Maternal Zygotic Transition Research Articles

Maternal DCAF13 Regulates Chromatin Tightness to Contribute to Embryonic Development

Maternal-zygotic transition (MZT) is critical for the developmental control handed from maternal products to newly synthesized zygotic genome in the earliest stage of embryogenesis. However, the spatiotemporal dynamic regulation of MZT by maternal factors is largely unknown. Here, we reported a novel maternal factor, DCAF13, which was highly expressed in growing oocyte nucleolus and had key maternal effects on oocyte and zygotic chromatin tightness during maternal to zygotic transition. DCAF13 specifically deleted in oocytes resulted in loose chromatin structure in fully grown germinal vesicle oocytes. Despite normal nuclear maturation in maternal DCAF13-deleted oocytes, the chromosomes at MII stage were not properly condensed. Consequently, the nuclear and nucleolar structure reorganized abnormally, and transcription was inactive in zygotic embryos. RNA-seq analysis of MII oocytes and 2-cell embryos demonstrated that the transcriptomes between knockout and control oocyte were similar, but the maternal DCAF13 deleted two-cell embryos showed a significant decrease in transcription. In addition, the maternal DCAF13-deleted embryos displayed arrest at the two-cell stage, which could not be rescued by injecting flag-Dcaf13 mRNA in the zygote. This revealed that DCAF13 was a unique maternal effect factor regulating the nucleolus.

DNA Methylation Patterns in the Early Human Embryo and the Epigenetic/Imprinting Problems: A Plea for a More Careful Approach to Human Assisted Reproductive Technology (ART).

An increasing number of publications indicate that babies born after IVF (in vitro fertilization) procedures have higher rates of anomalies related to imprinting/epigenetic changes, which may be attributed to suboptimal culture conditions. Appropriate maintenance of DNA methylation during the first few days of an in vitro culture requires a supply of methyl donors, which are lacking in current in vitro culture systems. The absence of protection against oxidative stress in the culture increases the risks for errors in methylation. A decrease in the methylation processes is sometimes observed immediately post fertilization, due to delays that occur during the maternal–zygotic transition period. Care should be exercised in ART (assisted reproductive technology) procedures in order to avoid the risk of generating errors in methylation during the in vitro culture period immediately post fertilization, which has an impact on imprinting/epigenetics. Formulation of IVF culture media needs to be re-assessed in the perspective of current knowledge regarding embryo physiology.

Alternative polyadenylation coordinates embryonic development, sexual dimorphism and longitudinal growth in Xenopus tropicalis.

RNA alternative polyadenylation contributes to the complexity of information transfer from genome to phenome, thus amplifying gene function. Here, we report the first X. tropicalis resource with 127,914 alternative polyadenylation (APA) sites derived from embryos and adults. Overall, APA networks play central roles in coordinating the maternal-zygotic transition (MZT) in embryos, sexual dimorphism in adults and longitudinal growth from embryos to adults. APA sites coordinate reprogramming in embryos before the MZT, but developmental events after the MZT due to zygotic genome activation. The APA transcriptomes of young adults are more variable than growing adults and male frog APA transcriptomes are more divergent than females. The APA profiles of young females were similar to embryos before the MZT. Enriched pathways in developing embryos were distinct across the MZT and noticeably segregated from adults. Briefly, our results suggest that the minimal functional units in genomes are alternative transcripts as opposed to genes.

On the Regulatory Evolution of New Genes Throughout Their Life History.

Every gene has a birthplace and an age, that is, a cis-regulatory environment and an evolution lifespan since its origination, yet how the two shape the evolution trajectories of genes remains unclear. Here, we address this basic question by comparing phylogenetically dated new genes in the context of both their ages and origination mechanisms. In both Drosophila and vertebrates, we confirm a clear "out of the testis" transition from the specifically expressed young genes to the broadly expressed old housekeeping genes, observed only in testis but not in other tissues. Many new genes have gained important functions during embryogenesis, manifested as either specific activation at maternal-zygotic transition, or different spatiotemporal expressions from their parental genes. These expression patterns are largely driven by an age-dependent evolution of cis-regulatory environment. We discover that retrogenes are more frequently born in a pre-existing repressive regulatory domain, and are more diverged in their enhancer repertoire than the DNA-based gene duplications. During evolution, new gene duplications gradually gain active histone modifications and undergo more enhancer turnovers when becoming older, but exhibit complex trends of gaining or losing repressive histone modifications in Drosophila or vertebrates, respectively. Interestingly, vertebrate new genes exhibit an "into the testis" epigenetic transition that older genes become more likely to be co-occupied by both active and repressive ("bivalent") histone modifications specifically in testis. Our results uncover the regulatory mechanisms underpinning the stepwise acquisition of novel and complex functions by new genes, and illuminate the general evolution trajectory of genes throughout their life history.

The dynamics of energy metabolism in the tick embryo.

The cattle tick Rhipicephalus (Boophilus) microplus is an ectoparasite capable of transmitting a large number of pathogens, causing considerable losses in the cattle industry, with substantial damage to livestock. Over the years, important stages of its life cycle, such as the embryo, have been largely ignored by researchers. Tick embryogenesis has been typically described as an energy-consuming process, sustaining cell proliferation, differentiation, and growth. During the embryonic stage of arthropods, there is mobilization of metabolites of maternal origin for the development of organs and tissues of the embryo. Glycogen resynthesis in late embryogenesis is considered as an effective indicator of embryonic integrity. In the cattle tick R.(B. (B.) microplus, glycogen resynthesis is sustained by protein degradation through the gluconeogenesis pathway at the end of the embryonic period. Despite recent advancements in research on tick energy metabolism at the molecular level, the dynamics of nutrient utilization during R. (B.) microplus embryogenesis is still poorly understood. The present review aims to describe the regulatory mechanisms of carbohydrate metabolism during maternal-zygotic transition and identify possible new targets for the development of novel drugs and other control measures against R. (B.) microplus infestations.

Epigenome in Early Mammalian Development: Inheritance, Reprogramming and Establishment.

Drastic epigenetic reprogramming takes place during preimplantation development, leading to the conversion of terminally differentiated gametes to a totipotent embryo. Deficiencies in remodeling of the epigenomes can cause severe developmental defects, including embryonic lethality. However, how chromatin modifications and chromatin organization are reprogrammed upon fertilization in mammals has long remained elusive. Here, we review recent progress in understanding how the epigenome is dynamically regulated during early mammalian development. The latest studies, including many from genome-wide perspectives, have revealed unusual principles of reprogramming for histone modifications, chromatin accessibility, and 3D chromatin architecture. These advances have shed light on the regulatory network controlling the earliest development and maternal-zygotic transition.

The maternal-zygotic transition and zygotic activation of the Mnemiopsis leidyi genome occurs within the first three cleavage cycles.

The maternal-zygotic transition (MZT) describes the developmental reprogramming of gene expression marked by the degradation of maternally supplied gene products and activation of the zygotic genome. While the timing and duration of the MZT vary among taxa, little is known about early-stage transcriptional dynamics in the non-bilaterian phylum Ctenophora. We sought to better understand the extent of maternal mRNA loading and subsequent differential transcript abundance during the earliest stages of development by performing comprehensive RNA-sequencing-based analyses of mRNA abundance in single- and eight-cell stage embryos in the lobate ctenophore Mnemiopsis leidyi. We found 1,908 contigs with significant differential abundance between single- and eight-cell stages, of which 1,208 contigs were more abundant at the single-cell stage and 700 contigs were more abundant at the eight-cell stage. Of the differentially abundant contigs, 267 were exclusively present in the eight-cell samples, providing strong evidence that both the MZT and zygotic genome activation (ZGA) have commenced by the eight-cell stage. Many highly abundant transcripts encode genes involved in molecular mechanisms critical to the MZT, such as maternal transcript degradation, serine/threonine kinase activity, and chromatin remodeling. Our results suggest that chromosomal restructuring, which is critical to ZGA and the initiation of transcriptional regulation necessary for normal development, begins by the third cleavage within 1.5 hr post-fertilization in M. leidyi.

CFP1 Regulates Histone H3K4 Trimethylation and Developmental Potential in Mouse Oocytes

Trimethylation of histone H3 at lysine-4 (H3K4me3) isassociated with eukaryotic gene promoters andpoises their transcriptional activation during development. To examine the invivo function of H3K4me3 in the absence of DNA replication, we deleted CXXC finger protein 1 (CFP1), the DNA-binding subunit of the SETD1 histone H3K4 methyltransferase, in developing oocytes. We find that CFP1 isrequired for H3K4me3 accumulation and the deposition of histone variants onto chromatin during oocyte maturation. Decreased H3K4me3 in oocytes caused global downregulation of transcription activity. Oocytes lacking CFP1 failed to complete maturation and were unable to gain developmental competence after fertilization, due to defects in cytoplasmic lattice formation, meiotic division, and maternal-zygotic transition. Our study highlights the importance of H3K4me3 in continuous histone replacement for transcriptional regulation, chromatin remodeling, and normal developmental progression in a non-replicative system.

Maternal SENP7 programs meiosis architecture and embryo survival in mouse

Understanding the mechanisms underlying abnormal egg production and pregnancy loss is significant for human fertility. SENP7, a SUMO poly-chain editing enzyme, has been regarded as a mitotic regulator of heterochromatin integrity and DNA repair. Herein, we report the roles of SENP7 in mammalian reproductive scenario. Mouse oocytes deficient in SENP7 experienced meiotic arrest at prophase I and metaphase I stages, causing a substantial decrease of mature eggs. Hyperaceylation and hypomethylation of histone H3 and up-regulation of Cdc14B/C accompanied by down-regulation of CyclinB1 and CyclinB2 were further recognized as contributors to defective M-phase entry and spindle assembly in oocytes. The spindle assembly checkpoint activated by defective spindle morphogenesis, which was also caused by mislocalization and ubiquitylation-mediated proteasomal degradation of γ-tubulin, blocked oocytes at meiosis I stage. SENP7-depleted embryos exhibited severely defective maternal-zygotic transition and progressive degeneration, resulting in nearly no blastocyst production. The disrupted epigenetic landscape on histone H3 restricted Rad51C loading onto DNA lesions due to elevated HP1α euchromatic deposition, and reduced DNA 5hmC challenged the permissive status for zygotic DNA repair, which induce embryo death. Our study pinpoints SENP7 as a novel determinant in epigenetic programming and major pathways that govern oocyte and embryo development programs in mammals.

Dual Functional Roles of Molecular Beacon as a MicroRNA Detector and Inhibitor

MicroRNAs are essential in many cellular processes. The ability to detect microRNAs is important for understanding its function and biogenesis. This study is aimed at using a molecular beacon to detect miR-430 in developing zebrafish embryos as a proof of principle. miR-430 is crucial for the clearance of maternal mRNA during maternal zygotic transition in embryonic development. Despite its known function, the temporal and spatial expression of miR-430 remains unclear. We used various imaging techniques, including laser scanning confocal microscopy, spinning disk, and lightsheet microscopy, to study the localization of miR-430 and any developmental defects possibly caused by the molecular beacon. Our results show that miR-430 is expressed early in development and is localized in distinct cytoplasmic granules where its target mRNA can be detected. We also show that the designed molecular beacon can inhibit the function of miR-430 and cause developmental defect in the brain, notochord, heart, and kidney, depending on the delivery site within the embryo, suggesting that miR-430 plays a diverse role in embryonic morphogenesis. When compared with morpholino, molecular beacon is 2 orders of magnitude more potent in inhibiting miR-430. Thus, our results reveal that in addition to being used as a valuable tool for the detection of microRNAs in vivo, molecular beacons can also be employed to inhibit microRNAs in a specific manner.

Epigenetic modifications and reprogramming in paternal pronucleus: sperm, preimplantation embryo, and beyond.

Pronuclear/zygotic stage is the very first stage of life. In this period, paternal pronucleus undergoes massive chromatin remodeling called "paternal reprogramming" including protamine-histone replacement and subsequent acquisition of epigenetic modifications. Although these consecutive events are required for the initiation of maternal-zygotic transition, the precise role of paternal reprogramming and its effect on subsequent embryonic development has been largely unknown to date. Recently, various new techniques, especially next-generation sequencing (NGS) and RNAi microinjection contribute to unveil the epigenetic transition from both paternal and maternal to early preimplantation embryos, suggesting not only the simple transcriptional regulation by transcription factors but also dynamic structural alteration of chromatin to initiate the wave of zygotic gene transcription. This review summarizes such recent progress for understanding the epigenetic transition in sperm and preimplantation embryos, and further argue about its transgenerational effect.

Maternal DCAF2 is crucial for maintenance of genome stability during the first cell cycle in mice.

Precise regulation of DNA replication and genome integrity is crucial for gametogenesis and early embryogenesis. Cullin ring-finger ubiquitin ligase 4 (CRL4) has multiple functions in the maintenance of germ cell survival, oocyte meiotic maturation, and maternal-zygotic transition in mammals. DDB1-cullin-4-associated factor-2 (DCAF2, also known as DTL or CDT2) is an evolutionarily conserved substrate receptor of CRL4. To determine whether DCAF2 is a key CRL4 substrate adaptor in mammalian oocytes, we generated a novel mouse strain that carries a Dcaf2 allele flanked by loxP sequences, and specifically deleted Dcaf2 in oocytes. Dcaf2 knockout in mouse oocytes leads to female infertility. Although Dcaf2-null oocytes were able to develop and mature normally, the embryos derived from them were arrested at one- to two-cell stage, owing to prolonged DNA replication and accumulation of massive DNA damage. These results indicate that DCAF2 is a previously unrecognized maternal factor that safeguards zygotic genome stability. Maternal DCAF2 protein is crucial for prevention of DNA re-replication in the first and unique mitotic cell cycle of the zygote.This article has an associated First Person interview with the first author of the paper.

85 REGULATION OF STEAROYL-CoENZYME A DESATURASE BY FATTY ACIDS IS ESSENTIAL TO PORCINE EARLY EMBRYO DEVELOPMENT

Various fatty acids are found in large amounts in follicular fluid and the uterus. These fatty acids are known to regulate lipid metabolism in a mammal’s embryo. Lipid metabolism provides a source of energy, and its importance during embryogenesis is being increasingly recognised. Especially, the pig has larger amounts of intercellular lipid bilayers in the embryo than do other species, which indicates the porcine embryo is more dependent on fatty acid on their metabolic pathway. This study investigated the transcriptome analysis data of in vivo embryos and the effect of oleic acid (C18:1n-9) and stearic acid (C18:0) on in vitro-produced porcine embryos. In transcriptome analysis of in vivo embryos, we found several genes were increasing before and after maternal zygotic transition stage, and interactions were mapped and given a significance score. Among these genes, stearoyl-coenzyme A desaturase (SCD) gene was significantly increased during 8-cell to blastocyst stage. Stearoyl-coenzyme A desaturase is responsible for converting saturated fatty acid (stearic acid) to monounsaturated fatty acid (oleic acid). Furthermore, we treated with oleic acid (50, 100, 250, and 500 μM) and stearic acid (50, 75, and 100 μM) on 2 day and 4 day after parthenogenetic embryos. Both oleic acid and stearic acid concentration over 100 μM had a negative effect on blastocyst formation rate and cell numbers because of exogenous fatty acid toxicity. In addition, there was no significant difference among all stearic acid treated groups and total cell number of oleic acid treated blastocyst. However, the 2-day oleic acid treated group (45.92 ± 4.01, n = 8, 2 day, 100 μM) had a significant increase of blastocyst formation rate (P < 0.05) compared with the control group (34.88 ± 2.93, n = 8). These data could support that porcine embryos can use exogenous oleic acid as a metabolic energy source. The data also demonstrate the important role of SCD in porcine early embryo development. To confirm the transcriptome analysis, we are investigating mRNA level of SCD on in vitro-produced embryos at 1-cell to morula and blastocyst stage on the control group, oleic acid treated group, fetal bovine serum treated group, and nontreated group. Furthermore, we are investigating SCD inhibition assay by A939572 (SCD inhibitor) on parthenogenetic embryos in a fetal bovine serum treated group and nontreated group for identifying blastocyst formation rate and total cell numbers. This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2014R1A1A2055199) and Next BioGreen 21 program (PJ0113002016), Rural Development Administration, Republic of Korea.

BTG4 is a meiotic cell cycle-coupled maternal-zygotic-transition licensing factor in oocytes.

The mRNAs stored in oocytes undergo general decay during the maternal-zygotic transition (MZT), and their stability is tightly interconnected with meiotic cell-cycle progression. However, the factors that trigger decay of maternal mRNA and couple this event to oocyte meiotic maturation remain elusive. Here, we identified B-cell translocation gene-4 (BTG4) as an MZT licensing factor in mice. BTG4 bridged CNOT7, a catalytic subunit of the CCR4-NOT deadenylase, to eIF4E, a key translation initiation factor, and facilitated decay of maternal mRNA. Btg4-null females produced morphologically normal oocytes but were infertile, owing to early developmental arrest. The intrinsic MAP kinase cascade in oocytes triggered translation of Btg4 mRNA stored in fully grown oocytes by targeting the 3' untranslated region, thereby coupling CCR4-NOT deadenylase-mediated decay of maternal mRNA with oocyte maturation and fertilization. This is a key step in oocyte cytoplasmic maturation that determines the developmental potential of mammalian embryos.

A MAPK cascade couples maternal mRNA translation and degradation to meiotic cell cycle progression in mouse oocytes.

Mammalian oocyte maturation depends on the translational activation of stored maternal mRNAs upon meiotic resumption. Cytoplasmic polyadenylation element binding protein 1 (CPEB1) is a key oocyte factor that regulates maternal mRNA translation. However, the signal that triggers CPEB1 activation at the onset of mammalian oocyte maturation is not known. We provide evidence that a mitogen-activated protein kinase (MAPK) cascade couples maternal mRNA translation to meiotic cell cycle progression in mouse oocytes by triggering CPEB1 phosphorylation and degradation. Mutations of the phosphorylation sites or ubiquitin E3 ligase binding sites in CPEB1 have a dominant-negative effect in oocytes, and mimic the phenotype of ERK1/2 knockout, by impairing spindle assembly and mRNA translation. Overexpression of the CPEB1 downstream translation activator DAZL in ERK1/2-deficient oocytes partially rescued the meiotic defects, indicating that ERK1/2 is essential for spindle assembly, metaphase II arrest and maternal-zygotic transition (MZT) primarily by triggering the translation of key maternal mRNAs. Taken together, ERK1/2-mediated CPEB1 phosphorylation/degradation is a major mechanism of maternal mRNA translational activation, and is crucial for mouse oocyte maturation and MZT.

What cell death does in development.

Cell death is prominent in gametogenesis and shapes and sculpts embryos. In non-mammalian embryos one sees little or no cell death prior to the maternal-zygotic transition, but, in mammalian embryos, characteristic deaths of one or two cells occur at the end of compaction and are apparently necessary for the separation of the trophoblast from the inner cell mass. Considerable sculpting of the embryo occurs by cell deaths during organogenesis, and appropriate cell numbers, especially in the CNS and in the immune system, are generated by massive overproduction of cells and selection of a few, with death of the rest. The timing, identity, and genetic control of specific cells that die have been well documented in Caenorhabditis, but in other embryos the stochastic nature of the deaths limit our ability to do more than identify the regions in which cells will die. Complete disruption of the cell death machinery can be lethal, but many mutations of the regulatory machinery yield only modest or no phenotypes, indicating substantial redundancy and compensation of regulatory mechanisms. Most of the deaths are apoptotic and are identified by techniques used to recognize apoptosis, but techniques identifying lysosomes (whether in dying or involuting cells or in the phagocytes that invade the tissue) also reveal patterns of cell death. Aberrant cell deaths that produce known phenotypes are typically localized, indicating that the mechanism of activating a programmed death in a specific region, rather than the mechanism of death, is aberrant. These results lead us to conclude that we need to know much more about the conversations among cells that lead cells to commit suicide.

Genome methylation in D. melanogaster is found at specific short motifs and is independent of DNMT2 activity

Cytosine methylation in the genome of Drosophila melanogaster has been elusive and controversial: Its location and function have not been established. We have used a novel and highly sensitive genomewide cytosine methylation assay to detect and map genome methylation in stage 5 Drosophila embryos. The methylation we observe with this method is highly localized and strand asymmetrical, limited to regions covering ∼1% of the genome, dynamic in early embryogenesis, and concentrated in specific 5-base sequence motifs that are CA- and CT-rich but depleted of guanine. Gene body methylation is associated with lower expression, and many genes containing methylated regions have developmental or transcriptional functions. The only known DNA methyltransferase in Drosophila is the DNMT2 homolog MT2, but lines deficient for MT2 retain genomic methylation, implying the presence of a novel methyltransferase. The association of methylation with a lower expression of specific developmental genes at stage 5 raises the possibility that it participates in controlling gene expression during the maternal-zygotic transition.

Development: The Maternal–Zygotic Transition Revisited

The handover from maternal to zygotic control has to be carefully orchestrated. In most animal embryos, maternal products drive early embryogenesis, and the genome of the zygote is only switched on later. However, in the nematode Ascaris the zygotic genome is never silent, and the maternal products are rapidly eliminated.

Could time-lapse embryo imaging reduce the need for biopsy and PGS?

To review relevant studies examining the relationship between embryo morpho-kinetics and aneuploidy. Search of Pubmed and Medline using relevant keywords pertaining to morphology, morphokinetics and embryonic aneuploidy, as well as examination of various reference lists and conference proceedings. An abundance of publications, both preliminary and peer-reviewed, have emerged regarding the usefulness of time-lapse imaging in tracking embryo development and improving embryo selection. Recently, these publications have explored ability to not only predict blastocyst formation and implantation, but also the ability to detect embryonic chromosomal aneuploidy. Of the two peer-reviewed retrospective studies on morpho-kinetics and embryonic aneuploidy, one demonstrates that early cleavage timings can indicate chromosomal complement, while the other demonstrates that key events following the maternal-zygotic transition can be markers of aneuploidy. A recent paper also demonstrates improved outcomes following IVF using a selection algorithm to identify embryos at "low risk" of chromosomal abnormalities. However, the predictive nature of these events and timings is far from ideal. Additionally, results may be dependent upon the day of biopsy and method utilized for chromosomal assessment. With continued effort, the combination of multiple morphologic endpoint assessments and developmental timings and refinement of modeling systems may improve the predictive ability to determine embryonic aneuploidy. This may help select a subset of embryos that are less likely to carry chromosomal abnormalities and improve assisted reproductive outcomes. However, embryo biopsy, followed by preimplantation genetic screening/comprehensive chromosomal screening still remains the most reliable method to assess chromosomal complement of preimplantation embryos.

Depleting Gene Activities in Early Drosophila Embryos with the “Maternal-Gal4–shRNA” System

In a developing Drosophila melanogaster embryo, mRNAs have a maternal origin, a zygotic origin, or both. During the maternal-zygotic transition, maternal products are degraded and gene expression comes under the control of the zygotic genome. To interrogate the function of mRNAs that are both maternally and zygotically expressed, it is common to examine the embryonic phenotypes derived from female germline mosaics. Recently, the development of RNAi vectors based on short hairpin RNAs (shRNAs) effective during oogenesis has provided an alternative to producing germline clones. Here, we evaluate the efficacies of: (1) maternally loaded shRNAs to knockdown zygotic transcripts and (2) maternally loaded Gal4 protein to drive zygotic shRNA expression. We show that, while Gal4-driven shRNAs in the female germline very effectively generate phenotypes for genes expressed maternally, maternally loaded shRNAs are not very effective at generating phenotypes for early zygotic genes. However, maternally loaded Gal4 protein is very efficient at generating phenotypes for zygotic genes expressed during mid-embryogenesis. We apply this powerful and simple method to unravel the embryonic functions of a number of pleiotropic genes.