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Extracellular signal-regulated kinase 5 (ERK5), a mitogen-activated protein kinase (MAPK) family member, plays an important role in various biological processes, such as proliferation, apoptosis, differentiation, survival, and cell regulation. However, studies on the effects of ERK5 on porcine preimplantation embryos are limited. In this study, to determine the function of ERK5 during porcine embryo development, ERK5 function was inhibited by adding the ERK5 inhibitor JWG-071. The ERK5 mRNA and protein expression levels tended to decrease from the 4-cell stage compared to the 1-cell and 2-cell stages, suggesting that ERK5 is the maternal gene. During embryonic development in pigs, adding 5 μM of JWG-071 significantly reduced the phosphorylation of ERK5 and the blastocyst development rate (control: 53.44 ± 8.38 %; treatment: 26.65 ± 3.40 %). Additionally, ERK5 inhibition increased the expression of UPR-related proteins, glucose-regulated protein (GRP78), and C/EBP homologous protein (CHOP) by inducing ER stress. Compared to the control group, the expression of the autophagy-related proteins LC3 and ATG7 was significantly increased in the ERK5 inhibition group, indicating that the inhibition of ERK5 induced autophagy. In addition, ERK5 inhibition increased the expression of BAX, a pro-apoptotic gene, resulting in apoptosis. In conclusion, the results show that ERK5 inhibition during porcine embryonic development induces autophagy and apoptosis by increasing ER stress, resulting in a negative effect on embryonic development in pigs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Vertebrate development is regulated by several complex well-characterized morphogen signaling pathways, transcription factors, and structural proteins, but less is known about the enzymatic pathways that regulate early development. We have identified that factors from the inflammation-mediating cyclooxygenase (COX) signaling pathway are expressed at early stages of development in avian embryos. Using Gallus gallus (chicken) as a research model, we characterized the spatiotemporal expression of a subset of genes and proteins in the COX pathway during early neural development stages. Specifically, here we show expression patterns of COX-1, COX-2, and microsomal prostaglandin E synthase-2 (mPGES-2) as well as the genes encoding these enzymes (PTGS1, PTGS2, and PTGES-2). Unique expression patterns of individual players within the COX pathway suggest that they may play non-canonical/non-traditional roles in the embryo compared to their roles in the adult. Future work should examine the function of the COX pathway in tissue specification and morphogenesis and determine if these expression patterns are conserved across species., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Studies using the embryos of the freshwater prawn Macrobrachium olfersii have reported changes in embryonic cells after exposure to ultraviolet B (UVB) radiation, such as DNA damage and apoptosis activation. Considering the importance of mitochondria in embryonic cells, this study aimed to characterize the aspects of mitochondrial morphofunctionality in M. olfersii embryos and mitochondrial responses to UVB radiation exposure. The coding sequences of genes Tfam, Nrf1, Mfn1, and Drp1 were identified from the transcriptome of M. olfersii embryos. The phylogenetic relationship showed strong amino acid identity and a highly conserved nature of the sequences. Additionally, the number of mitochondrial DNA (mtDNA) copies were higher in the early embryonic days. The results showed that the expression of the analyzed genes was highly regulated during embryonic development, increasing their levels near hatching. Furthermore, when embryos were exposed to UVB radiation, mitochondrial biogenesis was activated, recognized by higher levels of transcripts of genes Tfam and Nrf1, accompanied by mitochondrial fission. Additionally, these mitochondrial events were supported by an increase of mtDNA copies. Our results showed that UVB radiation was able to change the mitochondrial morphofunctionality, and under the current knowledge, certainly compromise embryonic cellular integrity. Additionally, mitochondria is an important cellular target of this radiation and its responses can be used to assess environmental stress caused by UVB radiation in embryos of aquatic species., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Thaline de Quadros reports financial support was provided by Coordination of Higher Education Personnel Improvement. Evelise Maria Nazari reports financial support was provided by National Council for Scientific and Technological Development. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Totipotent stem cells, the earliest cells in embryonic development, can differentiate into complete embryos and extra-embryonic tissues, making them essential for understanding both development and regenerative medicine. This review examines recent advances in the dynamic remodeling of nuclear structures during the transition between totipotency and pluripotency, as well as other cell fate transition processes. Additionally, we highlight innovative experimental and computational methods that elucidate the relationship between nuclear architecture and cell fate decisions. By integrating these insights, we aim to enhance our understanding of how nuclear remodeling influences totipotency and other cell fate transitions, paving the way for future research in this critical field., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Genomic instability is the main cause of abnormal embryo development and abortion. NLRP7 dysfunctions affect embryonic development and lead to Hydatidiform Moles, but the underlying mechanisms remain largely elusive. Here, we show that NLRP7 knockout affects the genetic stability, resulting in increased DNA damage in both human embryonic stem cells and blastoids, making embryonic cells in blastoids more susceptible to apoptosis. Mechanistically, NLRP7 can interact with factors related to alternative splicing and DNA damage response, including DDX39B, PRPF8, THRAP3 and PARP1. Moreover, NLRP7 dysfunction leads to abnormal alternative splicing of genes involved in homologous recombination in human embryonic stem cells, Such as Brca1 and Rad51. These results indicate that NLRP7-mediated Alternative splicing is potentially required for the maintenance of genome integrity during early human embryogenesis. Together, this study uncovers that NLRP7 plays an essential role in the maintenance of genetic stability during early human embryonic development by regulating alternative splicing of homologous recombination-related genes., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Recent studies support the influence of paternal lifestyle and diet before conception on the health of the offspring via epigenetic inheritance through sperm DNA methylation, histone modification, and small non-coding RNA (sncRNA) expression and regulation. Smoking may induce DNA hypermethylation in genes related to anti-oxidation and insulin resistance. Paternal diet and obesity are associated with greater risks of metabolic dysfunction in offspring via epigenetic alterations in the sperm. Metabolic changes, such as high blood glucose levels and increased body weight, are commonly observed in the offspring of fathers subjected to chronic stress, in addition to an enhanced risk of depressive-like behaviour and increased sensitivity to stress in both the F0 and F1 generations. DNA methylation is correlated with alterations in sperm quality and the ability to fertilise oocytes, possibly via a differentially regulated MAKP81IP3 signalling pathway. Paternal exposure to toxic endocrine-disrupting chemicals (EDCs) is also linked to the transgenerational transmission of increased predisposition to disease, infertility, testicular disorders, obesity, and polycystic ovarian syndrome (PCOS) in females through epigenetic changes during gametogenesis. As the success of assisted reproductive technology (ART) is also affected by paternal diet, BMI, and alcohol consumption, its outcomes could be improved by modifying factors that are dependent on male lifestyle choices and environmental factors. This review discusses the importance of epigenetic signatures in sperm-including DNA methylation, histone retention, and sncRNA-for sperm functionality, early embryo development, and offspring health. We also discuss the mechanisms by which paternal lifestyle and environmental factors (obesity, smoking, EDCs, and stress) may impact the sperm epigenome., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests. Ethics approval and Consent to participate: Not applicable, as this is a review article. Consent for publication: Not applicable, as this is a review article., (© 2025. The Author(s).)

While proliferating cells optimize their metabolism to produce biomass, the metabolic objectives of cells that perform non-proliferative tasks are unclear. The opposing requirements for optimizing each objective result in a trade-off that forces single cells to prioritize their metabolic needs and optimally allocate limited resources. Here, we present single-cell optimization objective and trade-off inference (SCOOTI), which infers metabolic objectives and trade-offs in biological systems by integrating bulk and single-cell omics data, using metabolic modeling and machine learning. We validated SCOOTI by identifying essential genes from CRISPR-Cas9 screens in embryonic stem cells, and by inferring the metabolic objectives of quiescent cells, during different cell-cycle phases. Applying this to embryonic cell states, we observed a decrease in metabolic entropy upon development. We further uncovered a trade-off between glutathione and biosynthetic precursors in one-cell zygote, two-cell embryo, and blastocyst cells, potentially representing a trade-off between pluripotency and proliferation. A record of this paper's transparent peer review process is included in the supplemental information., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mammalian pre-implantation development is entirely devoted to the specification of extra-embryonic lineages, which are fundamental for embryo morphogenesis and support. The second fate decision is taken just before implantation, as defined by the epiblast (EPI) and the primitive endoderm (PE) specification. Later, EPI forms the embryo proper and PE contributes to the formation of the yolk sac. The formation of EPI and PE as molecularly and morphologically distinct lineages is the final step of a multistage process, which begins when bipotent progenitor cells diverge into separate fates. Despite advances in uncovering the molecular mechanisms underlying the differential transcriptional patterns that dictate how apparently identical cells make fate decisions and how lineage integrity is maintained, a detailed overview of these mechanisms is still lacking. In this review, we dissect the EPI and PE formation process into four stages (initiation, specification, segregation, and maintenance) and we provide a comprehensive understanding of the molecular mechanisms involved in lineage establishment in the mouse. In addition, we discuss the conservation of key processes in humans, based on the most recent findings., (© 2025 Athanasouli et al.)

Female infertility is a significant healthcare burden that is frequently encountered among couples globally. While environmental factors, comorbidities, and lifestyle determine reproductive health, certain genetic variants in key reproductive genes can potentially cause unsuccessful pregnancies. Such crucial proteins have been identified within the subcortical maternal complex (SCMC) and play an integral role in the early stages of embryogenesis before embryo implantation. SCMC proteins are associated with crucial pathways during embryogenesis, causing changes that are necessary for the transition of an oocyte to an embryo. These vital processes include the formation of cytoplasmic spindles and lattices, accurate positioning of meiotic spindles, regulatory roles in various gene translations, organelle redistribution, and zygotic genome reprogramming. While these genes are well studied in animal models, often mice, translation to clinical studies is comparatively less. The present study elucidates the transition in genetic studies from animal to human models of SCMC proteins. The present literature review shows that the expression of various SCMC proteins impairs embryo development at different stages. The clinical translation of SCMC occurs via various pathways. Therefore, females experiencing multiple unsuccessful pregnancies after natural or assisted conception techniques are candidates for underlying SCMC mutations. Although the phenotype of affected individuals has been identified, the molecular mechanisms that lead to impaired pathways still require investigation. Therefore, the present study paves the way for future research leading to the early diagnosis of lethal variants and possible subsequent management., Competing Interests: Declarations. Ethical approval: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Nature B.V.)

Preimplantation embryonic development is orchestrated by dynamic changes in the proteome and transcriptome, regulated by mechanisms such as maternal-to-zygotic transition. Here, we employed label-free quantitative proteomics to comprehensively analyze proteome dynamics from germinal vesicle oocytes to blastocysts in mouse embryos. We identified 3490 proteins, including 715 consistently detected across all stages, revealing stage-specific changes in proteins associated with translation, protein modification, and mitochondrial metabolism. Comparison with transcriptomic data highlighted a low correlation between mRNA and protein levels, underscoring the significance of non-transcriptional regulatory mechanisms during early development. Additionally, we analyzed WD repeat-containing protein 74 (WDR74)-deficient embryos generated using CRISPR-Cas9 genome editing. WDR74, a pre-60S ribosome maturation factor, was found to be critical for ribosome biogenesis and cell division. Furthermore, WDR74 deficiency led to a significant reduction in ribosomal protein large subunit and impaired progression beyond the morula stage. Key ribosomal proteins such as ribosomal protein L24 (RPL24) and ribosomal protein L26 (RPL26), which influence cell division timing, were notably affected, while small subunit proteins remained largely unchanged. Taken together, our study demonstrates the utility of integrating genome editing with proteomic analysis to elucidate molecular mechanisms underlying early embryogenesis, and provides new insights into protein-level regulation of preimplantation development., (© 2025 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Epigenetic factors are crucial for ensuring proper chromatin dynamics during the initial stages of embryo development. Among these factors, the Polycomb group (PcG) of proteins plays a key role in establishing correct transcriptional programmes during mouse embryogenesis. PcG proteins are classified into two complexes: Polycomb repressive complex 1 (PRC1) and PRC2. Both complexes decorate histone proteins with distinct post-translational modifications (PTMs) that are predictive of a silent transcriptional chromatin state. In recent years, a critical adaptation of the classical techniques to analyse chromatin profiles and to study biochemical interactions at low-input resolution has allowed us to deeply explore PcG molecular mechanisms in the very early stages of mouse embryo development- from fertilisation to gastrulation, and from zygotic genome activation (ZGA) to specific lineages differentiation. These advancements provide a foundation for a deeper understanding of the fundamental role Polycomb complexes play in early development and have elucidated the mechanistic dynamics of PRC1 and PRC2. In this review, we discuss the functions and molecular mechanisms of both PRC1 and PRC2 during early mouse embryo development, integrating new studies with existing knowledge. Furthermore, we highlight the molecular functionality of Polycomb complexes from ZGA through gastrulation, with a particular focus on non-canonical imprinted and bivalent genes, and Hox cluster regulation., Competing Interests: Competing interests: The authors declare no competing interests, (© 2024. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

Abstract: Spermatozoa have a highly complex RNA profile. Several of these transcripts are suggested as biomarkers for male infertility and contribute to early development. To analyze the differences between sperm RNA quantity and expression of protamine ( PRM1 and PRM2 ) and testis-specific histone 2B ( TH2B ) genes, spermatozoa from 33 patients who enrolled in assisted reproduction treatment (ART) program were analyzed. Sperm RNA of teratozoospermic (T), oligoteratozoospermic (OT), and normozoospermic (N) samples was extracted, and the differences in transcript levels among the study groups were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). The correlations of total RNA per spermatozoon and the expression of the transcripts were evaluated in relation to sperm characteristics and preimplantation embryo development. The mean (±standard deviation) RNA amount per spermatozoon was 28.48 (±23.03) femtogram in the overall group and was significantly higher in the OT group than that in N and T groups. Total sperm RNA and gene expression of PRM1 and PRM2 genes were related to preimplantation embryo development and developmental arrest. Specific sperm characteristics were correlated with the expressions of PRM1 , PRM2 , or TH2B genes. We conclude that the sperm RNA amount and composition are important factors and might influence early embryonic development and also differ in different cases of male infertility., (Copyright ©The Author(s)(2024).)

PLK4 plays a crucial role in centriole duplication, which is essential for maintaining cellular processes such as cell division, cytoskeletal stability, and cilia formation. However, the mechanisms of PLK4 remain incompletely understood, especially in the embryonic development of vertebrate species. In this study, we observed that Plk4 dysfunction led to abnormal embryonic development in zebrafish, characterized by symptoms such as dark and wrinkled skin, microphthalmia, and body axis curvature. In plk4 mutants, defects in centriole duplication led to abnormal cell division, apoptosis, and ciliogenesis defects. Moreover, overexpression of plk4 in zebrafish embryos caused excessive centrosome amplification, disrupting embryonic gastrulation through abnormal cell division and ultimately resulting in embryonic lethality. Furthermore, we identified the "cryptic" polo box (CPB) domain, consisting of two PBs (PB1 and PB2), as the critical centrosome localization domain of Plk4. Surprisingly, overexpression of these two PB domains alone was sufficient to induce embryonic lethality. Additionally, we discovered a truncated form of CPB that localizes to the centrosome without causing defects in embryonic development. Our results demonstrate that Plk4 tightly controls centriole duplication, which is essential for early embryonic development in zebrafish., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Stem cell-based embryo models offer unprecedented experimental tools for studying early human development. The usefulness of embryo models hinges on their molecular, cellular and structural fidelities to their in vivo counterparts. To authenticate human embryo models, single-cell RNA sequencing has been utilized for unbiased transcriptional profiling. However, an organized and integrated human single-cell RNA-sequencing dataset, serving as a universal reference for benchmarking human embryo models, remains unavailable. Here we developed such a reference through the integration of six published human datasets covering development from the zygote to the gastrula. Lineage annotations are contrasted and validated with available human and nonhuman primate datasets. Using stabilized Uniform Manifold Approximation and Projection, we constructed an early embryogenesis prediction tool, where query datasets can be projected on the reference and annotated with predicted cell identities. Using this reference tool, we examined published human embryo models, highlighting the risk of misannotation when relevant references are not utilized for benchmarking and authentication., Competing Interests: Competing interests: J. Fu is an editor of NPJ Regenerative Medicine. The other authors declare no competing interests., (© 2024. The Author(s).)

Developmental abnormalities are more common in somatic cell nuclear transfer (SCNT) embryos due to epigenetic barriers that occur during the maternal-to-zygotic transition (MZT). N6-methyladenosine (m6A) is an RNA epigenetic modification that plays a significant role in numerous biological processes. However, the relationship between m6A and SCNT embryonic development is largely unexplored. In the present study, we found that the low expression of m6A methyltransferase-like 3 (METTL3) was associated with developmental arrest before zygotic genome activation (ZGA) in goat SCNT embryos and that karyokinesis defects were evident during their development. Notably, we demonstrated that METTL3 overexpression rescued the karyokinesis abnormalities, enhanced embryonic development and elevated the blastocyst formation rate. Further experiments revealed that METTL3 could mitigate the defects of maternal mRNA degradation, enhance the translation of Aurora kinase B (AURKB) and increase the phosphorylation of serine 10 on histone H3 (H3S10ph) to ensure the normal karyokinesis in SCNT embryos before ZGA in goats. Overall, our study highlights the essential role of METTL3 in enhancing the development of goat SCNT embryos. These findings indicate that METTL3 is critical for optimal SCNT efficiency and advance our understanding of m6A's role in embryonic development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Previous work indicated that the implantation and pregnancy rates of women with endometriosis are lower than those of healthy women during in-vitro fertilisation and embryonic transfer. And there are numerous microRNAs (miRNAs) in human uterine luminal fluid (ULF), some of which are associated with early preimplantation development of embryos. In our study, we sought to determine whether miRNAs in the ULF are differentially expressed between women with and without endometriosis and to uncover the association of miRNAs with the development potential of blastocysts. The implantation and clinical pregnancy rates significantly decreased in women with endometriosis than in those without endometriosis. Notably, hsa-miR-145-5p was upregulated in ULF samples from women with endometriosis (fold change > 2, false discovery rate < 0.001). Moreover, the ratios of mouse/human early embryos that developed into blastocyst-staged embryos (P = 0.0065 and P = 0.0098, respectively) were significantly affected via miR-145-5p upregulation in mouse/human early embryos. Notch signalling pathway components had abnormal expression levels in the mouse/human blastocyst-stage embryos in the miR-145-5p mimic-enriched extracellular vesicles (EVs) group. In conclusions, our study revealed that human extracellular vesicle-derived miRNAs in ULF impacted the developmental potential of blastocysts in women with endometriosis. Moreover, the upregulation of miR-145-5p-enriched EVs in mouse and human embryos negatively affected blastocyst development by suppressing the expression of components of the NOTCH signalling pathway, which may contribute to elucidate the cause of infertility in women with endometriosis., Competing Interests: Declarations. Ethics approval and consent to participate: This study adhered to the tenets of the Declaration of Helsinki. The studies involving human participants and animal experiments were reviewed and approved by the Institutional Review Committee of Fudan University, and all methods were carried out in accordance with its guidelines and regulations. Written informed consent for study participation was obtained from all subjects. And this study was also carried out in compliance with the ARRIVE guidelines for the reporting of animal experiments. Consent for publication: Consent for publication was obtained from each patient. Competing interests: The authors declare no competing interests. Capsule: miR-145-5p is upregulated in uterine luminal fluid in women with endometriosis. Upregulation of miR-145-5p-enriched extracellular vesicles negatively affected mouse and human blastocyst development by suppressing the NOTCH signalling pathway and may be one of the causes for compromised fertility in women with endometriosis., (© 2024. The Author(s).)

Cytosolic thiouridylase is a conserved cytoplasmic tRNA thiolase composed of two different subunits, CTU1 and CTU2. CTU2 serves as a scaffold protein, while CTU1 catalyzes the 2-thiolation at the 34th wobble uridine of the anticodon loop. tRNAGlnUUG, tRNAGluUUC, and tRNALysUUU are the tRNA substrates that are modified with a thiol group at the C2 positions (s2) by CTU1, and also with a methoxycarbonylmethyl group at the C5 positions (mcm5) by Elongator and ALKBH8. mcm5s2U34 modification of the three tRNAs, and their modifying enzymes are involved in human disease and development. Elongator mutant animals exhibit severe phenotypes, while the biological function of Ctu1 in vertebrate animal models remains poorly characterized. Here, we applied antisense morpholino oligonucleotides targeting cytosolic thiouridylase subunit1 (ctu1) transcripts in a zebrafish model and small interfereing RNA against CTU1 transcript in human endothelial cells to define the phenotypes. We found that deficiency of ctu1 causes impaired angiogenesis and development in zebrafish embryos, and CTU1 is involved in proliferation, migration, and tube formation of human endothelial cells. We employed single-cell RNA sequencing to acquire the transcriptomic atlas from ctu1 and control morphant zebrafish. Comprehensive bioinformatics analysis, including pseudo-time, RNA velocity, cell-cell communication, and gene regulatory network inference revealed that ctu1 deficiency leads to the arrest of cell cycle, and the defects of nerve development and erythrocyte differentiation and the attenuation of several pro-angiogenic signaling pathways, e.g., angpt-tek and dll4-notch. Our findings show for the first time that CTU1 is essential for angiogenesis and embryonic development in vertebrates., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Yu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The scarcity of zebrafish-specific motif databases presents a challenge to the analysis of transcription factor (TF) motif within zebrafish single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) data, thus hindering the identification of regulatory elements throughout zebrafish embryonic development. Here, we provide a protocol to analyze single-nucleus chromatin accessibility dataset during zebrafish early embryogenesis. We describe steps for fragment file retrieval, sample integration, quality control, Latent Semantic Indexing (LSI) clustering, and peak calling via ArchR. Crucially, we detail procedures for zebrafish-specific motif database construction, motif enrichment, and TF footprinting analysis. For complete details on the use and execution of this protocol, please refer to Lin et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

This study aimed to investigate the influence of sperm miRNAs on fertilization rates (FR) in in vitro fertilization (IVF) and to explore potential regulatory mechanisms in sperm-mediated fertilization and embryo development. Through high-throughput sequencing, we identified differentially expressed miRNAs in sperm, with miR-133a-3p significantly upregulated in samples associated with low FR and available embryo rate (AER). Key regulatory circRNAs and mRNAs were further identified via the Starbase database, intersected with differentially expressed RNA, and analyzed through GO, KEGG, and PPI analyses. The circMYH9/miR-133a-3p/CXCR4 axis emerged as a critical regulatory network. In vitro assays using the GC-2 spd mouse spermatogenic cell line revealed that miR-133a-3p inhibited cell growth and proliferation while promoting apoptosis. circMYH9, acting as a competing endogenous RNA (ceRNA) for miR-133a-3p, modulated CXCR4 expression, enhancing GC-2 spd cell growth and inhibiting apoptosis through the miR-133a-3p/CXCR4 axis. In vivo experiments using a mouse model confirmed that circMYH9 overexpression increased IVF success rates and promoted embryo development via this axis. Mechanistically, miR-133a-3p suppresses sperm fertilization and embryo development by targeting the circMYH9/miR-133a-3p/CXCR4 axis. These findings suggest that this regulatory network could serve as a novel biomarker for assessing fertilization potential and embryo quality in clinical settings and as a potential therapeutic target to improve IVF outcomes and address infertility. This study provides valuable insights into the molecular mechanisms governing sperm function and early embryonic development., Competing Interests: Declarations. Ethics approval and consent to participate: Consent for this study was granted by the Clinical Ethics Committee of Xuzhou Central Hospital (XZXY-LK-20230822–0147), and patients were informed and agreed to participate in adherence to the Helsinki Declaration guidelines. All experiments involving mice were approved by the Animal Ethics Committee of Xuzhou Medical University (No. 202302T006). Consent for publication: This work has not been published previously, and is approved by all authors for publication. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

In eukaryotes, defective mRNAs that impede the movement of the ribosome are subject to rapid decay via no-go decay (NGD). In this issue of PLOS Biology, Ishibashi and colleagues expand on the role of NGD and reveal new endogenous targets for the process in zebrafish., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Plastrik, Zaher. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Transcription factor lark has been demonstrated to play multiple functions in Drosophila, but the function of this gene in embryonic development remains to be elucidated., Results: In this study, the CRISPR/Cas9 gene-editing method was used to construct a Bmlark mutant strain of Bombyx mori to investigate the roles of this gene. The results showed that the homozygous mutant Bmlark -/- was lethal. The Bmlark -/- embryos showed obvious developmental defects, such as defective sclerotization and melanization of the exoskeleton. A transcriptomic comparison of Bmlark -/- and wild-type embryos showed that the differentially expressed genes were mainly enriched in the structure and metabolic processes of chitin and cuticles. While the expression levels of chitin metabolism-related enzyme genes did not significantly change, in the mutant embryos compared to the wild-type embryos, the expression levels of 63 putative cuticle protein genes showed significant differences. Among which, 35 genes were downregulated and 28 genes were upregulated. The expression levels of the transcription factor BmPOUM2 and eight wing disc cuticle protein genes (WCP) also changed. BmPOUM2, WCP5, WCP9, WCP10, WCP11 were downregulated and WCP1, WCP2, WCP3, WCP6 were upregulated in Bmlark -/- embryos. While the expression level of TH in the tyrosine-mediated pigmentation pathway was upregulated in the mutant embryos, the expression levels of the four key pigment synthesis genes DDC, aaNAT, Laccase2A, and yellow-f2 were significantly downregulated., Conclusions: The expression levels of 63 putative cuticle protein genes, eight WCP genes, and five pigment synthesis genes significantly changed in Bmlark mutant B. mori compared to those of the wildtype. These results suggest that Bmlark is essential for normal development of cuticle and tyrosine-mediated melanization in silkworm embryos., Competing Interests: Declarations. Ethics approval and consent to participate: Ethics approval is not applicable for experimental animals in this study. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Serotonin (5-HT), a versatile signaling molecule, plays a variety of roles in both neurotransmission and tissue regulation. The influence of serotonin on early development was first studied in marine invertebrate embryos and has since been documented in a variety of vertebrate species, including mammals. The present study investigates the expression and functional activity of serotonin components in mouse embryos, focusing on key receptors and transporters. Transcriptomic analyses revealed that mRNA transcripts related to serotonin show marked expression during the oogenesis and preimplantation stages. The results of the immunohistochemical studies show the presence of serotonin, the vesicular monoamine transporter VMAT2, and several membrane receptors (5-HT 1B , 5-HT 1D , 5-HT 2B , 5-HT 7 ) in the early stages of development. A functional analysis performed with the VMAT inhibitor reserpine revealed the crucial role of vesicular transport in the maintenance of serotonin signaling. The findings presented here support the hypothesis that serotonin plays a significant role in oocyte maturation and embryonic development, as well as in interblastomere interactions.

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., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

CMTR2 is an mRNA cap methyltransferase with poorly understood physiological functions. It catalyzes 2'-O-ribose methylation of the second transcribed nucleotide of mRNAs, potentially serving to mark RNAs as "self" to evade the cellular innate immune response. Here we analyze the consequences of Cmtr2 deficiency in mice. We discover that constitutive deletion of Cmtr2 results in mouse embryos that die during mid-gestation, exhibiting defects in embryo size, placental malformation and yolk sac vascularization. Endothelial cell deletion of Cmtr2 in mice results in vascular and hematopoietic defects, and perinatal lethality. Detailed characterization of the constitutive Cmtr2 KO phenotype shows an activation of the p53 pathway and decreased proliferation, but no evidence of interferon pathway activation. In summary, our study reveals the essential roles of Cmtr2 in mammalian cells beyond its immunoregulatory function., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

A largely stable genome is required for normal development, even as genetic change is an integral aspect of reproduction, genetic adaptation and evolution. Recent studies highlight a critical window of mammalian development with intrinsic DNA replication stress and genome instability in the first cell divisions after fertilization. Patterns of DNA replication and genome stability are established very early in mammals, alongside patterns of nuclear organization, and before the emergence of gene expression patterns, and prior to cell specification and germline formation. The study of DNA replication and genome stability in the mammalian embryo provides a unique cellular system due to the resetting of the epigenome to a totipotent state, and the de novo establishment of the patterns of nuclear organization, gene expression, DNA methylation, histone modifications and DNA replication. Studies on DNA replication and genome stability in the early mammalian embryo is relevant for understanding both normal and disease-causing genetic variation, and to uncover basic principles of genome regulation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The function of spargel/dPGC-1 in Drosophila oogenesis has been unequivocally established. Here, we sought to assess whether Spargel protein or RNA is essential for developmentally competent eggs. The trans-heterozygotic combination of two spargel mutant alleles allowed us to decrease Spargel expression to very low levels. Using this model, we now demonstrated the requirement for Spargel in eggshell patterning and embryonic development, which led us to establish that spargel is a maternal effect gene. Further examination of Spargel's potential mechanism of action in eggshell biogenesis revealed that low levels of Spargel in the adult ovary cause diminished Cyclin E activity, resulting in reduced chorion gene amplification levels, leading to eggshell biogenesis defects. Thus, another novel role for spargel/dPGC-1 is exposed whereby, through Cyclin E activity, this conserved transcriptional coactivator regulates the chorion gene amplification process., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

This study aimed to demonstrate the utilization value of 1PN embryos. The 1PN zygotes collected from December 2021 to September 2022 were included in this study. The embryo development, the pronuclear characteristics, and the genetic constitutions were investigated. The overall blastocyst formation and good-quality blastocyst rates in 1PN zygotes were 22.94 and 16.24%, significantly lower than those of 2PN zygotes (63.25 and 50.23%, respectively, P = 0.000). The pronuclear characteristics were found to be correlated with the developmental potential. When comparing 1PN zygotes that developed into blastocysts to those that arrested, the former exhibited a significantly larger area (749.49 ± 142.77 vs. 634.00 ± 119.05, P = 0.000), a longer diameter of pronuclear (29.81 ± 3.08 vs. 27.30 ± 3.00, P = 0.000), and a greater number of nucleolar precursor body (NPB) (11.56 ± 3.84 vs. 7.19 ± 2.73, P = 0.000). Among the tested embryos, the diploidy euploidy rate was significantly higher in blastocysts in comparison with the arrested embryos (66.67 vs. 11.76%, P = 0.000), which was also significantly higher in IVF-1PN blastocysts than in ICSI-1PN blastocysts (75.44 vs. 25.00%, P = 0.001). However, the pronuclear characteristics were not found to be linked to the chromosomal ploidy once they formed blastocysts.In summary, while the developmental potential of 1PN zygotes is reduced, our study shows that, in addition to the reported pronuclear area and diameter, the number of NPB is also associated with their developmental potential. The 1PN blastocysts exhibit a high diploidy euploidy rate, are recommend to be clinically used post genetic testing, especially for patients who do not have other 2PN embryos available.

LIF maintains self-renewal growth in mouse embryonic stem cells (mESC) by activating STAT3, which translocates into nucleus for pluripotent gene induction. However, the ERK signaling pathway activated by LIF at large counteract with pluripotent gene induction during self-renewal growth. Here, it is reported that in mESC STAT3 undergoes multivesicular endosomes (MVEs) translocation and subsequent secretion, LIF-activated STAT3 is acetylated on K177/180 and phosphorylated on Y293 residues within the N-terminal coiled-coil domain, which is responsible for the interaction between STAT3 and Secl5b, an exocyst complex component 6B (EXOC6B). STAT3 translocation into MVEs resulted in the downregulation of T202/Y204-ERK1/2 phosphorylation and up-regulation of S9-GSK3β phosphorylation for maintaining mESC self-renewal growth. STAT3 with K177R/K180R or Y293F substitution fails to execute MVEs translocation and Secl5b-dependent secretion. Mice expressing K177RK180R substitution (STAT3 mut/mut ) are partially embryonic lethal. In STAT3 mut/mut embryos, gene expressions related to hematological system function changed significantly and those living ones carry a series of abnormalities in the hematopoietic system. Furthermore, mice with Secl5b knockout exhibit embryonic lethality. Thus, Secl5b mediated STAT3 MVEs translocation regulates the balance of ERK and GSK3β signaling pathways and maintain mESC self-renewal growth, which is involved in regulating the stability of hematopoietic system., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Early embryonic arrest (EEA) is a critical impediment in assisted reproductive technology (ART), affecting 40% of infertile patients by halting the development of early embryos from the zygote to blastocyst stage, resulting in a lack of viable embryos for successful pregnancy. Despite its prevalence, the molecular mechanism underlying EEA remains elusive. This review synthesizes the latest research on the genetic and molecular factors contributing to EEA, with a focus on maternal, paternal, and embryonic factors. Maternal factors such as irregularities in follicular development and endometrial environment, along with mutations in genes like NLRP5, PADI6, KPNA7, IGF2, and TUBB8, have been implicated in EEA. Specifically, PATL2 mutations are hypothesized to disrupt the maternal-zygotic transition, impairing embryo development. Paternal contributions to EEA are linked to chromosomal variations, epigenetic modifications, and mutations in genes such as CFAP69, ACTL7A, and M1AP, which interfere with sperm development and lead to infertility. Aneuploidy may disrupt spindle assembly checkpoints and pathways including Wnt, MAPK, and Hippo signaling, thereby contributing to EEA. Additionally, key genes involved in embryonic genome activation-such as ZSCAN4, DUXB, DUXA, NANOGNB, DPPA4, GATA6, ARGFX, RBP7, and KLF5-alongside functional disruptions in epigenetic modifications, mitochondrial DNA, and small non-coding RNAs, play critical roles in the onset of EEA. This review provides a comprehensive understanding of the genetic and molecular underpinnings of EEA, offering a theoretical foundation for the diagnosis and potential therapeutic strategies aimed at improving pregnancy outcomes., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) play complex roles in liver health, influencing processes such as fibrosis, cancer development, and regeneration. WW domain binding protein-2 (WBP2) primarily enhances the co-translational activity of YAP/TAZ, which is crucial for the progression of liver diseases. Despite existing knowledge, the specific functions of WBP2 and its interactions with YAP remain inadequately understood. This study investigates the expression levels of WBP2 in zebrafish embryos and its molecular interaction with YAP. We employed morpholino-mediated knockdown of wbp2 and yap, followed by assessments of liver histology, immunofluorescence, and co-immunoprecipitation. Subsequently, RNA sequencing analyses were conducted to elucidate the signaling pathways and mechanisms underlying the interplay between YAP and WBP2 in liver injury. Our findings highlight the significant interaction between WBP2 and YAP, emphasizing their potential as therapeutic targets for liver diseases., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Alternative splicing (AS) is a pivotal posttranscriptional regulatory mechanism that is involved in embryonic development. However, the roles of AS in specific developmental events, especially the zygotic genome activation (ZGA) of porcine early embryos, remain unclear. In this study, we demonstrated that alternative splicing events (ASEs) were most prevalent in mammalian embryos during ZGA and that skipped exons were the predominant splicing pattern. When splicing factor 3B subunit 1 (SF3B1) was disrupted by the inhibitor pladienolide B (PlaB), we observed that porcine embryos were markedly arrested at the 4-cell stage. Concurrently, the main ZGA genes, namely, DPPA2, EIF6, and SORD, underwent aberrant splicing indicative of the failure of ZGA. Moreover, embryonic metabolic homeostasis was significantly disrupted at the 4-cell stage by SF3B1 inhibition, resulting in increases in the LDHA/LDHB ratio and lactate levels. Interestingly, the levels of the histone lactylation modifications pan-Kla and H4K5la also increased. Our findings enhance our understanding of early mammalian embryonic development, reveal the crucial role of porcine early embryogenesis, and help to resolve reproductive difficulties related to embryonic development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The human yolk sac (hYS) is essential for embryo nutrient biosynthesis/transport and development. However, there lacks a comprehensive study of hYS nutrient-gene interactions. Here we performed a secondary analysis of hYS transcript profiles (n = 9 samples) to identify nutrient-sensitive hYS genes and regulatory networks, including those that associate with adverse perinatal phenotypes with embryonic origins. Overall, 14.8% highly expressed hYS genes are nutrient-sensitive; the most common nutrient cofactors for hYS genes are metals and B vitamins. Functional analysis of highly expressed hYS genes reveals that nutrient-sensitive hYS genes are more likely to be involved in metabolic functions than hYS genes that are not nutrient-sensitive. Through nutrient-sensitive gene network analysis, we find that four nutrient-sensitive transcription regulators in the hYS (with zinc and/or magnesium cofactors) are predicted to collectively regulate 30.9% of highly expressed hYS genes. Lastly, we identify 117 nutrient-sensitive hYS genes that associate with an adverse perinatal outcome with embryonic origins. Among these, the greatest number of nutrient-sensitive hYS genes are linked to congenital heart defects (n = 54 genes), followed by microcephaly (n = 37). Collectively, our study characterises nutrient-sensitive hYS functions and improves understanding of the ways in which nutrient-gene interactions in the hYS may influence both typical and pathological development., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Poor-quality (PQ) embryos are commonly formed in assisted reproduction and often discarded, but emerging evidence suggests that some can develop into healthy children. Here, we performed single-embryo transcriptome profiling of day 3 human embryos of both good quality (GQ) and PQ. Our analyses revealed that day 3 PQ embryos can be categorized into two distinct subgroups: genuine PQ (gPQ) and morphological PQ (mPQ) embryos. This was further supported by additional transcriptomic data of GQ and PQ embryos donated by the same individuals. gPQ embryos showed greater impairments in both RNA decay and zygotic genome activation (ZGA) compared to mPQ embryos. Blastocysts that emerged from PQ embryos, more likely from mPQ embryos, demonstrated transcriptomic similarity to normal blastocysts. We also identified genes highly upregulated in day 3 gPQ embryos. These molecular insights may improve embryo selection and offer valuable clues for enhancing embryo quality in the future., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Forecasted increases in the prevalence and severity of extreme weather events accompanying changes in climatic behavior pose potential risk to the reproductive capacity of humans and animals of ecological and agricultural significance. While several studies have revealed that heat stress induced by challenges such as testicular insulation can elicit a marked negative effect on the male reproductive system, and particularly the production of spermatozoa, less is known about the immediate impact on male reproductive function following subchronic whole-body exposure to elevated ambient temperature. To address this knowledge gap, we exposed unrestrained male mice to heat stress conditions that emulate a heat wave (daily cycle of 8 h at 35 °C followed by 16 h at 25 °C) for a period of 7 d. Neither the testes or epididymides of heat-exposed male mice exhibited evidence of gross histological change, and similarly, spermatozoa of exposed males retained their functionality and ability to support embryonic development. However, the embryos generated from heat-exposed spermatozoa experienced pronounced changes in gene expression linked to acceleration of early embryo development, aberrant blastocyst hatching, and increased fetal:placental weight ratio. Such changes were causally associated with an altered sperm small noncoding RNA (sncRNA) profile, such that these developmental phenotypes were recapitulated by microinjection of wild-type embryos sired by control spermatozoa with RNAs extracted from heat-exposed spermatozoa. Such data highlight that even relatively modest excursions in ambient temperature can affect male reproductive function and identify the sperm sncRNA profile as a particular point of vulnerability to this imposed environmental stress., Competing Interests: Competing interests statement:The authors declare no competing interest.

Cellular proteins and the mRNAs that encode them are key factors in oocyte and sperm development, and the mechanisms that regulate their translation and degradation play an important role during early embryogenesis. There is abundant evidence that expression of microRNAs (miRNAs) is crucial for embryo development and are highly involved in regulating translation during oocyte and early embryo development. MiRNAs are a group of short (18-24 nucleotides) non-coding RNA molecules that regulate post-transcriptional gene silencing. The miRNAs are secreted outside the cell by embryos during preimplantation embryo development. Understanding regulatory mechanisms involving miRNAs during gametogenesis and embryogenesis will provide insights into molecular pathways active during gamete formation and early embryo development. This review summarizes recent findings regarding multiple roles of miRNAs in molecular signaling, plus their transport during gametogenesis and embryo preimplantation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Understanding how multicellular organisms reliably orchestrate cell-fate decisions is a central challenge in developmental biology, particularly in early mammalian development, where tissue-level differentiation arises from seemingly cell-autonomous mechanisms. In this study, we present a multi-scale, spatial-stochastic simulation framework for mouse embryogenesis, focusing on inner cell mass (ICM) differentiation into epiblast (EPI) and primitive endoderm (PRE) at the blastocyst stage. Our framework models key regulatory and tissue-scale interactions in a biophysically realistic fashion, capturing the inherent stochasticity of intracellular gene expression and intercellular signaling, while efficiently simulating these processes by advancing event-driven simulation techniques. Leveraging the power of Simulation-Based Inference (SBI) through the AI-driven Sequential Neural Posterior Estimation (SNPE) algorithm, we conduct a large-scale Bayesian inferential analysis to identify parameter sets that faithfully reproduce experimentally observed features of ICM specification. Our results reveal mechanistic insights into how the combined action of autocrine and paracrine FGF4 signaling coordinates stochastic gene expression at the cellular scale to achieve robust and reproducible ICM patterning at the tissue scale. We further demonstrate that the ICM exhibits a specific time window of sensitivity to exogenous FGF4, enabling lineage proportions to be adjusted based on timing and dosage, thereby extending current experimental findings and providing quantitative predictions for both mutant and wild-type ICM systems. Notably, FGF4 signaling not only ensures correct EPI-PRE lineage proportions but also enhances ICM resilience to perturbations, reducing fate-proportioning errors by 10-20% compared to a purely cell-autonomous system. Additionally, we uncover a surprising role for variability in intracellular initial conditions, showing that high gene-expression heterogeneity can improve both the accuracy and precision of cell-fate proportioning, which remains robust when fewer than 25% of the ICM population experiences perturbed initial conditions. Our work offers a comprehensive, spatial-stochastic description of the biochemical processes driving ICM differentiation and identifies the necessary conditions for its robust unfolding. It also provides a framework for future exploration of similar spatial-stochastic systems in developmental biology., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ramirez Sierra, Sokolowski. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Establishment of the 3 primordial germ layers (ectoderm, endoderm, and mesoderm) during early animal development represents an essential prerequisite for the emergence of properly patterned embryos. β-catenin is an ancient protein that is known to play essential roles in this process. However, these roles have chiefly been established through inhibition of β-catenin translation or function at the time of fertilization. Comprehensive analyses reporting the totality of functions played by nuclear β-catenin during early embryogenesis of a given animal, i.e., at different developmental stages and in different germ layers, are thus still lacking. In this study, we used an inducible, conditional knockdown system in the sea urchin to characterize all possible requirements of β-catenin for germ layer establishment and patterning. By blocking β-catenin protein production starting at 7 different time points of early development, between fertilization and 12 h post fertilization, we established a clear correlation between the position of a germ layer along the primary embryonic axis (the animal-vegetal axis) and its dependence on nuclear β-catenin activity. For example, in the vegetal hemisphere, we determined that the 3 germ layers (skeletogenic mesoderm, non-skeletogenic mesoderm, and endoderm) require distinct and highly specific durations of β-catenin production for their respective specification, with the most vegetal germ layer, the skeletogenic mesoderm, requiring the shortest duration. Likewise, for the 2 animal territories (ectoderm and anterior neuroectoderm), we established that their restriction, along the animal-vegetal axis, relies on different durations of β-catenin production and that the longest duration is required for the most animal territory, the anterior neuroectoderm. Moreover, we found that 2 of the vegetal germ layers, the non-skeletogenic mesoderm and the endoderm, further require a prolonged period of nuclear β-catenin activity after their specification to maintain their respective germ layer identities through time. Finally, we determined that restriction of the anterior neuroectoderm territory depends on at least 2 nuclear β-catenin-dependent inputs and a nuclear β-catenin-independent mechanism. Taken together, this work is the first to comprehensively define the spatiotemporal requirements of β-catenin during the early embryogenesis of a single animal, the sea urchin Paracentrotus lividus, thereby providing new experimental evidence for a better understanding of the roles played by this evolutionary conserved protein during animal development., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Lhomond et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The expression of a few key genes determines the body plan of the fruit fly. We show that the spatial expression patterns for several of these genes scale precisely with embryo size. Discrete positional markers such as the peaks in striped patterns or the boundaries of expression domains have positions along the embryo's major axis proportional to embryo length, accurate to within 1%. Further, the information (in bits) that graded patterns of expression provide about a cell's position can be decomposed into information about fractional or scaled position and information about absolute position or embryo length; all information available is about scaled position, with [Formula: see text]2% error. These findings imply that the underlying genetic network's behavior exhibits scale invariance in a more precise mathematical sense. We argue that models that can explain this scale invariance also have a "zero mode" in the dynamics of gene expression, and this connects to observations on the spatial correlation of fluctuations in expression levels., Competing Interests: Competing interests statement:The authors declare no competing interest.

Mammalian preimplantation development culminates in the formation of a blastocyst that undergoes extensive gene expression regulation to successfully implant into the maternal endometrium. Zinc-finger HIT domain-containing (ZNHIT) 1 and 2 are members of a highly conserved family, yet they have been identified as subunits of distinct complexes. Here, we report that knockout of either Znhit1 or Znhit2 results in embryonic lethality during peri-implantation stages. Znhit1 and Znhit2 mutant embryos have overlapping phenotypes, including reduced proportion of SOX2-positive inner cell mass cells, a lack of Fgf4 expression, and aberrant expression of NANOG and SOX17. Furthermore, we find that the similar phenotypes are caused by distinct mechanisms. Specifically, embryos lacking ZNHIT1 likely fail to incorporate sufficient H2A.Z at the promoter region of Fgf4 and other genes involved in cell projection organization resulting in impaired invasion of trophoblast cells during implantation. In contrast, Znhit2 mutant embryos display a complete lack of nuclear EFTUD2, a key component of U5 spliceosome, indicating a global splicing deficiency. Our findings unveil the indispensable yet distinct roles of ZNHIT1 and ZNHIT2 in early mammalian embryonic development., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

More and more studies have demonstrated that pseudogenes possess coding ability, and the functions of their transcripts in the development of diseases have been partially revealed. However, the role of pseudogenes in maintenance of normal physiological states and life activities has long been neglected. Here, we identify pseudogenes that are dynamically expressed during human early embryogenesis, showing different expression patterns from that of adult tissues. We explore the expression correlation between pseudogenes and the parent genes, partly due to their shared gene regulatory elements or the potential regulation network between them. The essential role of three pseudogenes, PI4KAP1, TMED10P1, and FBXW4P1, in maintaining self-renewal of human embryonic stem cells is demonstrated. We further find that the three pseudogenes might perform their regulatory functions by binding to proteins or microRNAs. The pseudogene-related single-nucleotide polymorphisms are significantly associated with human congenital disease, further illustrating their importance during early embryonic development. Overall, this study is an excavation and exploration of functional pseudogenes during early human embryonic development, suggesting that pseudogenes are not only capable of being specifically activated in pathological states, but also play crucial roles in the maintenance of normal physiological states., Competing Interests: Conflict of interest The authors declare that no competing interests exist., (Copyright © 2024 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)

Embryogenesis entails dramatic shifts in mRNA translation and turnover that reprogram gene expression during cellular proliferation and differentiation. Codon identity modulates mRNA stability during early vertebrate embryogenesis, but how the composition of tRNA pools is matched to translational demand is unknown. By quantitative profiling of tRNA repertoires in zebrafish embryos during the maternal-to-zygotic transition, we show that zygotic tRNA repertoires are established after the onset of gastrulation, succeeding the major wave of zygotic mRNA transcription. Maternal and zygotic tRNA pools are distinct, but their reprogramming does not result in a better match to the codon content of the zygotic transcriptome. Instead, we find that an increase in global translation at gastrulation sensitizes decoding rates to tRNA supply, thus destabilizing maternal mRNAs enriched in slowly translated codons. Translational activation and zygotic tRNA expression temporally coincide with an increase of TORC1 activity at gastrulation, which phosphorylates and inactivates the RNA polymerase III repressor Maf1a/b. Our data indicate that a switch in global translation, rather than tRNA reprogramming, determines the onset of codon-dependent maternal mRNA decay during zebrafish embryogenesis., Competing Interests: Disclosure and competing interests statement AB and DDN are inventors on a patent application filed by the Max Planck Society pertaining to the mim-tRNAseq technology. The other authors declare no competing interests., (© 2024. The Author(s).)

The growing variety of RNA classes, such as mRNAs, lncRNAs, and circRNAs, plays pivotal roles in both developmental processes and various pathophysiological conditions. Nonetheless, our comprehension of RNA functions in live organisms remains limited due to the absence of durable and effective strategies for directly influencing RNA levels. In this study, we combined the CRISPR-RfxCas13d system with sperm-like stem cell-mediated semi-cloning techniques, which enabled the suppressed expression of different RNA species. This approach was employed to interfere with the expression of three types of RNA molecules: Sfmbt2 mRNA, Fendrr lncRNA, and circMan1a2(2,3,4,5,6). The results confirmed the critical roles of these RNAs in embryonic development, as their loss led to observable phenotypes, including embryonic lethality, delayed embryonic development, and embryo resorption. In summary, our methodology offers a potent toolkit for silencing specific RNA targets in living organisms without introducing genetic alterations., (© 2024. Science China Press.)

Human embryonic bone and joint formation is determined by coordinated differentiation of progenitors in the nascent skeleton. The cell states, epigenetic processes and key regulatory factors that underlie lineage commitment of these cells remain elusive. Here we applied paired transcriptional and epigenetic profiling of approximately 336,000 nucleus droplets and spatial transcriptomics to establish a multi-omic atlas of human embryonic joint and cranium development between 5 and 11 weeks after conception. Using combined modelling of transcriptional and epigenetic data, we characterized regionally distinct limb and cranial osteoprogenitor trajectories across the embryonic skeleton and further described regulatory networks that govern intramembranous and endochondral ossification. Spatial localization of cell clusters in our in situ sequencing data using a new tool, ISS-Patcher, revealed mechanisms of progenitor zonation during bone and joint formation. Through trajectory analysis, we predicted potential non-canonical cellular origins for human chondrocytes from Schwann cells. We also introduce SNP2Cell, a tool to link cell-type-specific regulatory networks to polygenic traits such as osteoarthritis. Using osteolineage trajectories characterized here, we simulated in silico perturbations of genes that cause monogenic craniosynostosis and implicate potential cell states and disease mechanisms. This work forms a detailed and dynamic regulatory atlas of bone and cartilage maturation and advances our fundamental understanding of cell-fate determination in human skeletal development., Competing Interests: Competing interests: C.D.B. is a founder of Mestag Therapeutics. S.A.T. is a scientific advisory board member of ForeSite Labs, OMass Therapeutics, Qiagen, a co-founder and equity holder of TransitionBio and EnsoCell Therapeutics, a non-executive director of 10x Genomics and a part-time employee of GlaxoSmithKline. Sarah also holds equity in TransitionBio and options for equity in Element Biosciences, where she is a former SAB member.  All other authors declare no competing interests., (© 2024. The Author(s).)

Purpose: The prevalence of infertility in Canada has substantially increased over 30 years, and plateaued success rates of culture systems warrant further optimization for transfer outcomes. In clinical programs, embryos commonly undergo extended culture under 5% O 2 until the blastocyst stage. The aim of this study is to characterize the developmental competence and stress-related responses of embryos cultured under 5 versus 2% O 2 in comparison to in vivo-derived blastocysts. We hypothesized 2% O 2 compromises developmental competence through altered embryonic stress responses and induction of apoptosis-related genes relative to those cultured under 5% O 2 and in vivo-derived blastocysts., Methods: Quantitative measures of development and relative expressions of a cohort of stress-related genes in CD1 mouse zygotes cultured to blastocysts under 5 or 2% O 2 were compared to in vivo-derived embryos. Apoptotic responses were evaluated using an immunofluorescence assay for Caspase-3., Results: The mean percentage of blastocysts developed, and total cell number of embryos derived in vivo or cultured under 5% O 2 was significantly higher than those cultured under 2% O 2 . Blastocyst expansion was greatest in embryos cultured under 5% O 2 . Stress response genes were significantly upregulated in embryos cultured under 2% O 2 , and expression of antioxidant-related genes was significantly lower in cultured versus in vivo-derived embryos. Caspase-3 immunofluorescence was significantly higher in cultured embryos versus in vivo-derived embryos., Conclusion: We inferred that 5% O 2 systems better approximate physiologic oxygen availability for culture of mouse embryos, warranting re-evaluation of culturing embryos under threshold or sub-physiologic oxygen concentrations during clinical IVF programs., Competing Interests: Declarations. Conflict of interest: The authors of this manuscript have no conflicts of interest to disclose. Ethics approval was issued by the University of Calgary Health Sciences Animal Care Committee for the project #AC18-0084 in accordance with the Canadian Council on Animal Care guidelines originally approved on June 29, 2018., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The maternal-to-zygotic transition is crucial in embryonic development, marked by the degradation of maternally provided mRNAs and initiation of zygotic gene expression. However, the changes occurring at the protein level during this transition remain unclear. Here, we conducted protein profiling throughout zebrafish embryogenesis using quantitative mass spectrometry, integrating transcriptomics and translatomics datasets. Our data show that, unlike RNA changes, protein changes are less dynamic. Further, increases in protein levels correlate with mRNA translation, whereas declines in protein levels do not, suggesting active protein degradation processes. Interestingly, proteins from pure zygotic genes are present at fertilization, challenging existing mRNA-based gene classifications. As a proof of concept, we utilized CRISPR-Cas13d to target znf281b mRNA, a gene whose protein significantly accumulates within the first 2 h post-fertilization, demonstrating its crucial role in development. Consequently, our protein profiling, coupled with CRISPR-Cas13d, offers a complementary approach to unraveling maternal factor function during embryonic development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The early stages of embryonic development rely on maternal products for proper regulation. However, screening for functional maternal-specific factors is challenging due to the time- and labor-intensive nature of traditional approaches. Here, we combine a computational pipeline and F0 null mutant technology to screen for functional maternal-specific chromatin regulators in zebrafish embryogenesis and identify Mcm3l, Mcm6l, and Npm2a as playing essential roles in DNA replication and cell division. Our results contribute to understanding the molecular mechanisms underlying early embryo development and highlight the importance of maternal-specific chromatin regulators in this critical stage., (© 2024. The Author(s).)

The most common epigenetic modification of messenger RNAs (mRNAs) is N6-methyladenosine (m6A), which is mainly located near the 3' untranslated region of mRNAs, near the stop codons, and within internal exons. The biological effect of m6A is dynamically modulated by methyltransferases (writers), demethylases (erasers), and m6A-binding proteins (readers). By controlling post-transcriptional gene expression, m6A has a significant impact on numerous biological functions, including RNA transcription, translation, splicing, transport, and degradation. Hence, m6A influences various physiological and pathological processes, such as spermatogenesis, oogenesis, embryogenesis, placental function, and human reproductive system diseases. During gametogenesis and embryogenesis, genetic material undergoes significant changes, including epigenomic modifications such as m6A. From spermatogenesis and oogenesis to the formation of an oosperm and early embryogenesis, m6A changes occur at every step. m6A abnormalities can lead to gamete abnormalities, developmental delays, impaired fertilization, and maternal-to-zygotic transition blockage. Both mice and humans with abnormal m6A modifications exhibit impaired fertility. In this review, we discuss the dynamic biological effects of m6A and its regulators on gamete and embryonic development and review the possible mechanisms of infertility caused by m6A changes. We also discuss the drugs currently used to manipulate m6A and provide prospects for the prevention and treatment of infertility at the epigenetic level., (© The Author(s) 2024. Published by Oxford University Press and Science Press on behalf of the Beijing Institute of Genomics, Chinese Academy of Sciences / China National Center for Bioinformation and Genetics Society of China.)

Background: Juvenile hormone (JH) is an insect-exclusive hormone involved in regulating diverse aspects of insect physiology, and the evolution of its diverse function is widely interesting. Studying embryogenesis in basal wingless insects is important to understand the functional evolution of JH; however, experimental studies in this regard are scarce. In this study, we conducted CRISPR/Cas9-mediated knockout (KO) of genes involved in JH biosynthesis and signaling cascades in the ametabolous firebrat, Thermobia domestica. Additionally, we investigated whether the primitive action of JH is conserved in the hemimetabolous cricket, Gryllus bimaculatus., Results: We observed that KO of JHAMT, CYP15A1, Met, and Kr-h1 resulted in embryonic lethality in T. domestica. Deprivation of JH or JH signaling arrested the progression of extraembryonic fluid resorption after dorsal closure and hatching, which is consistent with the gene expression pattern showing high Kr-h1 expression in the late embryos of T. domestica. The embryos deficient in JH signaling displayed wrinkled and weak legs. Comparative transcriptome analysis revealed that JH signaling promotes embryonic leg maturation through inducing energy supply and muscle activity, as validated by transmission electron microscopy (TEM). In addition, JH signaling exhibited similar embryonic effects in G. bimaculatus., Conclusions: This study reveals the indispensable role of JH signaling in facilitating the maturation of terminal tissues during late embryogenesis, as demonstrated by the regulation of leg development, in ametabolous and hemimetabolous insects. These findings further indicate that the embryonic functions of JH evolved earlier than its anti-metamorphic functions during postembryonic development., (© 2024. The Author(s).)

The Wnt/β-catenin signaling pathway is crucial for embryonic development and adult tissue homeostasis. Dysregulation of Wnt signaling is linked to various developmental anomalies and diseases, notably cancer. Although numerous regulators of the Wnt signaling pathway have been identified, their precise function during mouse embryo development remains unclear. Here, we revealed that TMEM132A is a crucial regulator of canonical Wnt/β-catenin signaling in mouse development. Mouse embryos lacking Tmem132a displayed a range of malformations, including open spina bifida, caudal truncation, syndactyly, and renal defects, similar to the phenotypes of Wnt/β-catenin mutants. Tmem132a knockdown in cultured cells suppressed canonical Wnt/β-catenin signaling. In developing mice, loss of Tmem132a also led to diminished Wnt/β-catenin signaling. Mechanistically, we showed that TMEM132A interacts with the Wnt co-receptor LRP6, thereby stabilizing it and preventing its lysosomal degradation. These findings shed light on a novel role for TMEM132A in regulating LRP6 stability and canonical Wnt/β-catenin signaling during mouse embryo development. This study provides valuable insights into the molecular intricacies of the Wnt signaling pathway. Further research may deepen our understanding of Wnt pathway regulation and offer its potential therapeutic applications., (© 2024. The Author(s).)

The structural organization of eukaryotic genomes is contingent upon the fractionation of DNA into transcriptionally permissive euchromatin and repressive heterochromatin. However, we have a limited understanding of how these distinct states are first established during animal embryogenesis. Histone 3 lysine 9 trimethylation (H3K9me3) is critical to heterochromatin formation, and bulk establishment of this mark is thought to help drive large-scale remodeling of an initially naive chromatin state during animal embryogenesis. However, a detailed understanding of this process is lacking. Here, we leverage CUT&RUN to define the emerging H3K9me3 landscape of the zebrafish embryo with high sensitivity and temporal resolution. Despite the prevalence of DNA transposons in the zebrafish genome, we found that LTR transposons are preferentially targeted for embryonic H3K9me3 deposition, with different families exhibiting distinct establishment timelines. High signal-to-noise ratios afforded by CUT&RUN revealed new, emerging sites of low-amplitude H3K9me3 that initiated before the major wave of zygotic genome activation (ZGA). Early sites of establishment predominated at specific subsets of transposons and were particularly enriched for transposon sequences with maternal piRNAs and pericentromeric localization. Notably, the number of H3K9me3 enriched sites increased linearly across blastula development, while quantitative comparison revealed a >10-fold genome-wide increase in H3K9me3 signal at established sites over just 30 min at the onset of major ZGA. Continued maturation of the H3K9me3 landscape was observed beyond the initial wave of bulk establishment., Competing Interests: Conflicts of interest The author(s) declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)