Expression data from early Zebrafish embryos after knockdown of mir-34
ABSTRACT: microRNAs play crucial roles in the early development of an organism. However the regulation of transcription through the action of microRNAs during the initial embyonic development has not been studied. We used microarrays to detail the effect of maternal microRNA mir-34 in regulation of zygotic trancription during the initial stages of Zebrafish embryonic development from one cell stage through the maternal-zygotic transition up to 24 hours post fertilization. Zebrafish embryos were selected at specific stages of early embryonic development (1, 7, and 24 hours post fertilization). RNA was extracted and hybridized to Affymetrix microarrays. The embryos were injected with anti-microRNA LNA and were kept for constant monitoring for the indicated time points. A mockLNA was injected as a control. The embryos were visualized for any visible developmental abnormalities.
Project description:Sox31 is a member of the zebrafish SoxB1 subfamily, and its expression can be detected both pre- and post-MBT. To distinguish the function of its maternal and zygotic transcripts, a splice blocking morpholino (Sb MO) was designed to interfere with the processing of new, zygotically synthesised mRNAs without interfering with mRNAs of maternal origin. Developmental arrest was observed in Sb MO which could not bypass MBT. Mid-Blastula Transition (MBT) functions as a time window for zygotic genome activation and maternal mRNA degradation. To uncover whether the “zygotic up” and “maternal down” event during MBT is retarded in Sb morphants, we performed microarray experiment at the end of MBT (about 4.3 hours post fertilication/4.3 hpf) to compare mRNAs from Sb morphants and control embryos. In one experiment, three flocks of zebrafish eggs were injected with the Sox19b morpholino immediately after fertilization, while another three control populations were injected with placebo. At 4.3 hpf, these six flocks of embryos were sent for gene expression profiling with six Affymetrix Zebrafish Genome Arrays. In another experiment, we compared two wildtype embryo samples at 4h (post-MBT) against two wildtype samples at 2.5 h (pre-MBT).
Project description:microRNAs play crucial roles in the early development of an organism. However the regulation of transcription through the action of microRNAs during the initial embyonic development has not been studied. We used microarrays to detail the effect of maternal microRNA mir-34 in regulation of zygotic trancription during the initial stages of Zebrafish embryonic development from one cell stage through the maternal-zygotic transition up to 24 hours post fertilization. Overall design: Zebrafish embryos were selected at specific stages of early embryonic development (1, 7, and 24 hours post fertilization). RNA was extracted and hybridized to Affymetrix microarrays. The embryos were injected with anti-microRNA LNA and were kept for constant monitoring for the indicated time points. A mockLNA was injected as a control. The embryos were visualized for any visible developmental abnormalities.
Project description:Upon fertilization, maternal factors direct development in a transcriptionally silent embryo. At the maternal-to-zygotic transition (MZT), a universal step in animal development, unknown maternal factors trigger zygotic genome activation (ZGA). In zebrafish, ZGA is required for gastrulation and clearance of maternal mRNAs, which is achieved in part by the conserved microRNA miR-430. However, the precise factors that activate the zygotic program remain largely unknown. Here we show that Nanog, Pou5f1 and SoxB1 are required for genome activation in zebrafish. We identified several hundred genes directly activated by maternal factors, thus constituting the first wave of zygotic transcription in zebrafish. Ribosome profiling in the pre-MZT embryo revealed that nanog, sox19b and pou5f1 are the most highly translated transcription factor mRNAs. Combined loss of function for Nanog, SoxB1 and Pou5f1 resulted in developmental arrest prior to gastrulation, and a failure to activate >75% of zygotic genes. Furthermore, we found that Nanog binds the miR-430 locus and together with Pou5f1 and SoxB1 initiate miR-430 expression and activity. Our results demonstrate that maternal Nanog, Pou5f1 and SoxB1 are required to initiate the zygotic developmental program and in turn trigger the clearance of the maternal program by activating miR-430 expression. Wild type and loss-of-function total mRNA sequencing of embryonic transcriptomes pre- and post-MZT; ribosome profiling pre-MZT
Project description:During development histone modifying enzymes are required for cell identity and lineage commitment, however little is known about the regulatory origins of the epigenome during embryonic development. Here we generate a comprehensive set of embryonic epigenome reference maps, which we use to determine the extent to which maternal factors shape chromatin state in Xenopus embryos. Using α-amanitin to inhibit zygotic transcription, we find that the majority of H3K4me3 and H3K27me3-enriched regions form a maternally defined epigenetic regulatory space with an underlying logic of hypomethylated islands. This maternal regulatory space extends to a substantial proportion of neurula stage-activated promoters. In contrast, p300-recruitment to distal regulatory regions requires embryonic transcription at most loci. The results show that H3K4me3 and H3K27me3 are part of a regulatory space that exerts an extended maternal control well into post-gastrulation development, and highlight the combinatorial action of maternal and zygotic factors through proximal and distal regulatory sequences. We have performed ChIP-sequencing of eight histone modifications, RNA polymerase II (RNAPII) and the enhancer protein p300 at five stages of development: blastula (st. 9), gastrula (st. 10.5, 12.5), neurula (st. 16) and tailbud (st. 30). These experiments allow identification of enhancers (H3K4me1, p300), promoters (H3K4me3, H3K9ac), transcribed regions (H3K36me3, RNAPII) and repressed and heterochromatic domains (H3K27me3, H3K9me2, H3K9me3, H4K20me3). In addition we generated pre-MBT (st. 8) maps for three histone modifications (H3K4me3, H3K9ac, H3K27me3) and single-base resolution DNA methylome maps using whole genome bisulfite sequencing of blastula and gastrula (st. 9 and 10.5) embryos. To determine the maternal and zygotic contributions to chromatin state, we used alpha-amanitin to block embryonic transcription. Fertilised eggs were injected with 2.3 nl of 2.67 ng/ul alpha-amanitin and developed until the control embryos reached mid-gastrulation. Alpha-amanitin and control embryos were used for RNA-seq and ChIP-seq of RNAPII, H3K4me3, H3K27me3 and p300. For all ChIP-seq samples of the epigenome reference maps and RNAPII ChIP-seq samples of the α-amanitin experiments three biological replicates of different chromatin isolations of 45 embryos were pooled. Two biological replicates for H3K4me3 (α-amanitin injected: resp. 90 and 56 embryo equivalents (eeq); control: resp. 45 and 67 eeq), H3K27me3 (α-amanitin injected: resp. 90 and 180 eeq; control: resp. 45 and 202 eeq) and p300 (α-amanitin injected: resp. 112 and 56 eeq; control: resp. 112 and 67 eeq) ChIP-seq samples of the α-amanitin experiments were generated. For RNA-seq samples of the α-amanitin experiments RNA from 5 embryos from one biological replicate was isolated and depleted of ribosomal RNA
Project description:We report the m6A methylation maps of zebrafish embryos during early development, and transcriptome-wide changes occured in ythdf2 LOF mutant embryos. Our study reveals the m6A-dependent RNA decay as a previously unidentified maternal mode mechanism to regulate maternal mRNA clearance during zebrafish MZT, highlighting the critical role of the m6A mRNA methylation in animal development. Overall design: m6A-seq of mRNA from zebrafish embryos at 0, 2, 4, 6, 8 h.p.f.; mRNA-seq of wild-type and maternal ythdf2-null mutant embryos at 0, 2, 4, 6, 8 h.p.f.; mRNA-seq of embryos injected with control or ythdf2-MO at 4 h.p.f.; mRNA-seq of wild-type and maternal-zygotic ythdf2-null mutant embryos at 6, 8, 12, 24 h.p.f; and mRNA-seq of wild-type and maternal ythdf2-null mutant embryos injected with control or ythdf2-MO at 24, 36, 48 h.p.f. Replicate of each seq is also included.
Project description:In zebrafish, vitellogenic oocytes can incorporate significant amounts of 17β-estradiol released from nearby granulosa cells according to a first-order kinetics, since the steroid low polarity ensures high permeability and affinity for yolk lipids. Estrogen bioactivity is likely, because the maternal mRNA for the estrogen receptor-β2 (ers2a) is highly expressed in ovulated oocytes. This transcript is available for translation in the embryo until its sharp decline from 4 to 8 hours post-fertilization (hpf), being replaced by low levels of zygotic ers2a mRNA from 24 hpf to hatching at 48 hpf, as determined by qRT-PCR. Estrogen receptors-α and -β1 are only expressed zygotically at low levels from 24 hpf onwards. To test the functional role of maternal ers2a mRNA, 1- or 2-cell embryos were injected with 10.3 ng each of morpholino to knockdown translation (MO2-ers2a) of both maternal and zygotic ers2a transcripts, missplicing morpholino (MO3-ers2a) to block post-transcriptionally the zygotic transcript alone, and a nonspecific morpholino (MO-control) as a control. Treatment with MO2-ers2a caused severe malformations in 63% of 1-5 dpf larvae, as compared to 10-11% in those treated with MO3-ers2a and MO2-control. Defects included body growth delay and curved shape, abnormal brain and splanchnocranium development, enlarged and hemorrhagic pericardial cavity, uninflated swim bladder and rudimentary caudal fin with aberrant circular motion. Affected larvae could survive for only 12-14 days. Co-injection of an anti-p53 MO failed to rescue the MO2-ers2a-phenotypes, eliminating the possibility of off-target effects. Pangenomic microarray analysis revealed that 240 and 219 significantly expressed transcripts were up- and down-regulated, respectively, by maternal Ers2a protein deficiency in 8-hpf MO2-ers2a-embryos. Also at 48 hpf, 162 and 120 presumably zygotic transcripts were up- and down-regulated, respectively, but only 18 were in common with each of the 8-hpf sets. Whole-mount in situ hybridization revealed an intensified expression of the genes six3.1 and emx1 in MO2-ers2a-embryos at 24-48 hpf, as compared to controls. These findings suggest the involvement of maternal ers2a mRNA in the epigenetic programming of zebrafish development. MO2-ers2a morphants were compared with MO-control at 8 hpf and 48 hpf. MO2-ers2a is a morpholinos selected to knockdown translation of ers2a mRNA
Project description:The maternal-to-zygotic transition (MZT) is a process that occurs in animal embryos at the earliest developmental stages, during which maternally deposited mRNAs and other molecules are degraded and replaced by products of the zygotic genome. The zygotic genome is not activated immediately upon fertilization, therefore post-transcriptional mechanisms control the first steps of development in the early, pre-MZT embryo. To perform unbiased organism-wide identification of Drosophila RNA binding proteins (RPBs), crucial players of post-transcriptional control, we applied the recently developed RNA interactome capture method, which involves cross-linking of RNAs and their direct protein partners by UV light, purification of RNA under stringent conditions and identification of proteins by mass spectrometry. Our analysis yielded 523 high confidence RBP hits, half of which were not previously reported to bind RNA. Our comparison of the RNA interactomes of pre- and post-MZT embryos reveals a highly dynamic behavior of the RNA-bound proteome during early development, and suggests active regulation of RNA binding of some RBPs. This resource provides the first evidence of RNA binding for hundreds of Drosophila proteins, and opens new avenues for study of molecular mechanisms of early development.
Project description:Prenatal exposure to ethanol leads to a myriad of developmental disorders known as fetal alcohol spectrum disorder, often characterized by growth and mental retardation, central nervous system damage and specific craniofacial dysmorphic features. Although the exact mechanisms of ethanol toxicity are not well understood it is known that ethanol exposure during development affects the expression of several genes involved in cell cycle control, apoptosis and transcription. MicroRNAs (miRNAs) are implicated in some of these processes however it is unclear if they are involved in ethanol-induced toxicity. Here we tested whether ethanol deregulates miRNA expression in zebrafish embryos and if a miRNA deregulation signature could be inferred. For this, zebrafish embryos were exposed to two different ethanol concentrations (1% and 1.5%) from 4 hours post-fertilization (hpf) to 24hpf. MicroRNA expression profiles revealed that ethanol exposure induces deregulation of miRNA expression significantly. Seven miRNAs are commonly up-regulated after both ethanol treatments, namely miR-153a, miR-725, miR-30d, let-7k, miR-100, miR-738 and miR-732, whereas downregulation of miR-23a, miR-203, let-7c, miR-128 and miR-193b is detected after 1% ethanol exposure only. Target prediction of deregulated miRNAs shows that putative targets are involved in cell cycle control, apoptosis and transcription, which are the main processes affected by ethanol toxicity. The overall study shows that the effects of ethanol on miRNA deregulation are dose-dependent and that miRNAs are relevant in the context of alcohol toxicity. Moreover, a miRNA toxicity signature for embryonic ethanol exposure was obtained. Zebrafish embryos were obtained from spawning adults in groups of about 10 males and 10 females. Zebrafish embryos were collected and Petri dishes with approximately 250 eggs each were incubated at 28ºC to allow normal zebrafish development until 4hpf, when blastula is reached. At this stage, embryos were examined under a dissecting microscope and those that had developed normally were selected for EtOH exposure (approximately 200 eggs). Briefly, 200 embryos were randomly distributed into plastic Petri dishes containing 20 mL of EtOH test solutions (1% EtOH, 1.5% EtOH). All solutions were made by dilution of absolute EtOH in system water. Exposure was from 4hpf to 24hpf. At this stage, solutions were changed by system water and embryos were allowed to grow until 24hpf. The control group was allowed to grow in plain system water. Zebrafish embryos were collected at 24hpf for microarray analysis. Two biological replicates were performed for each assay.
Project description:mRNA seq based approach to determine the transcriptome dynamics during early development To study the mechanisms regulating this developmental event in zebrafish, we applied RNA deep sequencing technology and generated comprehensive transcriptome profiles of 6 developmental stages from oocyte to early gastrulation. We determined the expression levels of maternal and zygotic transcripts and clustered them based on expression pattern. We identified a large number of novel transcribed regions in un-annotated regions of the genome, as well as splice variants with an estimated frequency of 40-75% during early zebrafish embryogenesis. Our data constitute a useful resource for developmental studies, gene discovery, and genome annotation. RNA was extracted from pooled embryos of desired stages and one RNA seq library was generated for each sample. Totally 6 stages were selected: Maternal, 1cell, 16/32 cells, 128/256 cells, 3.5hpf and 5.3hpf
Project description:We compared Agilent custom made expression microarrays with Illumina deep sequencing for RNA analysis of zebrafish embryos 5 days post fertilization, showing as expected a high degree of correlation of expression of a common set of 15,927 genes for untreated fish. The transcriptomes were also compared for fish injected in the yolk with Mycobacterium marinum This RNA deep sequencing study was designed to determine the gene expression profile of zebrafish embryos 5 days post fertilization. We also have compared expression with embryos that were injected with Mycobacterium marinum in the yolk at 2 hours post fertilization. After injections embryos were transferred into fresh egg water and incubated at 28°C. 150 embryos of mock-injected embryos or 200 embryos injected with 12 CFU bacteria were snap-frozen in liquid nitrogen, and total RNA was isolated using TRIZOL reagent.