Zygotic Gene Products Research Articles

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.

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.

Early zygotic gene product Dunk interacts with anillin to regulate Myosin II during Drosophila cleavage.

Drosophila melanogaster cellularization is a special form of cleavage that converts syncytial embryos into cellular blastoderms by partitioning the peripherally localized nuclei into individual cells. An early event in cellularization is the recruitment of nonmuscle myosin II ("myosin") to the leading edge of cleavage furrows, where myosin forms an interconnected basal array before reorganizing into individual cytokinetic rings. The initial recruitment and organization of basal myosin are regulated by a cellularization-specific gene, dunk, but the underlying mechanism is unclear. Through a genome-wide yeast two-hybrid screen, we identified anillin (Scraps in Drosophila), a conserved scaffolding protein in cytokinesis, as the primary binding partner of Dunk. Dunk colocalizes with anillin and regulates its cortical localization during the formation of cleavage furrows, while the localization of Dunk is independent of anillin. Furthermore, Dunk genetically interacts with anillin to regulate the basal myosin array during cellularization. Similar to Dunk, anillin colocalizes with myosin since the very early stage of cellularization and is required for myosin retention at the basal array, before the well-documented function of anillin in regulating cytokinetic ring assembly. Based on these results, we propose that Dunk regulates myosin recruitment and spatial organization during early cellularization by interacting with and regulating anillin.

Reorganization, specialization, and degradation of oocyte maternal components for early development.

Oocyte components are maternally provided, solely determine oocyte quality, and coordinately determine embryo quality with zygotic gene expression. During oocyte maturation, maternal organelles are drastically reorganized and specialized to support oocyte characteristics. A large number of maternal components are actively degraded after fertilization and gradually replaced by zygotic gene products. The molecular basis and the significance of these processes on oocyte/embryo quality are not fully understood. Firstly, recent findings in organelle characteristics of other cells or oocytes from model organisms are introduced for further understanding of oocyte organelle reorganization/specialization. Secondly, recent progress in studies on maternal components degradation and their molecular mechanisms are introduced. Finally, future applications of these advancements for predicting mammalian oocyte/embryo quality are discussed. The significance of cellular surface protein degradation via endocytosis for embryonic development, and involvement of biogenesis of lipid droplets in embryonic quality, were recently reported using mammalian model organisms. Identifying key oocyte component characteristics and understanding their dynamics may lead to new applications in oocyte/embryo quality prediction and improvement. To implement these multidimensional concepts, development of new technical approaches that allow us to address the complexity and efficient studies using model organisms are required.

STAT3 Is an Upstream Regulator of Granzyme G in the Maternal-To-Zygotic Transition of Mouse Embryos.

The maternal-to-zygotic transition (MZT), which controls maternal signaling to synthesize zygotic gene products, promotes the preimplantation development of mouse zygotes to the two-cell stage. Our previous study reported that mouse granzyme g (Gzmg), a serine-type protease, is required for the MZT. In this study, we further identified the maternal factors that regulate the Gzmg promoter activity in the zygote to the two-cell stage of mouse embryos. A full-length Gzmg promoter from mouse genomic DNA, FL-pGzmg (−1696~+28 nt), was cloned, and four deletion constructs of this Gzmg promoter, Δ1-pGzmg (−1369~+28 nt), Δ2-pGzmg (−939~+28 nt), Δ3-pGzmg (−711~+28 nt) and Δ4-pGzmg (−417~+28 nt), were subsequently generated. Different-sized Gzmg promoters were used to perform promoter assays of mouse zygotes and two-cell stage embryos. The results showed that Δ4-pGzmg promoted the highest expression level of the enhanced green fluorescent protein (EGFP) reporter in the zygotes and two-cell embryos. The data suggested that time-specific transcription factors upregulated Gzmg by binding cis-elements in the −417~+28-nt Gzmg promoter region. According to the results of the promoter assay, the transcription factor binding sites were predicted and analyzed with the JASPAR database, and two transcription factors, signal transducer and activator of transcription 3 (STAT3) and GA-binding protein alpha (GABPα), were identified. Furthermore, STAT3 and GABPα are expressed and located in zygote pronuclei and two-cell nuclei were confirmed by immunofluorescence staining; however, only STAT3 was recruited to the mouse zygote pronuclei and two-cell nuclei injected with the Δ4-pGzmg reporter construct. These data indicated that STAT3 is a maternal transcription factor and may upregulate Gzmg to promote the MZT. Furthermore, treatment with a STAT3 inhibitor, S3I-201, caused mouse embryonic arrest at the zygote and two-cell stages. These results suggest that STAT3, a maternal protein, is a critical transcription factor and regulates Gzmg transcription activity in preimplantation mouse embryos. It plays an important role in the maternal-to-zygotic transition during early embryonic development.

Precise Temporal Regulation of Post-transcriptional Repressors Is Required for an Orderly Drosophila Maternal-to-Zygotic Transition

SUMMARYIn animal embryos, the maternal-to-zygotic transition (MZT) hands developmental control from maternal to zygotic gene products. We show that the maternal proteome represents more than half of the protein-coding capacity of Drosophila melanogaster’s genome, and that 2% of this proteome is rapidly degraded during the MZT. Cleared proteins include the post-transcriptional repressors Cup, Trailer hitch (TRAL), Maternal expression at 31B (ME31B), and Smaug (SMG). Although the ubiquitin-proteasome system is necessary for clearance of these repressors, distinct E3 ligase complexes target them: the C-terminal to Lis1 Homology (CTLH) complex targets Cup, TRAL, and ME31B for degradation early in the MZT and the Skp/Cullin/F-box-containing (SCF) complex targets SMG at the end of the MZT. Deleting the C-terminal 233 amino acids of SMG abrogates F-box protein interaction and confers immunity to degradation. Persistent SMG downregulates zygotic re-expression of mRNAs whose maternal contribution is degraded by SMG. Thus, clearance of SMG permits an orderly MZT.

Excess dNTPs Trigger Oscillatory Surface Flow in the Early Drosophila Embryo

During the first 2 hours of Drosophila development, precisely orchestrated nuclear cleavages, cytoskeletal rearrangements, and directed membrane growth lead to the formation of an epithelial sheet around the yolk. The newly formed epithelium remains relatively quiescent during the next hour as it is patterned by maternal inductive signals and zygotic gene products. We discovered that this mechanically quiet period is disrupted in embryos with high levels of dNTPs, which have been recently shown to cause abnormally fast nuclear cleavages and interfere with zygotic transcription. High levels of dNTPs are associated with robust onset of oscillatory two-dimensional flows during the third hour of development. Tissue cartography, particle image velocimetry, and dimensionality reduction techniques reveal that these oscillatory flows are low dimensional and are characterized by the presence of spiral vortices. We speculate that these aberrant flows emerge through an instability triggered by deregulated mechanical coupling between the nascent epithelium and three-dimensional yolk. These results highlight an unexplored connection between a core metabolic process and large-scale mechanics in a rapidly developing embryo.

Maternal contributions to gastrulation in zebrafish.

Gastrulation is a critical early morphogenetic process of animal development, during which the three germ layers; mesoderm, endoderm and ectoderm, are rearranged by internalization movements. Concurrent epiboly movements spread and thin the germ layers while convergence and extension movements shape them into an anteroposteriorly elongated body with head, trunk, tail and organ rudiments. In zebrafish, gastrulation follows the proliferative and inductive events that establish the embryonic and extraembryonic tissues and the embryonic axis. Specification of these tissues and embryonic axes are controlled by the maternal gene products deposited in the egg. These early maternally controlled processes need to generate sufficient cell numbers and establish the embryonic polarity to ensure normal gastrulation. Subsequently, after activation of the zygotic genome, the zygotic gene products govern mesoderm and endoderm induction and germ layer patterning. Gastrulation is initiated during the maternal-to-zygotic transition, a process that entails both activation of the zygotic genome and downregulation of the maternal transcripts. Genomic studies indicate that gastrulation is largely controlled by the zygotic genome. Nonetheless, genetic studies that investigate the relative contributions of maternal and zygotic gene function by comparing zygotic, maternal and maternal zygotic mutant phenotypes, reveal significant contribution of maternal gene products, transcripts and/or proteins, that persist through gastrulation, to the control of gastrulation movements. Therefore, in zebrafish, the maternally expressed gene products not only set the stage for, but they also actively participate in gastrulation morphogenesis.

Early genome activation in Drosophila is extensive with an initial tendency for aborted transcripts and retained introns.

Control of metazoan embryogenesis shifts from maternal to zygotic gene products as the zygotic genome becomes transcriptionally activated. In Drosophila, zygotic genome activation (ZGA) has been thought to occur in two phases, starting with a minor wave, in which a small number of genes become expressed, and progressing to the major wave, in which many more genes are activated. However, technical challenges have hampered the identification of early transcripts or obscured the onset of their transcription. Here, we develop an approach to isolate transcribed mRNAs and apply it over the course of Drosophila early genome activation. Our results increase by 10-fold the genes reported to be activated during what has been thought of as the minor wave and show that early genome activation is continuous and gradual. Transposable-element mRNAs are also produced, but discontinuously. Genes transcribed in the early and middle part of ZGA are short with few if any introns, and their transcripts are frequently aborted and tend to have retained introns, suggesting that inefficient splicing as well as rapid cell divisions constrain the lengths of early transcripts.

Metabolic Regulation of Developmental Cell Cycles and Zygotic Transcription

The thirteen nuclear cleavages that give rise to the Drosophila blastoderm are some of the fastest known cell cycles [1]. Surprisingly, the fertilized egg is provided with at most one-third of the dNTPs needed to complete the thirteen rounds of DNA replication [2]. The rest must be synthesized by the embryo, concurrent with cleavage divisions. What is the reason for the limited supply of DNA building blocks? We propose that frugal control of dNTP synthesis contributes to the well-characterized deceleration of the cleavage cycles and is needed for robust accumulation of zygotic gene products. In support of this model, we demonstrate that when the levels of dNTPs are abnormally high, nuclear cleavages fail to sufficiently decelerate, the levels of zygotic transcription are dramatically reduced, and the embryo catastrophically fails early in gastrulation. Our work reveals a direct connection between metabolism, the cell cycle, and zygotic transcription.

Histone Demethylase UTX is an Essential Factor for Zygotic Genome Activation and Regulates Zscan4 Expression in Mouse Embryos.

Following fertilization, the zygotic genome is activated through a process termed zygotic genome activation (ZGA), which enables zygotic gene products to replace the maternal products and initiates early embryonic development. During the ZGA period, the embryonic epigenome experiences extensive recodifications. The H3K27me3 demethylase UTX is essential for post-implantation embryonic development. However, it remains unclear whether UTX participates in preimplantation development, especially during the ZGA process. In the present study, we showed that either knockdown or overexpression of UTX led to embryonic development retardation, whereas simultaneous depletion of UTX and overexpression of ZSCAN4D rescued the embryonic development, indicating that UTX positively regulated Zscan4d expression. Using a transgenic mice model, we also found that UTX was required for preimplantation embryonic development. In conclusion, these results indicate that UTX functions as a novel regulator and plays critical roles during ZGA in addition to early embryonic development.

Mechanisms regulating zygotic genome activation

Following fertilization, the two specified gametes must unite to create an entirely new organism. The genome is initially transcriptionally quiescent, allowing the zygote to be reprogrammed into a totipotent state. Gradually, the genome is activated through a process known as the maternal-to-zygotic transition, which enables zygotic gene products to replace the maternal supply that initiated development. This essential transition has been broadly characterized through decades of research in several model organisms. However, we still lack a full mechanistic understanding of how genome activation is executed and how this activation relates to the reprogramming of the zygotic chromatin architecture. Recent work highlights the central role of transcriptional activators and suggests that these factors may coordinate transcriptional activation with other developmental changes.

Dynamics and Function of Nuclear Bodies during Embryogenesis.

Nuclear bodies are RNA-rich membraneless organelles in the cell nucleus that concentrate specific sets of nuclear proteins and RNA-protein complexes. Nuclear bodies such as the nucleolus, Cajal body (CB), and the histone locus body (HLB) concentrate factors required for nuclear steps of RNA processing. Formation of these nuclear bodies occurs on genomic loci and is frequently associated with active sites of transcription. Whether nuclear body formation is dependent on a particular gene element, an active process such as transcription, or the nascent RNA present at gene loci is a topic of debate. Recently, this question has been addressed through studies in model organisms and their embryos. The switch from maternally provided RNA and protein to zygotic gene products in early embryos has been well characterized in a variety of organisms. This process, termed maternal-to-zygotic transition, provides an excellent model for studying formation of nuclear bodies before, during, and after the transcriptional activation of the zygotic genome. Here, we review findings in embryos that reveal key principles in the study of the formation and function of nucleoli, CBs, and HLBs. We propose that while particular gene elements may contribute to formation of these nuclear bodies, active transcription promotes maturation of nuclear bodies and efficient RNA processing within them.

Developmental transition to bilaterally symmetric cell divisions is regulated by Pax-mediated transcription in embryos of the leech Helobdella austinensis

The leech embryo develops by spiral cleavage, and establishes the symmetry properties of its adult body plan through the bilaterally symmetric divisions of mesodermal proteloblast DM″ and ectodermal proteloblast DNOPQ‴. We here show that transcriptional inhibitors α-amanitin and actinomycin D specifically disrupt the symmetry and orientation of these two proteloblast cell divisions while having no apparent effect on the timing or geometry of other divisions. Transcriptional inhibition had a similar effect on both proteloblasts, i.e. cytokinesis was highly asymmetric and the cleavage plane roughly orthogonal to that seen during normal development. These findings suggest that zygotic gene product(s) are required, either directly or indirectly, for the correct placement of the proteloblast cleavage furrow. The same phenotypes were also observed following in vivo expression of dominant-negative Pax gene constructs. These dominant-negative phenotypes depended on protein/DNA interaction, and could be rescued by coexpression of full length Pax proteins. However, symmetric cleavage of the mesodermal proteloblast was rescued by full length constructs of either Hau-Paxβ1 or Hau-Pax2/5/8, while only Hau-Paxβ1 rescued the symmetry of ectodermal cleavage. We conclude that both proteloblasts need Pax-mediated transcription to adopt their normally symmetric cleavage patterns, but differ in terms of the specific Pax proteins required. The implication of these findings for the evolution of spiral cleavage is discussed.

A Genomewide Survey Argues That Every Zygotic Gene Product Is Dispensable for the Initiation of Somatic Homolog Pairing in Drosophila

Studies from diverse organisms show that distinct interchromosomal interactions are associated with many developmental events. Despite recent advances in uncovering such phenomena, our understanding of how interchromosomal interactions are initiated and regulated is incomplete. During the maternal-to-zygotic transition (MZT) of Drosophila embryogenesis, stable interchromosomal contacts form between maternal and paternal homologous chromosomes, a phenomenon known as somatic homolog pairing. To better understand the events that initiate pairing, we performed a genomewide assessment of the zygotic contribution to this process. Specifically, we took advantage of the segregational properties of compound chromosomes to generate embryos lacking entire chromosome arms and, thus, all zygotic gene products derived from those arms. Using DNA fluorescence in situ hybridization (FISH) to assess the initiation of pairing at five separate loci, this approach allowed us to survey the entire zygotic genome using just a handful of crosses. Remarkably, we found no defect in pairing in embryos lacking any chromosome arm, indicating that no zygotic gene product is essential for pairing to initiate. From these data, we conclude that the initiation of pairing can occur independently of zygotic control and may therefore be part of the developmental program encoded by the maternal genome.

Zygotically controlled F-actin establishes cortical compartments to stabilize furrows duringDrosophilacellularization

Cortical compartments partition proteins and membrane at the cell surface to define regions of specialized function. Here we ask how cortical compartments arise along the plasma membrane furrows that cellularize the early Drosophila embryo, and investigate the influence that this compartmentalization has on furrow ingression. We find that the zygotic gene product Nullo aids the establishment of discrete cortical compartments, called furrow canals, which form at the tip of incipient furrows. Upon nullo loss-of-function, proteins that are normally restricted to adjacent lateral regions of the furrow, such as Neurotactin and Discs large, spread into the furrow canals. At the same time, cortical components that should concentrate in furrow canals, such as Myosin 2 (Zipper) and Anillin (Scraps), are missing from some furrows. Depletion of these cortical components from the furrow canal compartments precipitates furrow regression. Contrary to previous models, we find that furrow compartmentalization does not require cell-cell junctions that border the furrow canals. Instead, compartmentalization is disrupted by treatments that reduce levels of cortical F-actin. Because the earliest uniform phenotype detected in nullo mutants is reduced levels of F-actin at furrow canals, we propose that Nullo compartmentalizes furrows via its regulation of F-actin, thus stabilizing furrows and insuring their ingression to complete cellularization.

Local Actin-Dependent Endocytosis Is Zygotically Controlled to Initiate Drosophila Cellularization

In early Drosophila embryos, several mitotic cycles proceed with aborted cytokinesis before a modified cytokinesis, called cellularization, finally divides the syncytium into individual cells. Here, we find that scission of endocytic vesicles from the plasma membrane (PM) provides a control point to regulate the furrowing events that accompany this development. At early mitotic cycles, local furrow-associated endocytosis is controlled by cell cycle progression, whereas at cellularization, which occurs in a prolonged interphase, it is controlled by expression of the zygotic gene nullo. nullo mutations impair cortical F-actin accumulation and scission of endocytic vesicles, such that membrane tubules remain tethered to the PM and deplete structural components from the furrows, precipitating furrow regression. Thus, Nullo regulates scission to restrain endocytosis of proteins essential for furrow stabilization at the onset of cellularization. We propose that developmentally regulated endocytosis can coordinate actin/PM remodeling to directly drive furrow dynamics during morphogenesis.

Specification and development of the germline in Caenorhabditis elegans.

Maternal-effect sterile (mes) genes encode maternal components that are required for establishment and development of the germline. Five such genes have been identified in the nematode Caenorhabditis elegans. Mutations in one of the genes result in defects in the asymmetric division and cytoplasmic partitioning that generate the primordial germ cell P4 at the 16-24-cell stage of embryogenesis. As a result of these defects, the P4 cell is transformed into a muscle progenitor and mutant embryos develop into sterile adults with extra body muscles. Mutations in the other four mes genes do not affect formation of the germline during embryogenesis, but result in drastically reduced proliferation of the germline during post-embryonic stages and in an absence of gametes in adults. The failure to form gametes may reflect a defect in germline specification or may be a consequence of reduced germline proliferation. We are currently testing these two possibilities. In addition to the mes gene products, wild-type function of the zygotic gene glp-4 is required for normal post-embryonic proliferation of the germline. Germ cells in glp-4 mutant worms are arrested in prophase of the mitotic cell cycle and are unable to enter meiosis and form gametes. Thus, following establishment of the germ lineage in the early embryo, both maternal and zygotic gene products work in concert to promote the extensive proliferation of the germline and to enable germ cells to generate functional gametes.

Germ line specific expression of a protein phosphatase Y interacting protein (PPYR1) in Drosophila

PPYR1, the product of the CG15031 gene, was identified as a protein phosphatase Y (PPY) interacting protein in Drosophila melanogaster using a yeast two-hybrid screen. PPYR1 displays a biphasic expression pattern: the maternal protein is abundant in the developing egg chambers and in the early embryos, while the zygotic protein appears later in development and is localized specifically in the testes of the males. The maternal and zygotic gene products differ from each other in their size having apparent molecular masses of 47 and 66 kDa, respectively. The maternal PPYR1 is localized in the cytoplasm of the follicular and nurse cells and is deposited as a ribonucleoprotein complex in the oocyte. In the early embryos, the PPYR1 is distributed evenly, and it gradually diminishes during embryonic development. Zygotic PPYR1 is expressed exclusively in the testes, predominantly in the cytoplasm of the spermatocytes. PPY is localized in the nuclei of the same cells. Our results suggest that PPYR1 has two distinct developmental isoforms: a maternal protein the expression of which is independent of PPY and a zygotic protein which is co-expressed with PPY.

A major role for zygotichunchbackin patterning theNasoniaembryo

Developmental genetic analysis has shown that embryos of the parasitoid wasp Nasonia vitripennis depend more on zygotic gene products to direct axial patterning than do Drosophila embryos. In Drosophila, anterior axial patterning is largely established by bicoid, a rapidly evolving maternal-effect gene, working with hunchback, which is expressed both maternally and zygotically. Here, we focus on a comparative analysis of Nasonia hunchback function and expression. We find that a lesion in Nasonia hunchback is responsible for the severe zygotic headless mutant phenotype, in which most head structures and the thorax are deleted, as are the three most posterior abdominal segments. This defines a major role for zygotic Nasonia hunchback in anterior patterning, more extensive than the functions described for hunchback in Drosophila or Tribolium. Despite the major zygotic role of Nasonia hunchback, we find that it is strongly expressed maternally, as well as zygotically. Nasonia Hunchback embryonic expression appears to be generally conserved; however, the mRNA expression differs from that of Drosophila hunchback in the early blastoderm. We also find that the maternal hunchback message decays at an earlier developmental stage in Nasonia than in Drosophila, which could reduce the relative influence of maternal products in Nasonia embryos. Finally, we extend the comparisons of Nasonia and Drosophila hunchback mutant phenotypes, and propose that the more severe Nasonia hunchback mutant phenotype may be a consequence of differences in functionally overlapping regulatory circuitry.