An FGF timer for zygotic genome activation.

Zygotic genome activation has been extensively studied in a variety of systems including flies, frogs, and mammals. However, there is comparatively little known about the precise timing of gene induction during the earliest phases of embryogenesis. Here we used high-resolution in situ detection methods, along with genetic and experimental manipulations, to study the timing of zygotic activation in the simple model chordate Ciona with minute-scale temporal precision. We found that two Prdm1 homologs in Ciona are the earliest genes that respond to FGF signaling. We present evidence for a FGF timing mechanism that is driven by ERK-mediated derepression of the ERF repressor. Depletion of ERF results in ectopic activation of FGF target genes throughout the embryo. A highlight of this timer is the sharp transition in FGF responsiveness between the eight- and 16-cell stages of development. We propose that this timer is an innovation of chordates that is also used by vertebrates.

Dynamics of the epigenetic landscape during the maternal-to-zygotic transition.

A remarkable epigenetic remodelling process occurs shortly after fertilization, which restores totipotency to the zygote. This involves global DNA demethylation, chromatin remodelling, genome spatial reorganization and substantial transcriptional changes. Key to these changes is the transition from the maternal environment of the oocyte to an embryonic-driven developmental expression programme, a process termed the maternal-to-zygotic transition (MZT). Zygotic genome activation occurs predominantly at the two-cell stage in mice and the eight-cell stage in humans, yet the dynamics of its control are still mostly obscure. In recent years, partly due to single-cell and low-cell number epigenomic studies, our understanding of the epigenetic and chromatin landscape of preimplantation development has improved considerably. In this Review, we discuss the latest advances in the study of the MZT, focusing on DNA methylation, histone post-translational modifications, local chromatin structure and higher-order genome organization. We also discuss key mechanistic studies that investigate the mode of action of chromatin regulators, transcription factors and non-coding RNAs during preimplantation development. Finally, we highlight areas requiring additional research, as well as new technological advances that could assist in eventually completing our understanding of the MZT.

Analysis of Genome Architecture during SCNT Reveals a Role of Cohesin in Impeding Minor ZGA.

Somatic cell nuclear transfer (SCNT) can reprogram a somatic nucleus to a totipotent state. However, the re-organization of 3D chromatin structure in this process remains poorly understood. Using low-input Hi-C, we revealed that, during SCNT, the transferred nucleus first enters a mitotic-like state (premature chromatin condensation). Unlike fertilized embryos, SCNT embryos show stronger topologically associating domains (TADs) at the 1-cell stage. TADs become weaker at the 2-cell stage, followed by gradual consolidation. Compartments A/B are markedly weak in 1-cell SCNT embryos and become increasingly strengthened afterward. By the 8-cell stage, somatic chromatin architecture is largely reset to embryonic patterns. Unexpectedly, we found cohesin represses minor zygotic genome activation (ZGA) genes (2-cell-specific genes) in pluripotent and differentiated cells, and pre-depleting cohesin in donor cells facilitates minor ZGA and SCNT. These data reveal multi-step reprogramming of 3D chromatin architecture during SCNT and support dual roles of cohesin in TAD formation and minor ZGA repression.

Widespread Enhancer Dememorization and Promoter Priming during Parental-to-Zygotic Transition.

The epigenome plays critical roles in controlling gene expression and development. However, how the parental epigenomes transit to the zygotic epigenome in early development remains elusive. Here we show that parental-to-zygotic transition in zebrafish involves extensive erasure of parental epigenetic memory, starting with methylating gametic enhancers. Surprisingly, this occurs even prior to fertilization for sperm. Both parental enhancers lose histone marks by the 4-cell stage, and zygotic enhancers are not activated until around zygotic genome activation (ZGA). By contrast, many promoters remain hypomethylated and, unexpectedly, acquire histone acetylation before ZGA at as early as the 4-cell stage. They then resolve into either activated or repressed promoters upon ZGA. Maternal depletion of histone acetyltransferases results in aberrant ZGA and early embryonic lethality. Finally, such reprogramming is largely driven by maternal factors, with zygotic products mainly contributing to embryonic enhancer activation. These data reveal widespread enhancer dememorization and promoter priming during parental-to-zygotic transition.

Enzymatic modules of the SAGA chromatin-modifying complex play distinct roles in Drosophila gene expression and development.

The Spt-Ada-Gcn5-acetyltransferase (SAGA) chromatin-modifying complex is a transcriptional coactivator that contains four different modules of subunits. The intact SAGA complex has been well characterized for its function in transcription regulation and development. However, little is known about the roles of individual modules within SAGA and whether they have any SAGA-independent functions. Here we demonstrate that the two enzymatic modules of Drosophila SAGA are differently required in oogenesis. Loss of the histone acetyltransferase (HAT) activity blocks oogenesis, while loss of the H2B deubiquitinase (DUB) activity does not. However, the DUB module regulates a subset of genes in early embryogenesis, and loss of the DUB subunits causes defects in embryogenesis. ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) analysis revealed that both the DUB and HAT modules bind most SAGA target genes even though many of these targets do not require the DUB module for expression. Furthermore, we found that the DUB module can bind to chromatin and regulate transcription independently of the HAT module. Our results suggest that the DUB module has functions within SAGA and independent functions.

Dynamics of hunchback translation in real-time and at single-mRNA resolution in the Drosophila embryo.

The Hunchback (Hb) transcription factor is crucial for anterior-posterior patterning of the Drosophila embryo. The maternal hb mRNA acts as a paradigm for translational regulation due to its repression in the posterior of the embryo. However, little is known about the translatability of zygotically transcribed hb mRNAs. Here, we adapt the SunTag system, developed for imaging translation at single-mRNA resolution in tissue culture cells, to the Drosophila embryo to study the translation dynamics of zygotic hb mRNAs. Using single-molecule imaging in fixed and live embryos, we provide evidence for translational repression of zygotic SunTag-hb mRNAs. Whereas the proportion of SunTag-hb mRNAs translated is initially uniform, translation declines from the anterior over time until it becomes restricted to a posterior band in the expression domain. We discuss how regulated hb mRNA translation may help establish the sharp Hb expression boundary, which is a model for precision and noise during developmental patterning. Overall, our data show how use of the SunTag method on fixed and live embryos is a powerful combination for elucidating spatiotemporal regulation of mRNA translation in Drosophila.

Cell cycle-dependent and independent mating blocks ensure fungal zygote survival and ploidy maintenance.

To ensure genome stability, sexually reproducing organisms require that mating brings together exactly 2 haploid gametes and that meiosis occurs only in diploid zygotes. In the fission yeast Schizosaccharomyces pombe, fertilization triggers the Mei3-Pat1-Mei2 signaling cascade, which represses subsequent mating and initiates meiosis. Here, we establish a degron system to specifically degrade proteins postfusion and demonstrate that mating blocks not only safeguard zygote ploidy but also prevent lysis caused by aberrant fusion attempts. Using long-term imaging and flow-cytometry approaches, we identify previously unrecognized and independent roles for Mei3 and Mei2 in zygotes. We show that Mei3 promotes premeiotic S-phase independently of Mei2 and that cell cycle progression is both necessary and sufficient to reduce zygotic mating behaviors. Mei2 not only imposes the meiotic program and promotes the meiotic cycle, but also blocks mating behaviors independently of Mei3 and cell cycle progression. Thus, we find that fungi preserve zygote ploidy and survival by at least 2 mechanisms where the zygotic fate imposed by Mei2 and the cell cycle reentry triggered by Mei3 synergize to prevent zygotic mating.

Dynamic methylation pattern of H19DMR and KvDMR1 in bovine oocytes and preimplantation embryos.

PURPOSE: This study aimed to evaluate the epigenetic reprogramming of ICR1 (KvDMR1) and ICR2 (H19DMR) and expression of genes controlled by them as well as those involved in methylation, demethylation, and pluripotency. METHODS: We collected germinal vesicle (GV) and metaphase II (MII) oocytes, and preimplantation embryos at five stages [zygote, 4-8 cells, 8-16 cells, morula, and expanded blastocysts (ExB)]. DNA methylation was assessed by BiSeq, and the gene expression was evaluated using qPCR. RESULTS: H19DMR showed an increased DNA methylation from GV to MII oocytes (68.04% and 98.05%, respectively), decreasing in zygotes (85.83%) until morula (61.65%), and ExB (63.63%). H19 and IGF2 showed increased expression in zygotes, which decreased in further stages. KvDMR1 was hypermethylated in both GV (71.82%) and MII (69.43%) and in zygotes (73.70%) up to morula (77.84%), with a loss of methylation at the ExB (36.64%). The zygote had higher expression of most genes, except for CDKN1C and PHLDA2, which were highly expressed in MII and GV oocytes, respectively. DNMTs showed increased expression in oocytes, followed by a reduction in the earliest stages of embryo development. TET1 was downregulated until 4-8-cell and upregulated in 8-16-cell embryos. TET2 and TET3 showed higher expression in oocytes, and a downregulation in MII oocytes and 4-8-cell embryo. CONCLUSION: We highlighted the heterogeneity in the DNA methylation of H19DMR and KvDMR1 and a dynamic expression pattern of genes controlled by them. The expression of DNMTs and TETs genes was also dynamic owing to epigenetic reprogramming.

Birth of Thirty-Two Healthy Babies Following Transfer of Fresh and Frozen-Thawed Embryos Derived from Monopronuclear Zygotes: A Retrospective Study.

Background and Objectives: Fertilized zygotes normally display two pronuclei (PN), but abnormal fertilization patterns (0, 1 or >2PN) are observed daily in IVF labs. Multiple PN zygotes (>2) are generally discarded due to an increased risk of aneuploidy. However, the decision to transfer or not transfer 1PN-derived embryos remains controversial. The aims of our study were to analyze the neonatal outcomes of fresh or frozen-thawed embryos derived from 1PN zygotes, and to evaluate the influence of the fertilization method. Materials and Methods: Data were retrospectively collected from cycles performed between January 2018 and December 2022. Fresh cycles were analyzed for the comparative fate of 1PN zygotes (n = 1234) following conventional in vitro fertilization (cIVF; n = 648) or intracytoplasmic sperm injection (ICSI; n = 586), as well as the results of the 64 transfers of 1PN-derived embryos (pregnancy rate (PR) and neonatal outcomes). This pregnancy follow-up was also applied to 167 transfers of frozen-thawed 1PN-derived embryos. Results: In fresh cycles, 46% of the 1PN zygotes in the cIVF group developed into embryos of sufficient quality to be transferred or frozen (day 3 or 5/6). This rate was lower in the fresh ICSI cycles (33%). Blastulation rate was also significantly higher in the cIVF group (44%) in comparison to the ICSI group (20%). The fresh single embryo transfers (32 per group) allowed seven pregnancies in the cIVF group (PR = 21.9%) as compared to four pregnancies in the ICSI group (PR = 12.5%). In the cIVF group, five deliveries of healthy newborns were achieved, but only one in the ICSI group. In frozen/thawed cycles, 36 pregnancies were obtained out of the 167 transfers. A non-significant difference was observed between embryos derived from cIVF cycles (PR = 26%) and ICSI cycles (PR = 16%) with 18 and 8 healthy babies born, respectively. Conclusions: We observed better outcomes for 1PN zygotes in cIVF cycles in comparison to ICSI cycles. Our center policy to transfer good-quality 1PN-derived embryos allowed the birth of 32 healthy babies.

The Lid/KDM5 histone demethylase complex activates a critical effector of the oocyte-to-zygote transition.

Following fertilization of a mature oocyte, the formation of a diploid zygote involves a series of coordinated cellular events that ends with the first embryonic mitosis. In animals, this complex developmental transition is almost entirely controlled by maternal gene products. How such a crucial transcriptional program is established during oogenesis remains poorly understood. Here, we have performed an shRNA-based genetic screen in Drosophila to identify genes required to form a diploid zygote. We found that the Lid/KDM5 histone demethylase and its partner, the Sin3A-HDAC1 deacetylase complex, are necessary for sperm nuclear decompaction and karyogamy. Surprisingly, transcriptomic analyses revealed that these histone modifiers are required for the massive transcriptional activation of deadhead (dhd), which encodes a maternal thioredoxin involved in sperm chromatin remodeling. Unexpectedly, while lid knock-down tends to slightly favor the accumulation of its target, H3K4me3, on the genome, this mark was lost at the dhd locus. We propose that Lid/KDM5 and Sin3A cooperate to establish a local chromatin environment facilitating the unusually high expression of dhd, a key effector of the oocyte-to-zygote transition.

Dual role of Fam208a during zygotic cleavage and early embryonic development.

Maintenance of genome stability is essential for every living cell as genetic information is repeatedly challenged during DNA replication in each cell division event. Errors, defects, delays, and mistakes that arise during mitosis or meiosis lead to an activation of DNA repair processes and in case of their failure, programmed cell death, i.e. apoptosis, could be initiated. Fam208a is a protein whose importance in heterochromatin maintenance has been described recently. In this work, we describe the crucial role of Fam208a in sustaining genome stability during cellular division. The targeted depletion of Fam208a in mice using CRISPR/Cas9 led to embryonic lethality before E12.5. We also used the siRNA approach to downregulate Fam208a in zygotes to avoid the influence of maternal RNA in the early stages of development. This early downregulation increased arresting of the embryonal development at the two-cell stage and the occurrence of multipolar spindles formation. To investigate this further, we used the yeast two-hybrid (Y2H) system and identified new putative interaction partners Gpsm2, Svil, and Itgb3bp. Their co-expression with Fam208a was assessed by RT-qPCR profiling and in situ hybridization [1] in multiple murine tissues. Based on these results we proposed that Fam208a functions within the HUSH complex by interaction with Mphosph8 as these proteins are not only able to physically interact but also co-localise. We are bringing new evidence that Fam208a is a multi-interacting protein affecting genome stability on the cell division level at the earliest stages of development and by interaction with methylation complex in adult tissues. In addition to its epigenetic functions, Fam208a appears to have an important role in the zygotic division, possibly via interaction with newly identified putative partners Gpsm2, Svil, and Itgb3bp.

Maternal and zygotic gene regulatory effects of endogenous RNAi pathways.

Endogenous small RNAs (sRNAs) and Argonaute proteins are ubiquitous regulators of gene expression in germline and somatic tissues. sRNA-Argonaute complexes are often expressed in gametes and are consequently inherited by the next generation upon fertilization. In Caenorhabditis elegans, 26G-RNAs are primary endogenous sRNAs that trigger the expression of downstream secondary sRNAs. Two subpopulations of 26G-RNAs exist, each of which displaying strongly compartmentalized expression: one is expressed in the spermatogenic gonad and associates with the Argonautes ALG-3/4; plus another expressed in oocytes and in embryos, which associates with the Argonaute ERGO-1. The determinants and dynamics of gene silencing elicited by 26G-RNAs are largely unknown. Here, we provide diverse new insights into these endogenous sRNA pathways of C. elegans. Using genetics and deep sequencing, we dissect a maternal effect of the ERGO-1 branch of the 26G-RNA pathway. We find that maternal primary sRNAs can trigger the production of zygotic secondary sRNAs that are able to silence targets, even in the absence of zygotic primary triggers. Thus, the interaction of maternal and zygotic sRNA populations, assures target gene silencing throughout animal development. Furthermore, we explore other facets of 26G-RNA biology related to the ALG-3/4 branch. We find that sRNA abundance, sRNA pattern of origin and the 3' UTR length of target transcripts are predictors of the regulatory outcome by the Argonautes ALG-3/4. Lastly, we provide evidence suggesting that ALG-3 and ALG-4 regulate their own mRNAs in a negative feedback loop. Altogether, we provide several new regulatory insights on the dynamics, target regulation and self-regulation of the endogenous RNAi pathways of C. elegans.

Constitutive signaling activity of a receptor-associated protein links fertilization with embryonic patterning in Arabidopsis thaliana.

In flowering plants, the asymmetrical division of the zygote is the first hallmark of apical-basal polarity of the embryo and is controlled by a MAP kinase pathway that includes the MAPKKK YODA (YDA). In Arabidopsis, YDA is activated by the membrane-associated pseudokinase SHORT SUSPENSOR (SSP) through an unusual parent-of-origin effect: SSP transcripts accumulate specifically in sperm cells but are translationally silent. Only after fertilization is SSP protein transiently produced in the zygote, presumably from paternally inherited transcripts. SSP is a recently diverged, Brassicaceae-specific member of the BRASSINOSTEROID SIGNALING KINASE (BSK) family. BSK proteins typically play broadly overlapping roles as receptor-associated signaling partners in various receptor kinase pathways involved in growth and innate immunity. This raises two questions: How did a protein with generic function involved in signal relay acquire the property of a signal-like patterning cue, and how is the early patterning process activated in plants outside the Brassicaceae family, where SSP orthologs are absent? Here, we show that Arabidopsis BSK1 and BSK2, two close paralogs of SSP that are conserved in flowering plants, are involved in several YDA-dependent signaling events, including embryogenesis. However, the contribution of SSP to YDA activation in the early embryo does not overlap with the contributions of BSK1 and BSK2. The loss of an intramolecular regulatory interaction enables SSP to constitutively activate the YDA signaling pathway, and thus initiates apical-basal patterning as soon as SSP protein is translated after fertilization and without the necessity of invoking canonical receptor activation.

Five multicopy gene family genes expressed during the maternal-to-zygotic transition are not essential for mouse development.

Upon fertilization, oocytes transform into totipotent and pluripotent cleavage stage cells through the maternal-to-zygotic transition (MZT), which is regulated by maternal factors and zygotic genome activation (ZGA). Here, we investigated the in vivo function of 16 genes expressed with strong biases in oocytes and cleavage stage embryos by generating knockout (KO) mice. These MZT-associated genes are conserved across many mammalian species and include five multicopy gene family genes: the Nlrp9, Khdc1, Rfpl4, Trim43, and Zscan5 genes. Intercrosses between female KO and male KO mice, including Nlrp9a/b/c triple KO (TKO), Khdc1a/b/c TKO, Rfpl4a/b double KO (DKO), Trim43a/b/c TKO, and Zscan5b KO mice led to the birth to healthy offspring that in turn produced healthy offspring. Our study not only demonstrated that these MZT-associated genes are not essential for mouse development, but also provides valuable resources for analyzing the functions of these genes in other genetic backgrounds, in the presence of stressors, and under pathogenic conditions.

First mitotic spindle formation is led by sperm centrosome-dependent MTOCs in humans.

Unlike in mice, multinucleated blastomeres appear at a high frequency in the two-cell-stage embryos in humans. In this Point of View article, we demonstrate that the first mitotic spindle formation led by sperm centrosome-dependent microtubule organizing centers may cause a high incidence of zygotic division errors using human tripronuclear zygotes.

HIRA contributes to zygote formation in mice and is implicated in human 1PN zygote phenotype.

Elucidating the mechanisms underpinning fertilisation is essential to optimising IVF procedures. One of the critical steps involves paternal chromatin reprogramming, in which compacted sperm chromatin packed by protamines is removed by oocyte factors and new histones, including histone H3.3, are incorporated. HIRA is the main H3.3 chaperone governing this protamine-to-histone exchange. Failure of this step results in abnormally fertilised zygotes containing only one pronucleus (1PN), in contrast to normal two-pronuclei (2PN) zygotes. 1PN zygotes are frequently observed in IVF treatments, but the genotype-phenotype correlation remains elusive. We investigated the maternal functions of two other molecules of the HIRA complex, Cabin1 and Ubn1, in mouse. Loss-of-function Cabin1 and Ubn1 mouse models were developed: their zygotes displayed an abnormal 1PN zygote phenotype. We then studied human 1PN zygotes and found that the HIRA complex was absent in 1PN zygotes that lacked the male pronucleus. This shows that the role of the HIRA complex in male pronucleus formation potentially has coherence from mice to humans. Furthermore, rescue experiments in mouse showed that the abnormal 1PN phenotype derived from Hira mutants could be resolved by overexpression of HIRA. We have demonstrated that HIRA complex regulates male pronucleus formation in mice and is implicated in humans, that both CABIN1 and UBN1 components of the HIRA complex are equally essential for male pronucleus formation, and that rescue is feasible.

The molecular foundations of zygosis.

Zygosis is the generation of new biological individuals by the sexual fusion of gamete cells. Our current understanding of eukaryotic phylogeny indicates that sex is ancestral to all extant eukaryotes. Although sexual development is extremely diverse, common molecular elements have been retained. HAP2-GCS1, a protein that promotes the fusion of gamete cell membranes that is related in structure to certain viral fusogens, is conserved in many eukaryotic lineages, even though gametes vary considerably in form and behaviour between species. Similarly, although zygotes have dramatically different forms and fates in different organisms, diverse eukaryotes share a common developmental programme in which homeodomain-containing transcription factors play a central role. These common mechanistic elements suggest possible common evolutionary histories that, if correct, would have profound implications for our understanding of eukaryogenesis.

Differentially-dimensioned furrow formation by zygotic gene expression and the MBT.

Despite extensive work on the mechanisms that generate plasma membrane furrows, understanding how cells are able to dynamically regulate furrow dimensions is an unresolved question. Here, we present an in-depth characterization of furrow behaviors and their regulation in vivo during early Drosophila morphogenesis. We show that the deepening in furrow dimensions with successive nuclear cycles is largely due to the introduction of a new, rapid ingression phase (Ingression II). Blocking the midblastula transition (MBT) by suppressing zygotic transcription through pharmacological or genetic means causes the absence of Ingression II, and consequently reduces furrow dimensions. The analysis of compound chromosomes that produce chromosomal aneuploidies suggests that multiple loci on the X, II, and III chromosomes contribute to the production of differentially-dimensioned furrows, and we track the X-chromosomal contribution to furrow lengthening to the nullo gene product. We further show that checkpoint proteins are required for furrow lengthening; however, mitotic phases of the cell cycle are not strictly deterministic for furrow dimensions, as a decoupling of mitotic phases with periods of active ingression occurs as syncytial furrow cycles progress. Finally, we examined the turnover of maternal gene products and find that this is a minor contributor to the developmental regulation of furrow morphologies. Our results suggest that cellularization dynamics during cycle 14 are a continuation of dynamics established during the syncytial cycles and provide a more nuanced view of developmental- and MBT-driven morphogenesis.

Inhibition of LINE-1 retrotransposition represses telomere reprogramming during mouse 2-cell embryo development.

PURPOSE: To investigate whether inhibition of LINE-1 affects telomere reprogramming during 2-cell embryo development. METHODS: Mouse zygotes were cultured with or without 1 µM azidothymidine (AZT) for up to 15 h (early 2-cell, G1/S) or 24 h (late 2-cell, S/G2). Gene expression and DNA copy number were determined by RT-qPCR and qPCR respectively. Immunostaining and telomeric PNA-FISH were performed for co-localization between telomeres and ZSCAN4 or LINE-1-Orf1p. RESULTS: LINE-1 copy number was remarkably reduced in later 2-cell embryos by exposure to 1 µM AZT, and telomere lengths in late 2-cell embryos with AZT were significantly shorter compared to control embryos (P = 0.0002). Additionally, in the absence of LINE-1 inhibition, Dux, Zscan4, and LINE-1 were highly transcribed in early 2-cell embryos, as compared to late 2-cell embryos (P < 0.0001), suggesting that these 2-cell genes are activated at the early 2-cell stage. However, in early 2-cell embryos with AZT treatment, mRNA levels of Dux, Zscan4, and LINE-1 were significantly decreased. Furthermore, both Zscan4 and LINE-1 encoded proteins localized to telomere regions in 2-cell embryos, but this co-localization was dramatically reduced after AZT treatment (P < 0.001). CONCLUSIONS: Upon inhibition of LINE-1 retrotransposition in mouse 2-cell embryos, Dux, Zscan4, and LINE-1 were significantly downregulated, and telomere elongation was blocked. ZSCAN4 foci and their co-localization with telomeres were also significantly decreased, indicating that ZSCAN4 is an essential component of the telomere reprogramming that occurs in mice at the 2-cell stage. Our findings also suggest that LINE-1 may directly contribute to telomere reprogramming in addition to regulating gene expression.