Gene Expression and Phenotypic Assessment of Egg Quality across Developmental Stages of Atlantic Cod throughout the Spawning Season.

Egg quality in fishes is commonly determined by fertilisation success and cleavage patterns as a phenotypic outcome of underlying regulatory mechanisms. Although these phenotypic estimators of egg quality are useful in farming conditions, these "good quality" egg batches do not always translate to good larval growth and survival. The identification of genes involved in embryonic development may help find links between genetic factors of maternal origin and egg quality. Herein, the relative expression of seven stage-specific developmental genes of Atlantic cod was analysed using quantitative PCR to understand the function during embryogenesis and its relationship with egg quality. Genes ccnb2 and pvalb1 showed significant differential expression between developmental stages and significant upregulation from blastula and somite stages, respectively. The comparison of spawning batches showed that the relative gene expression of genes ccnb2, acta, tnnt3 and pvalb1 was significantly higher from the middle of the spawning season where phenotypic quality estimators establish the best egg quality. Moreover, a positive significant correlation was observed between quality estimators based on egg morphology and the genetic expression of genes acta and acta1 during somitogenesis. This study suggests that the combination of quality estimators, genetics and batch timing could help optimise reproductive protocols for commercial stocks of Atlantic cod.

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.

Transient chromatin decompaction at the start of D. melanogaster male embryonic germline development.

Embryonic germ cells develop rapidly to establish the foundation for future developmental trajectories, and in this process, they make critical lineage choices including the configuration of their unique identity and a decision on sex. Here, we use single-cell genomics patterns for the entire embryonic germline in Drosophila melanogaster along with the somatic gonadal precursors after embryonic gonad coalescence to investigate molecular mechanisms involved in the setting up and regulation of the germline program. Profiling of the early germline chromatin landscape revealed sex- and stage-specific features. In the male germline immediately after zygotic activation, the chromatin structure underwent a brief remodeling phase during which nucleosome density was lower and deconcentrated from promoter regions. These findings echoed enrichment analysis results of our genomics data in which top candidates were factors with the ability to mediate large-scale chromatin reorganization. Together, they point to the importance of chromatin regulation in the early germline and raise the possibility of a conserved epigenetic reprogramming-like process required for proper initiation of germline development.

Telomere Elongation During Pre-Implantation Embryo Development.

The primary mechanism of telomere elongation in mammals is reverse transcription by telomerase. An alternative (ALT) pathway elongates telomeres by homologous recombination in some cancer cells and during pre-implantation embryo development, when telomere length increases rapidly within a few cell cycles. The maternal and paternal telomeres in the zygote are genetically and epigenetically distinct, with differences in telomere length and in chromatin packaging. We discuss models for how these asymmetries may contribute to telomere regulation during the earliest embryonic cell cycles and suggest directions for future research.

Zygote cryobanking applied to CRISPR/Cas9 microinjection in mice.

Microinjection of CRISPR/Cas9 requires the availability of zygotes that implies animal breeding, superovulation schemes, and embryo collection. Vitrification of zygotes may allow having ready-to-use embryos and to temporally dissociate the workload of embryo production from microinjection. In this study, fresh (F group) or vitrified (V group) zygotes were microinjected with CRISPR/Cas9 system to test the hypothesis that vitrified zygotes could be a suitable source of embryos for microinjection. In Experiment 1 (in vitro evaluation), B6D2F1/J zygotes were microinjected and cultured until blastocyst stage. Embryo survival and cleavage rates after microinjection were similar between groups (~50% and ~80% respectively; P = NS). Development rate was significantly higher for F than V group (55.0% vs. 32.6%, respectively; P<0.05). Mutation rate did not show statistical differences among groups (P = NS). In Experiment 2 (in vivo evaluation), C57BL/6J zygotes were microinjected and transferred to recipient females. Embryo survival was significantly lower in fresh than in vitrified zygotes (49.2% vs. 62.7%, respectively; P<0.05). Cleavage rate did not show statistical differences (~70%; P = NS). Pregnancy rate (70.0% vs. 58.3%) and birth rate (11.9% vs. 11.2%) were not different between groups (F vs. V group; P = NS). Offspring mutation rate was higher for F than V group, in both heterodimer analysis (73.7% vs. 33.3%, respectively; P = 0.015) and Sanger sequencing (89.5% vs. 41.7%, respectively; P = 0.006). In conclusion, vitrified-warmed zygotes present a viable alternative source for CRISPR/Cas9 microinjection when the production of fresh embryos is impeded by limited technical support. The possibility of zygote cryobanking to perform microinjection sessions on demand seems to be a suitable alternative to avoid the breeding and maintenance of animals all over the year, enhancing the implementation of CRISPR technology.

Multi-labeling Live Imaging to Quantify Gene Expression Dynamics During Drosophila Embryonic Development.

Transcription in developing metazoans is inherently stochastic, involving transient and dynamic interactions among transcriptional machinery. A fundamental challenge with traditional techniques, including fixed-tissue protein and RNA staining, is the lack of temporal resolution. Quantifying kinetic changes in transcription can elucidate underlying mechanisms of interaction among regulatory modules. In this protocol, we describe the successful implementation of a combination of MS2/MCP and PP7/PCP systems in living Drosophila embryos to further our understanding of transcriptional dynamics during development. Our technique can be extended to visualize transcriptional activities of multiple genes or alleles simultaneously, characterize allele-specific expression of a target gene, and quantitatively analyze RNA polymerase II activity in a single-cell resolution.

Mapping the chromatin accessibility landscape of zebrafish embryogenesis at single-cell resolution by SPATAC-seq.

Currently, the dynamic accessible elements that determine regulatory programs responsible for the unique identity and function of each cell type during zebrafish embryogenesis lack detailed study. Here we present SPATAC-seq: a split-pool ligation-based assay for transposase-accessible chromatin using sequencing. Using SPATAC-seq, we profiled chromatin accessibility in more than 800,000 individual nuclei across 20 developmental stages spanning the sphere stage to the early larval protruding mouth stage. Using this chromatin accessibility map, we identified 604 cell states and inferred their developmental relationships. We also identified 959,040 candidate cis-regulatory elements (cCREs) and delineated development-specific cCREs, as well as transcription factors defining diverse cell identities. Importantly, enhancer reporter assays confirmed that the majority of tested cCREs exhibited robust enhanced green fluorescent protein expression in restricted cell types or tissues. Finally, we explored gene regulatory programs that drive pigment and notochord cell differentiation. Our work provides a valuable open resource for exploring driver regulators of cell fate decisions in zebrafish embryogenesis.

Expression analysis of thg1l during Xenopus laevis development.

The tRNA-histidine guanylyltransferase 1-like (THG1L), also known as induced in high glucose-1 (IHG-1), encodes for an essential mitochondria-associated protein highly conserved throughout evolution, that catalyses the 3'-5' addition of a guanine to the 5'-end of tRNA-histidine (tRNAHis). Previous data indicated that THG1L plays a crucial role in the regulation of mitochondrial biogenesis and dynamics, in ATP production, and is critically involved in the modulation of apoptosis, cell-cycle progression and survival, as well as in cellular stress responses and redox homeostasis. Dysregulations of THG1L expression play a central role in various pathologies, including nephropathies, and neurodevelopmental disorders often characterized by developmental delay and cerebellar ataxia. Despite the essential role of THG1L, little is known about its expression during vertebrate development. Herein, we examined the detailed spatio-temporal expression of this gene in the developing Xenopus laevis. Our results show that thg1l is maternally inherited and its temporal expression suggests a role during the earliest stages of embryogenesis. Spatially, thg1l mRNA localizes in the ectoderm and marginal zone mesoderm during early stages of development. Then, at tadpole stages, thg1l transcripts mostly localise in neural crests and their derivatives, somites, developing kidney and central nervous system, therefore largely coinciding with territories displaying intense energy metabolism during organogenesis in Xenopus.

Early transcriptional similarities between two distinct neural lineages during ascidian embryogenesis.

In chordates, the central nervous system arises from precursors that have distinct developmental and transcriptional trajectories. Anterior nervous systems are ontogenically associated with ectodermal lineages while posterior nervous systems are associated with mesoderm. Taking advantage of the well-documented cell lineage of ascidian embryos, we asked to what extent the transcriptional states of the different neural lineages become similar during the course of progressive lineage restriction. We performed single-cell RNA sequencing (scRNA-seq) analyses on hand-dissected neural precursor cells of the two distinct lineages, together with those of their sister cell lineages, with a high temporal resolution covering five successive cell cycles from the 16-cell to neural plate stages. A transcription factor binding site enrichment analysis of neural specific genes at the neural plate stage revealed limited evidence for shared transcriptional control between the two neural lineages, consistent with their different ontogenies. Nevertheless, PCA analysis and hierarchical clustering showed that, by neural plate stages, the two neural lineages cluster together. Consistent with this, we identified a set of genes enriched in both neural lineages at the neural plate stage, including miR-124, Celf3.a, Zic.r-b, and Ets1/2. Altogether, the current study has revealed genome-wide transcriptional dynamics of neural progenitor cells of two distinct developmental origins. Our scRNA-seq dataset is unique and provides a valuable resource for future analyses, enabling a precise temporal resolution of cell types not previously described from dissociated embryos.

Pioneer Transcription Factors: The First Domino in Zygotic Genome Activation.

Zygotic genome activation (ZGA) is a pivotal event in mammalian embryogenesis, marking the transition from maternal to zygotic control of development. During the ZGA process that is characterized by the intricate cascade of gene expression, who tipped the first domino in a meticulously arranged sequence is a subject of paramount interest. Recently, Dux, Obox and Nr5a2 were identified as pioneer transcription factors that reside at the top of transcriptional hierarchy. Through co-option of retrotransposon elements as hubs for transcriptional activation, these pioneer transcription factors rewire the gene regulatory network, thus initiating ZGA. In this review, we provide a snapshot of the mechanisms underlying the functions of these pioneer transcription factors. We propose that ZGA is the starting point where the embryo's own genome begins to influence development trajectory, therefore in-depth dissecting the functions of pioneer transcription factors during ZGA will form a cornerstone of our understanding for early embryonic development, which will pave the way for advancing our grasp of mammalian developmental biology and optimizing in vitro production (IVP) techniques.

The first embryo, the origin of cancer and animal phylogeny. III. The totipotency as revealed by morphogenesis and the neoplasia controlled by cellular differentiation.

We have extensively described that the neoplastic process (NP) has deep evolutionary roots and we have made specific predictions about the connection between cancer and the formation of the first embryo, which allowed for the evolutionary radiation of metazoans. My main hypothesis is that the NP is at the heart of cellular mechanisms responsible for animal morphogenesis, and given its embryological basis, also at the center of cell differentiation-one of the most interesting and relevant aspects of embryogenesis. In this article, I take forward the idea of the role of physics in the modeling of the neoplastic functional module (NFM) and its contribution to morphogenesis to reveal the totipotency of the zygote. In my consideration of these arguments, I examine mechanical and biophysical clues and their intimate connection with cellular differentiation. I expound on how cancer biology is perfectly intertwined with embryonic differentiation and why it is considered a disease of cell differentiation. The neoplasia is controlled by textural gradients that lead to cell differentiation within the embryo. Thus, the embryo would be a benign tumor. Finally, inspired by evolutionary history and by what the nervous system represents for current biology and based on the impressive nervous system of ctenophores as seen in fossil records, I propose a hypothesis with physical foundations (mechanical morphogenesis) for the formation of a preneural pattern of the nervous system of the first animal embryo.

IκBα controls dormancy in hematopoietic stem cells via retinoic acid during embryonic development.

Recent findings suggest that Hematopoietic Stem Cells (HSC) and progenitors arise simultaneously and independently of each other already in the embryonic aorta-gonad mesonephros region, but it is still unknown how their different features are established. Here, we uncover IκBα (Nfkbia, the inhibitor of NF-κB) as a critical regulator of HSC proliferation throughout development. IκBα balances retinoic acid signaling levels together with the epigenetic silencer, PRC2, specifically in HSCs. Loss of IκBα decreases proliferation of HSC and induces a dormancy related gene expression signature instead. Also, IκBα deficient HSCs respond with superior activation to in vitro culture and in serial transplantation. At the molecular level, chromatin regions harboring binding motifs for retinoic acid signaling are hypo-methylated for the PRC2 dependent H3K27me3 mark in IκBα deficient HSCs. Overall, we show that the proliferation index in the developing HSCs is regulated by a IκBα-PRC2 axis, which controls retinoic acid signaling.

High-resolution transcriptome datasets during embryogenesis of plant-parasitic nematodes.

Understanding the transcriptional regulatory characteristics throughout the embryogenesis of plant-parasitic nematodes is crucial for elucidating their developmental processes' uniqueness. However, a challenge arises due to the lack of suitable technical methods for synchronizing the age of plant-parasitic nematodes embryo, it is difficult to collect detailed transcriptome data at each stage of embryonic development. Here, we recorded the 11 embryonic developmental time-points of endophytic nematode Meloidogyne incognita (isolated from Wuhan, China), Heterodera glycines (isolated from Wuhan, China), and Ditylenchus destructor (isolated from Jinan, China) species, and constructed transcriptome datasets of single embryos of these three species utilizing low-input smart-seq2 technology. The datasets encompassed 11 complete embryonic development stages, including Zygote, 2-cell, 4-cell, 8-cell, 24-44 cell, 64-78 cell, Comma, 1.5-fold, 2-fold, Moving, and L1, each stage generated four to five replicates, resulting in a total of 162 high-resolution transcriptome libraries. This high-resolution cross-species dataset serves as a crucial resource for comprehending the embryonic developmental properties of plant-parasitic nematodes and for identifying functional regulatory genes during embryogenesis.

Obox4 promotes zygotic genome activation upon loss of Dux.

Once fertilized, mouse zygotes rapidly proceed to zygotic genome activation (ZGA), during which long terminal repeats (LTRs) of murine endogenous retroviruses with leucine tRNA primer (MERVL) are activated by a conserved homeodomain-containing transcription factor, DUX. However, Dux-knockout embryos produce fertile mice, suggesting that ZGA is redundantly driven by an unknown factor(s). Here, we present multiple lines of evidence that the multicopy homeobox gene, Obox4, encodes a transcription factor that is highly expressed in mouse two-cell embryos and redundantly drives ZGA. Genome-wide profiling revealed that OBOX4 specifically binds and activates MERVL LTRs as well as a subset of murine endogenous retroviruses with lysine tRNA primer (MERVK) LTRs. Depletion of Obox4 is tolerated by embryogenesis, whereas concomitant Obox4/Dux depletion markedly compromises embryonic development. Our study identified OBOX4 as a transcription factor that provides genetic redundancy to preimplantation development.

Mapping putative enhancers in mouse oocytes and early embryos reveals TCF3/12 as key folliculogenesis regulators.

Dynamic epigenomic reprogramming occurs during mammalian oocyte maturation and early development. However, the underlying transcription circuitry remains poorly characterized. By mapping cis-regulatory elements using H3K27ac, we identified putative enhancers in mouse oocytes and early embryos distinct from those in adult tissues, enabling global transitions of regulatory landscapes around fertilization and implantation. Gene deserts harbour prevalent putative enhancers in fully grown oocytes linked to oocyte-specific genes and repeat activation. Embryo-specific enhancers are primed before zygotic genome activation and are restricted by oocyte-inherited H3K27me3. Putative enhancers in oocytes often manifest H3K4me3, bidirectional transcription, Pol II binding and can drive transcription in STARR-seq and a reporter assay. Finally, motif analysis of these elements identified crucial regulators of oogenesis, TCF3 and TCF12, the deficiency of which impairs activation of key oocyte genes and folliculogenesis. These data reveal distinctive regulatory landscapes and their interacting transcription factors that underpin the development of mammalian oocytes and early embryos.

Embryos from Prepubertal Hyperglycemic Female Mice Respond Differentially to Oxygen Tension In Vitro.

Reduced oxygen during embryo culture in human ART prevents embryo oxidative stress. Oxidative stress is also the major mechanism by which maternal diabetes impairs embryonic development. This study employed induced hyperglycemia prepubertal mice to mimic childhood diabetes to understand the effects of varying oxygen tension during in vitro embryonic development. The oocytes were fertilized and cultured at low (≈5%) oxygen (LOT) or atmospheric (≈20%) oxygen tension (HOT) for up to 96 h. Embryo development, apoptosis in blastocysts, inner cell mass (ICM) outgrowth proliferation, and Hif1α expression were assessed. Though the oocyte quality and meiotic spindle were not affected, the fertilization rate (94.86 ± 1.18 vs. 85.17 ± 2.81), blastocyst rate (80.92 ± 2.92 vs. 69.32 ± 2.54), and ICM proliferation ability (51.04 ± 9.22 vs. 17.08 ± 3.05) of the hyperglycemic embryos were significantly higher in the LOT compared to the HOT group. On the other hand, blastocysts from the hyperglycemic group, cultured at HOT, had a 1.5-fold increase in apoptotic cells compared to the control and lower Hif1α transcripts in ICM outgrowths compared to the LOT. Increased susceptibility of embryos from hyperglycemic mice to higher oxygen tension warrants the need to individualize the conditions for embryo culture systems in ART clinics, particularly when an endogenous maternal pathology affects the ovarian environment.

The Effect of Cryopreserved Sperm on the Early Development, Survival, and Growth of Intergeneric Sterbel Hybrids (Acipenser ruthenus × Huso huso).

The aim of this study was to analyze the survival and growth of intergeneric (Acispenser ruthenus × Huso huso L.) sterbel hybrids obtained by fertilizing sterlet eggs with cryopreserved beluga semen. The rate of embryonic development did not differ between sterbel hybrids (experimental groups) and sterlets (control groups), and the hatching period was identical in all groups. The survival rate of hybrid larvae was higher in the experimental groups than in the control groups. Body weight and body length measurements revealed that sterbel hybrids grew at a faster rate than the control group sterlets. The hybrid origin of sterbels produced with the use of cryopreserved beluga semen was confirmed in a genetic analysis based on species-specific DNA fragments. To the best of the authors' knowledge, this is the first study to analyze the growth of sterbel hybrids derived from cryopreserved semen. The research findings indicate that this type of intergeneric hybridization delivers satisfactory results and can be applied in sturgeon aquaculture.

miRNAs in Follicular and Oviductal Fluids Support Global DNA Demethylation in Early-Stage Embryos.

Global methylation levels differ in in vitro- and in vivo-developed embryos. Follicular fluid (FF) contains extracellular vesicles (EVs) containing miRNAs that affect embryonic development. Here, we examined our hypothesis that components in FF affect global DNA methylation and embryonic development. Oocytes and FF were collected from bovine ovaries. Treatment of zygotes with a low concentration of FF induced global DNA demethylation, improved embryonic development, and reduced DNMT1/3A levels. We show that embryos take up EVs containing labeled miRNA secreted from granulosa cells and the treatment of zygotes with EVs derived from FF reduces global DNA methylation in embryos. Furthermore, the methylation levels of in vitro-developed blastocysts were higher than those of in their vivo counterparts. Based on small RNA-sequencing and in silico analysis, we predicted miR-29b, -199a-3p, and -148a to target DNMTs and to induce DNA demethylation, thereby improving embryonic development. Moreover, among FF from 30 cows, FF with a high content of these miRNAs demethylated more DNA in the embryos than FF with a lower miRNA content. Thus, miRNAs in FF play a role in early embryonic development.

A mathematical framework for understanding the spontaneous emergence of complexity applicable to growing multicellular systems.

In embryonic development and organogenesis, cells sharing identical genetic codes acquire diverse gene expression states in a highly reproducible spatial distribution, crucial for multicellular formation and quantifiable through positional information. To understand the spontaneous growth of complexity, we constructed a one-dimensional division-decision model, simulating the growth of cells with identical genetic networks from a single cell. Our findings highlight the pivotal role of cell division in providing positional cues, escorting the system toward states rich in information. Moreover, we pinpointed lateral inhibition as a critical mechanism translating spatial contacts into gene expression. Our model demonstrates that the spatial arrangement resulting from cell division, combined with cell lineages, imparts positional information, specifying multiple cell states with increased complexity-illustrated through examples in C.elegans. This study constitutes a foundational step in comprehending developmental intricacies, paving the way for future quantitative formulations to construct synthetic multicellular patterns.

DNA replication in early mammalian embryos is patterned, predisposing lamina-associated regions to fragility.

DNA replication in differentiated cells follows a defined program, but when and how it is established during mammalian development is not known. Here we show using single-cell sequencing, that late replicating regions are established in association with the B compartment and the nuclear lamina from the first cell cycle after fertilization on both maternal and paternal genomes. Late replicating regions contain a relative paucity of active origins and few but long genes and low G/C content. In both bovine and mouse embryos, replication timing patterns are established prior to embryonic genome activation. Chromosome breaks, which form spontaneously in bovine embryos at sites concordant with human embryos, preferentially locate to late replicating regions. In mice, late replicating regions show enhanced fragility due to a sparsity of dormant origins that can be activated under conditions of replication stress. This pattern predisposes regions with long neuronal genes to fragility and genetic change prior to separation of soma and germ cell lineages. Our studies show that the segregation of early and late replicating regions is among the first layers of genome organization established after fertilization.