and that they are produced by a Dicer independent mechanism. Many ovarian piRNAs are derived from transposons, implicating a role for these molecules in the silencing of these repetitive elements in vertebrates. Keywords: high-throughput 454 sequencing
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Project description:Small RNAs exert an effect through diverse RNA interference pathways to transcriptionally or post-transcriptionally silence their targets. The Piwi-interacting RNAs (piRNAs) represent a germline-specific small RNA pathway where Piwi proteins themselves are thought to mediate piRNA biosynthesis. Here, we provide strong evidence for a piRNA amplification loop in zebrafish, in which Ziwi and Zili bind piRNAs of opposite polarity. Furthermore, we describe a function for Zili in transposon defense and germ cell differentiation, as well as a crucial function in meiosis, significantly extending the function of Piwi proteins beyond the control of transposable elements in vertebrates. small RNA from total gonads or Argonaute IPs were cloned and sequenced using 454 GS FLX system.
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Project description:We have identified eukaryotic translation initiation factor 4E family member 1B (Eif4e1b) as a regulator of maternal RNA translation, whose maternal deletion leads to 2-cell arrest phenotype in mouse embryos. eIF4E1b protein binds mRNA selectively in oocytes and controls their translation in early embryos. eIF4E1b expresses specifically in mouse ovary and we have generated a knock-in mouse line in which HA tag is conjugated to the C terminus of eIF4E1b. We aim to identify eIF4E1b co-factors using mass spectrometry after anit-HA immunoprecipitation (IP) using mouse ovaries.
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Project description:Whereas in ovo exposure of genetically male (ZZ) chicken embryos to exogenous estrogens temporarily feminizes gonads at the time of hatching, the morphologically ovarian ZZ-gonads (FemZZs for feminized ZZ gonads) are masculinized back to testes within one year. To identify the feminization-resistant “memory” of genetic male sex, FemZZs showing varying degrees of feminization were subjected to transcriptomic, DNA methylome, and immunofluorescence analyses. Protein-coding genes were classified based on their relative mRNA expression across normal ZZ-testes, genetically female (ZW) ovaries, and FemZZs. We identified a group of 25 genes that were strongly expressed in both ZZ-testes and FemZZs but dramatically suppressed in ZW-ovaries. Interestingly, 84% (21/25) of these feminization-resistant testicular marker genes, including the DMRT1 master masculinizing gene, were located in chromosome Z. Expression of representative marker genes of germline cells (e.g., DAZL or DDX4/VASA) was stronger in FemZZs than normal ZZ-testes or ZW-ovaries. We also identified 231 repetitive sequences (RSs) that were strongly expressed in both ZZ-testes and FemZZs, but these RSs were not enriched in chromosome Z. Although 94% (165/176) of RSs exclusively expressed in ZW-ovaries were located in chromosome W, no feminization-inducible RS was detected in FemZZs. DNA methylome analysis distinguished FemZZs from normal ZZ- and ZW-gonads. Immunofluorescence analysis of FemZZ gonads revealed expression of DMRT1 protein in medullary SOX9+ somatic cells and apparent germline cell populations in both medulla and cortex. Taken together, our study provides evidence that both somatic and germline cell populations in morphologically feminized FemZZs maintain significant transcriptomic and epigenetic memories of genetic sex.
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Project description:Following sex determination, XY and XX gonads develop into a testis and an ovary, respectively. Depending on the sex of the gonad, resident germ cells will subsequently be committed to either spermatogenesis or oogenesis. In this study we took advantage of the Wv/Wv mouse genetic model, in which gonads are almost devoided of germ cells, to uncover gene expression underlying fetal germ cell development. Male and Female gonads were collected at 12, 14 and 16 days post coitum (dpc) from wild-type and Wv/Wv mouse embryos. Total RNAs were extracted and hybridized on Affymetrix microarrays. Expression signals from male and female gonads or from wild-type and mutant gonads were compared to identify sexually dimorphic genes as well as genes expressed in germ cell during fetal gonad development.
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