Zygotic Gene Research Articles

Effect of introducing somatic mitochondria into an early embryo on zygotic gene activation†.

Unlike differentiated somatic cells, which possess elongated mitochondria, undifferentiated cells, such as those of preimplantation embryos, possess round, immature mitochondria. Mitochondrial morphology changes dynamically during cell differentiation in a process called mitochondrial maturation. The significance of the alignment between cell differentiation and mitochondrial maturity in preimplantation development remains unclear. In this study, we analyzed mouse embryos into which liver-derived somatic mitochondria were introduced (SM-embryos). Most SM-embryos were arrested at the two-cell stage. Some of the introduced somatic mitochondria became round, while others remained elongated and large. RNA-sequencing revealed a disruption of both minor and major zygotic gene activation (ZGA) in SM-embryos. Minor ZGA did not terminate before major ZGA, and the onset of major ZGA was inhibited, as shown by histone modification analyses of histone H3 lysine 4 trimethylation and histone H3 lysine 27 acetylation. Further analysis of metabolites involved in histone modification regulation in SM-embryos showed a significantly lower NAD+/NADH ratio in SM-embryos than in control embryos. Additionally, the mitochondrial membrane potential, an indicator of mitochondrial function, was lower in SM-embryos than in control embryos. Our results demonstrated that introducing somatic mitochondria into an embryo induces mitochondrial dysfunction, thereby disrupting metabolite production, leading to a disruption in ZGA and inducing developmental arrest. Our findings reveal that the alignment between cell differentiation and mitochondrial maturity is essential for early embryonic development.

3-methyl-4-nitrophenol disturbs the maternal-to-zygotic transition of early embryos by damaging mitochondrial function and histone modification.

3-methyl-4-nitrophenol (PNMC), a chemical prevalent in various industries for drug, dye, and leather production, also serves as a primary byproduct of organophosphate insecticides. Despite its global recognition as an endocrine disruptor with documented reproductive toxicity, its detrimental impact on preimplantation embryonic development has yet to be thoroughly investigated. In this study, through the in vitro culture of mice embryos, it was initially observed that even low concentrations of PNMC exposure led to a significant reduction in blastocyst formation and a sharp decline in the ratio of inner cell mass within the blastocysts. SMART-seq2 transcriptome sequencing further confirmed that PNMC treatment disrupted global gene expression in 2-cell embryos, with differentially expressed genes enriched in multiple signaling pathways, including those related to autophagy, apoptosis, fertilization, embryonic development, transcription, and mRNA processing. Integration of transcriptome data with open databases revealed that both zygotic genome activation genes and maternal factors experienced significant transcript-level disruptions. Moreover, the study demonstrated that these gene expression changes were closely associated with mitochondrial dysfunction, evidenced by diminished mitochondrial membrane potential, reduced ATP production, aberrant expression of mitochondria-related genes, increased ROS accumulation, and heightened DNA damage in PNMC-treated embryos. Additionally, PNMC exposure induced defects in histone modification, as shown by altered levels of H3K9me3 and H3K27me3, H3K9ac and H3K27ac. Lastly, the findings indicated that PNMC triggered apoptosis in embryos, validated by elevated BAX and CASPASE3 expression, alongside positive TUNEL staining. In summary, PNMC exposure impairs the maternal-to-zygotic transition, likely through mitochondrial dysfunction and histone modification, culminating in developmental arrest and apoptosis in mouse preimplantation embryos.

Zelda is dispensable for Drosophila melanogaster histone gene regulation.

To ensure that the embryo can package exponentially increasing amounts of DNA, replication-dependent histones are some of the earliest transcribed genes from the zygotic genome. However, how the histone genes are identified is not known. The Drosophila melanogaster pioneer factor CLAMP regulates the embryonic histone genes and helps establish the histone locus body, a suite of factors that controls histone mRNA biosynthesis, but CLAMP is not unique to the histone genes. Zelda collaborates with CLAMP across the genome to regulate zygotic genome activation and target early activated genes. We hypothesized that Zelda helps identify histone genes for early embryonic expression. We found that Zelda targets the histone gene locus early during embryogenesis, prior to histone gene expression. However, depletion of zelda in the early embryo does not affect histone mRNA levels or prevent the recruitment of other factors. These results suggest the earliest events responsible for specifying the zygotic histone genes remain undiscovered.

HIRA and dPCIF1 coordinately establish totipotent chromatin and control orderly ZGA in Drosophila embryos

Early embryos undergo profound changes in their genomic architecture to establish the totipotent state, enabling pioneer factors to access chromatin and drive zygotic genome activation (ZGA). However, the mechanisms by which the totipotent state is established and properly interpreted by pioneer factors to allow orderly ZGA remain unknown. Here, we identify the H3.3-specific chaperone HIRA as a factor involving establishing totipotent-state chromatin in Drosophila early embryos. Through cophase separation with HIRA, the pioneer factor GAGA factor (GAF) efficiently binds to H3.3-marked nucleosomes to activate major-wave zygotic genes. Importantly, dPCIF1, a chromatin-associated protein, antagonized the GAF-HIRA interaction by competitively binding to HIRA, thereby restricting GAF on earlier chromatin and avoiding premature ZGA. Hence, the coordinated action of HIRA and dPCIF1 ensures sequential ZGA from the minor to major wave in early embryos. This study provides insights into understanding how a totipotent state is established and properly controlled during ZGA.

Comparison Between Electroporation at Different Voltage Levels and Microinjection to Generate Porcine Embryos with Multiple Xenoantigen Knock-Outs.

In the context of xenotransplantation, the production of genetically modified pigs is essential. For several years, knock-out pigs were generated through somatic cell nuclear transfer employing donor cells with the desired genetic modifications, which resulted in a lengthy and cumbersome procedure. The CRISPR/Cas9 system enables direct targeting of specific genes in zygotes directly through microinjection or electroporation. However, these techniques require improvement to minimize mosaicism and low mutation rates without compromising embryo survival. This study aimed to determine the gene editing potential of these two techniques to deliver multiplexed ribonucleotide proteins (RNPs) to generate triple-knock-out porcine embryos with a multi-transgenic background. We designed RNP complexes targeting the major porcine xenoantigens GGTA1, CMAH, and B4GALNT2. We then compared the development of mosaicism and gene editing efficiencies between electroporation and microinjection. Our results indicated a significant effect of voltage increase on molecule intake in electroporated embryos, without it notably affecting the blastocyst formation rate. Our gene editing analysis revealed differences among delivery approaches and gene loci. Notably, employing electroporation at 35 V yielded the highest frequency of biallelic disruptions. However, mosaicism was the predominant genetic variant in all RNP delivery methods, underscoring the need for further research to optimize multiplex genome editing in porcine zygotes.

Efficient gene editing of pig embryos by combining electroporation and lipofection

Background and Aim: Mosaicism, which is characterized by the presence of wild-type and more than one mutant allele, poses a serious problem in zygotic gene modification through the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 system. Therefore, we used pig embryos to compare the gene editing efficiencies achieved by combining electroporation and lipofection using different aminopeptidase N (APN)-targeting guide RNA (gRNA) sequences. Materials and Methods: Six gRNAs (gRNA1–6) with different target sequences were designed to target APN. Zona pellucida (ZP)-intact zygotes collected 10 h after the start of in vitro fertilization (IVF) were electroporated with each gRNA to compare their gene editing efficiency. The gRNA sequences that achieved the lowest and highest mutation rates (gRNA4 and gRNA6, respectively) were selected for additional lipofection to assess gene editing efficiency following combined treatment. As ZP removal is essential for lipofection, ZP-free zygotes were electroporated with gRNA4 or gRNA6 10 h after IVF initiation, followed by lipofection with the same gRNAs 24 or 29 h after IVF initiation. The electroporated ZP-intact and ZP-free zygotes were used as controls. Results: gRNA4 and gRNA6 exhibited the lowest and highest mutation rates, respectively. gRNA4-targeted ZP-free embryos subjected to additional lipofection 29 h after IVF initiation exhibited significantly higher total and biallelic mutation rates than ZP-intact embryos that received only electroporation. Additional lipofection of gRNA6-targeted embryos had no obvious effect on mutation rates. Conclusion: Electroporation combined with lipofection using gRNAs with low mutation rates may improve gene editing efficiency in pig embryos. However, the effects may vary based on the timing of gene editing. Keywords: electroporation, guide RNA sequence, lipofection, pig embryo.

Single-cell analysis of preimplantation embryonic development in guinea pigs

BackgroundGuinea pigs exhibit numerous physiological similarities to humans, yet the details of their preimplantation embryonic development remain largely unexplored.ResultsTo address this, we conducted single-cell sequencing on the transcriptomes of cells isolated from the zygote stage through preimplantation stages in guinea pigs. This study identified seven distinct cell types within guinea pig preimplantation embryos and pinpointed the timing of zygotic gene activation (ZGA). Trajectory analysis revealed a bifurcation into two lineage-specific branches, accompanied by alterations in specific pathways, including oxidative phosphorylation and vascular endothelial growth factor (VEGF). Additionally, co-expressed gene network analysis highlighted the most enriched functional modules for the epiblast (EPI), primitive endoderm (PrE), and inner cell mass (ICM). Finally, we compared the similarities and differences between human and guinea pig epiblasts (EPIs).ConclusionThis study systematically constructs a cell atlas of guinea pig preimplantation embryonic development, offering fresh insights into mammalian embryonic development and providing alternative experimental models for studying human embryonic development.

Comparison of the developmental competence of in vitro-produced mouse embryos cultured under 5 versus 2% O2 with in vivo-derived blastocysts.

The prevalence of infertility in Canada has substantially increased over 30 years, and plateaued success rates of culture systems warrant further optimization for transfer outcomes. In clinical programs, embryos commonly undergo extended culture under 5% O2 until the blastocyst stage. The aim of this study is to characterize the developmental competence and stress-related responses of embryos cultured under 5 versus 2% O2 in comparison to in vivo-derived blastocysts. We hypothesized 2% O2 compromises developmental competence through altered embryonic stress responses and induction of apoptosis-related genes relative to those cultured under 5% O2 and in vivo-derived blastocysts. Quantitative measures of development and relative expressions of a cohort of stress-related genes in CD1 mouse zygotes cultured to blastocysts under 5 or 2% O2 were compared to in vivo-derived embryos. Apoptotic responses were evaluated using an immunofluorescence assay for Caspase-3. The mean percentage of blastocysts developed, and total cell number of embryos derived in vivo or cultured under 5% O2 was significantly higher than those cultured under 2% O2. Blastocyst expansion was greatest in embryos cultured under 5% O2. Stress response genes were significantly upregulated in embryos cultured under 2% O2, and expression of antioxidant-related genes was significantly lower in cultured versus in vivo-derived embryos. Caspase-3 immunofluorescence was significantly higher in cultured embryos versus in vivo-derived embryos. We inferred that 5% O2 systems better approximate physiologic oxygen availability for culture of mouse embryos, warranting re-evaluation of culturing embryos under threshold or sub-physiologic oxygen concentrations during clinical IVF programs.

Embryonic Genome Activation (EGA) Occurred at 1-Cell Stage of Embryonic Development in the Mud Crab, Scylla paramamosain, Revealed by RNA-Seq.

As a prerequisite for the success of embryo development, embryonic genome activation (EGA) is an important biological event in which zygotic gene products in the embryo are activated to replace maternal-derived transcripts. Although EGA has been extensively studied in a large number of vertebrates and invertebrates, there is a lack of information regarding this event in crustacean crab. In this study, the timing of EGA was confirmed by examining a transcriptomic dataset of early embryonic development, including mature oocytes and embryos through six early developmental stages, and signaling pathways associated with EGA were identified in the mud crab, S. paramamosain. The comprehensive transcriptomic data identified a total of 53,915 transcripts from these sequencing samples. Notable transcriptomic change was evident at the 1-cell stage, indicated by a 36% transcript number shift and a reduction in transcript fragment length, compared to those present in the mature oocytes. Concurrently, a substantial increase in the expression of newly transcribed transcripts was observed, with gene counts reaching 3485 at the 1-cell stage, indicative of the onset of EGA. GO functional enrichment revealed key biological processes initiated at the 1-cell stage, such as protein complex formation, protein metabolism, and various biosynthetic processes. KEGG analysis identified several critical signaling pathways activated during EGA, including the "cell cycle," "spliceosome," "RNA degradation", and "RNA polymerase", pathways. Furthermore, transcription factor families, including zinc finger, T-box, Nrf1, and Tub were predominantly enriched at the 1-cell stage, suggesting their pivotal roles in regulating embryonic development through the targeting of specific DNA sequences during the EGA process. This groundbreaking study not only addresses a significant knowledge gap regarding the developmental biology of S. paramamosain, especially for the understanding of the mechanism underlying EGA, but also provides scientific data crucial for the research on the individual synchronization of seed breeding within S. paramamosain aquaculture. Additionally, it serves as a reference basis for the study of early embryonic development in other crustacean species.

Knockout, Knockdown, and the Schrödinger Paradox: Genetic Immunity to Phenotypic Recapitulation in Zebrafish.

Previous research has highlighted significant phenotypic discrepancies between knockout and knockdown approaches in zebrafish, raising concerns about the reliability of these methods. However, our study suggests that these differences are not as pronounced as was once believed. By carefully examining the roles of maternal and zygotic gene contributions, we demonstrate that these factors significantly influence phenotypic outcomes, often accounting for the observed discrepancies. Our findings emphasize that morpholinos, despite their potential off-target effects, can be effective tools when used with rigorous controls. We introduce the concept of graded maternal contribution, which explains how the uneven distribution of maternal mRNA and proteins during gametogenesis impacts phenotypic variability. Our research categorizes genes into three types-susceptible, immune, and "Schrödinger" (conditional)-based on their phenotypic expression and interaction with genetic compensation mechanisms. This distinction provides new insights into the paradoxical outcomes observed in genetic studies. Ultimately, our work underscores the importance of considering both maternal and zygotic contributions, alongside rigorous experimental controls, to accurately interpret gene function and the mechanisms underlying disease. This study advocates for the continued use of morpholinos in conjunction with advanced genetic tools like CRISPR/Cas9, stressing the need for a meticulous experimental design to optimize the utility of zebrafish in genetic research and therapeutic development.

Heat tolerance, oxidative stress response tuning and robust gene activation in early-stage Drosophila melanogaster embryos.

In organisms with complex life cycles, life stages that are most susceptible to environmental stress may determine species persistence in the face of climate change. Early embryos of Drosophila melanogaster are particularly sensitive to acute heat stress, yet tropical embryos have higher heat tolerance than temperate embryos, suggesting adaptive variation in embryonic heat tolerance. We compared transcriptomic responses to heat stress among tropical and temperate embryos to elucidate the gene regulatory basis of divergence in embryonic heat tolerance. The transcriptomes of tropical and temperate embryos differed in both constitutive and heat-stress-induced responses of the expression of relatively few genes, including genes involved in oxidative stress. Most of the transcriptomic response to heat stress was shared among all embryos. Embryos shifted the expression of thousands of genes, including increases in the expression of heat shock genes, suggesting robust zygotic gene activation and demonstrating that, contrary to previous reports, early embryos are not transcriptionally silent. The involvement of oxidative stress genes corroborates recent reports on the critical role of redox homeostasis in coordinating developmental transitions. By characterizing adaptive variation in the transcriptomic basis of embryonic heat tolerance, this study is a novel contribution to the literature on developmental physiology and developmental genetics.

Lactate modulates zygotic genome activation through H3K18 lactylation rather than H3K27 acetylation

In spite of its essential role in culture media, the precise influence of lactate on early mouse embryonic development remains elusive. Previous studies have implicated lactate accumulation in medium affecting histone acetylation. Recent research has underscored lactate-derived histone lactylation as a novel epigenetic modification in diverse cellular processes and diseases. Our investigation demonstrated that the absence of sodium lactate in the medium resulted in a pronounced 2-cell arrest at the late G2 phase in embryos. RNA-seq analysis revealed that the absence of sodium lactate significantly impaired the maternal-to-zygotic transition (MZT), particularly in zygotic gene activation (ZGA). Investigations were conducted employing Cut&Tag assays targeting the well-studied histone acetylation and lactylation sites, H3K18la and H3K27ac, respectively. The findings revealed a noticeable reduction in H3K18la modification under lactate deficiency, and this alteration showed a significant correlation with changes in gene expression. In contrast, H3K27ac exhibited minimal correlation. These results suggest that lactate may preferentially influence early embryonic development through H3K18la rather than H3K27ac modifications.

P-232 EIF4E1B Contributes to Early Translation Burst for Embryogenesis Whose Deficiency Causes Embryo Development Arrest at Four-Cell Stage during Oocyte to Embryo Transition in Women

Abstract Study question What are the effects and corresponding mechanisms of the early burst of translation inhibited during the oocyte-to-embryo transition (OET), which is essential for human embryogenesis? Summary answer EIF4E1B contributes to the early burst of translation for embryogenesis whose deficiency causes embryo development arrest at four-cell stage during OET in women. What is known already The first records of human zygotic genes are observed in late one-cell zygotes, and translation in mammals happens before the activation of zygotic genes. Recently, we identified an oocyte specific role of eIF4E1b in selecting and regulating the translation initiation of individual mRNAs that are involved in cell differentiation, cell cycle regulation, and cell fate determination during OET. The genetic ablation of Eif4e1b in mice resulted in a developmental arrest in the 2-cell phase of embryogenesis. How is the early burst of translation regulated in human embryogenesis remains unknown. Study design, size, duration A total of 236 female Chinese participants who underwent assisted reproductive technology (ART) and experienced at least one cycle of OET defects were recruited from our university-affiliated center from January 2017 to May 2023. 60 frozen human 3 pronuclei (3PN) embryos have been used for this work. The use of human embryos donated for research was allowed by the the Ethics Committee of the Reproductive and Genetic Hospital of CITIC–Xiangya (reference LL-SC-2018-009). Participants/materials, setting, methods All recruited individuals underwent whole-exome sequencing. In vitro transcription and micro-injection were employed to validate the embryo development pattern under different translation inhibition as well as potential effects of the EIF4E1B variant in mouse oocytes. Immunoprecipitation was conducted to investigate the causes of OET failure and identify the ability of eIF4E1B bind with PABPC1L. Additionally, a knock-in (KI) mouse model carrying homozygous Eif4e1bA192T was generated to evaluate female fertility and a more comprehensive mechanism. Main results and the role of chance Translation inhibitor cycloheximide (CHX) and knockdown of different translation initiation factors eIF4Es were administered to human 3 pronuclei (3PN) embryos, starting from the 1C stage. The absence of eIF4E1B was identified as a specific phenotype, leading to developmental arrest at the 1-4C stage, which is consistent with the effects of Translation inhibitor CHX. Additionally, a novel homozygous mutation (c.565G>A; p. Ala189Thr) in EIF4E1B was found within a consanguineous family. This mutation was associated with recurrent four-cell arrests, suggesting a translational regulation role for eIF4E1B from the 1C to 4C stage during OET. The mutated eIF4E1B disrupted the expression and condensate distribution of eIF4E1B. We also confirmed the interaction between eIF4E1B and PABPC1L, with the c.565G>A variant in EIF4E1B impairing their binding ability. Furthermore, KI mice carrying homozygous Eif4e1bA192T partially recapitulated the phenotype resulting from EIF4E1B deficiency. Limitations, reasons for caution More cases are required to demonstrate the association between EIF4E1B variants and phenotypes. Wider implications of the findings By delving deeper into the intricate interactions between basic physiology and clinical characteristics, this research elucidates the crucial role of maternal EIF4E1B in ensuring the early burst of translation for embryogenesis and provide a diagnostic genetic marker for women with OET defects. Trial registration number Not applicable

P-127 The RNA m6A landscape of mouse oocytes and preimplantation embryos

Abstract Study question Despite the significance of N6-methyladenosine (m6A) in gene regulation, the requirement for large amounts of RNA has hindered m6A profiling in mammalian early embryos. Summary answer We apply picoMeRIP-seq to map m6A in mouse oocytes and preimplantation embryos. We define the landscape of m6A during the maternal-to-zygotic transition. What is known already N6-methyladenosine (m6A), the most prevalent internal modification in eukaryotic messenger RNA (mRNA), plays key regulatory roles in many biological processes (for example, RNA stability, splicing, transport and translation) and is involved in a variety of physiological processes (for example, cell differentiation and reprogramming, embryonic development and stress responses). Study design, size, duration We recently developed an m6A mapping assay for low RNA input, low-input methyl RNA immunoprecipitation and sequencing (picoMeRIP-seq), which enables mapping of the m6A methylome using a low number of cells. In this article, we applied picoMeRIP-seq to multiple developmental stages of mouse oocytes and early embryos to profile their m6A landscapes. Participants/materials, setting, methods We use mouse as study materials.Using picoMeRIP-seq, we generated transcriptome-wide m6A maps for six stages (GV, MII, zygote, two-cell, eight-cell and blastocyst), with two biological replicates per stage and 49–110 oocytes/embryos per replicate. Main results and the role of chance We define the landscape of m6A during the maternal-to-zygotic transition, including stage-specifically expressed transcription factors essential for cell fate determination. Both the maternally inherited transcripts to be degraded post fertilization and the zygotically activated genes during zygotic genome activation are widely marked by m6A. In contrast to m6A-marked zygotic ally-activated genes, m6A-marked maternally inherited transcripts have a higher tendency to be targeted by microRNAs. Moreover, RNAs derived from retrotransposons, such as MTA that is maternally expressed and MERVL that is transcriptionally activated at the two-cell stage, are largely marked by m6A. Our results provide a foundation for future studies exploring the regulatory roles of m6A in mammalian early embryonic development. Limitations, reasons for caution The effect of m6A on the turnover of retrotransposon-derived transcripts abundant in oocytes and early embryos needs to be further addressed in future studies. Wider implications of the findings Our results provide a foundation for future studies exploring the regulatory roles of m6A in mammalian early embryonic development. Trial registration number not applicable

Muskelin acts as a substrate receptor of the highly regulated Drosophila CTLH E3 ligase during the maternal-to-zygotic transition.

The maternal-to-zygotic transition (MZT) is a conserved developmental process where the maternally-derived protein and mRNA cache is replaced with newly made zygotic gene products. We have previously shown that in Drosophila the deposited RNA-binding proteins ME31B, Cup, and Trailer Hitch (TRAL) are ubiquitylated by the CTLH E3 ligase and cleared. However, the organization and regulation of the CTLH complex remain poorly understood in flies. In particular, Drosophila lacks an identifiable substrate adaptor, and the mechanisms restricting degradation of ME31B and its cofactors to the MZT are unknown. Here, we show that the developmental specificity of the CTLH complex is mediated by multipronged regulation, including transcriptional control by the transcription factor OVO and autoinhibition of the E3 ligase. One major regulatory target is the subunit Muskelin, which we demonstrate acts as a substrate adaptor for the Drosophila CTLH complex. Although conserved, Muskelin has structural roles in other species, suggesting a surprising functional plasticity. Finally, we find that Muskelin has few targets beyond the three known RNA binding proteins, showing exquisite target specificity. Thus, multiple levels of integrated regulation restrict the activity of the embryonic CTLH complex to early embryogenesis, seemingly with the goal of regulating three important RNA binding proteins.

ULI-ssDRIP-seq revealed R-loop dynamics during vertebrate early embryogenesis

R-loop, a chromatin structure containing one RNA:DNA hybrid and one unpaired single-stranded DNA, plays multiple biological roles. However, due to technical limitations, the landscapes and potential functions of R-loops during embryogenesis remain elusive. Here, we developed a quantitative and high-resolution ultra-low input R-loop profiling method, named ULI-ssDRIP-seq, which can map global R-loops with as few as 1000 cells. By using ULI-ssDRIP-seq, we reveal the R-loop dynamics in the zebrafish from gametes to early embryos. In oocytes, the R-loop level is relatively low in most regions of the nuclear genome, except maternal-inherited rDNA and mitochondrial genome. The correlation between R-loop and CG methylation dynamics during early development is relatively weak. Furthermore, either up- or down-regulation of global R-loops by knockdown or overexpression of RNase H1 causes a delay of embryonic development with dramatic expression changes in zygotic and maternal genes. This study provides comprehensive R-loop landscapes during early vertebrate embryogenesis and demonstrates the implication of R-loops in embryonic development.

Cellular remodeling and JAK inhibition promote zygotic gene expression in the Ciona germline

Transcription control is a major determinant of cell fate decisions in somatic tissues. By contrast, early germline fate specification in numerous vertebrate and invertebrate species relies extensively on RNA-level regulation, exerted on asymmetrically inherited maternal supplies, with little-to-no zygotic transcription. However delayed, a maternal-to-zygotic transition is nevertheless poised to complete the deployment of pre-gametic programs in the germline. Here, we focus on early germline specification in the tunicate Ciona to study zygotic genome activation. We first demonstrate that a peculiar cellular remodeling event excludes localized postplasmic Pem-1 mRNA, which encodes the general inhibitor of transcription. Subsequently, zygotic transcription begins in Pem-1-negative primordial germ cells (PGCs), as revealed by histochemical detection of elongating RNA Polymerase II, and nascent Mef2 transcripts. In addition, we uncover a provisional antagonism between JAK and MEK/BMPRI/GSK3 signaling, which controls the onset of zygotic gene expression, following cellular remodeling of PGCs. We propose a 2-step model for the onset of zygotic transcription in the Ciona germline and discuss the significance of germ plasm dislocation and remodeling in the context of developmental fate specification.

DNA methylation remodeling and the functional implication during male gametogenesis in rice

BackgroundEpigenetic marks are reprogrammed during sexual reproduction. In flowering plants, DNA methylation is only partially remodeled in the gametes and the zygote. However, the timing and functional significance of the remodeling during plant gametogenesis remain obscure.ResultsHere we show that DNA methylation remodeling starts after male meiosis in rice, with non-CG methylation, particularly at CHG sites, being first enhanced in the microspore and subsequently decreased in sperm. Functional analysis of rice CHG methyltransferase genes CMT3a and CMT3b indicates that CMT3a functions as the major CHG methyltransferase in rice meiocyte, while CMT3b is responsible for the increase of CHG methylation in microspore. The function of the two histone demethylases JMJ706 and JMJ707 that remove H3K9me2 may contribute to the decreased CHG methylation in sperm. During male gametogenesis CMT3a mainly silences TE and TE-related genes while CMT3b is required for repression of genes encoding factors involved in transcriptional and translational activities. In addition, CMT3b functions to repress zygotic gene expression in egg and participates in establishing the zygotic epigenome upon fertilization.ConclusionCollectively, the results indicate that DNA methylation is dynamically remodeled during male gametogenesis, distinguish the function of CMT3a and CMT3b in sex cells, and underpin the functional significance of DNA methylation remodeling during rice reproduction.

MiR-430 regulates zygotic mRNA during zebrafish embryogenesis

BackgroundEarly embryonic developmental programs are guided by the coordinated interplay between maternally inherited and zygotically manufactured RNAs and proteins. Although these processes happen concomitantly and affecting gene function during this period is bound to affect both pools of mRNAs, it has been challenging to study their expression dynamics separately.ResultsBy employing SLAM-seq, a nascent mRNA labeling transcriptomic approach, in a developmental time series we observe that over half of the early zebrafish embryo transcriptome consists of maternal-zygotic genes, emphasizing their pivotal role in early embryogenesis. We provide an hourly resolution of de novo transcriptional activation events and follow nascent mRNA trajectories, finding that most de novo transcriptional events are stable throughout this period. Additionally, by blocking microRNA-430 function, a key post transcriptional regulator during zebrafish embryogenesis, we directly show that it destabilizes hundreds of de novo transcribed mRNAs from pure zygotic as well as maternal-zygotic genes. This unveils a novel miR-430 function during embryogenesis, fine-tuning zygotic gene expression.ConclusionThese insights into zebrafish early embryo transcriptome dynamics emphasize the significance of post-transcriptional regulators in zygotic genome activation. The findings pave the way for future investigations into the coordinated interplay between transcriptional and post-transcriptional landscapes required for the establishment of animal cell identities and functions.

Transcriptomic Profiling at the Maternal-to-Zygotic Transition in Leech, Helobdella austinensis.

The glossiphoniid leech, Helobdella austinensis, is an experimentally tractable member of the superphylum, Lophotrochozoa. Its large embryonic cells, stereotyped asymmetric cell divisions and ex vivo development capabilities makes it a favorable model for studying the molecular and cellular events of a representative spiralian. In this study, we focused on a narrow developmental time window of ~6-8 h, comprising stages just prior to and immediately following zygote deposition. Employing RNA-Seq methodology, we identified differentially expressed transcripts at this fundamental ontogenic boundary, known as the maternal-to-zygotic transition (MZT). Gene expression changes were characterized by the massive degradation of maternal RNAs (~45%) coupled with the rapid transcription of ~5000 zygotic genes (~20% of the genome) in the first mitotic cell cycle. The latter transcripts encoded a mixture of cell maintenance and regulatory proteins that predictably influence downstream developmental events.