Embryo Patterning Research Articles

Rescue Potential of Supportive Embryo Culture Conditions on Bovine Embryos Derived from Metabolically Compromised Oocytes.

Elevated non-esterified fatty acid (NEFA), predominantly palmitic acid (PA), concentrations in blood and follicular fluid are a common feature in maternal metabolic disorders such as obesity. This has a direct negative impact on oocyte developmental competence and the resulting blastocyst quality. We use NEFA-exposure during bovine oocyte in vitro maturation (IVM) as a model to mimic oocyte maturation under maternal metabolic stress conditions. However, the impact of supportive embryo culture conditions on these metabolically compromised zygotes are not known yet. We investigated if the addition of anti-apoptotic, antioxidative and mitogenic factors (namely, Insulin-Transferrin-Selenium (ITS) or serum) to embryo culture media would rescue development and important embryo quality parameters (cell proliferation, apoptosis, cellular metabolism and gene expression patterns) of bovine embryos derived from high PA- or high NEFA-exposed oocytes when compared to controls (exposed to basal NEFA concentrations). ITS supplementation during in vitro culture of PA-exposed oocytes supported the development of lower quality embryos during earlier development. However, surviving blastocysts were of inferior quality. In contrast, addition of serum to the culture medium did not improve developmental competence of PA-exposed oocytes. Furthermore, surviving embryos displayed higher apoptotic cell indices and an aberrant cellular metabolism. We conclude that some supportive embryo culture supplements like ITS and serum may increase IVF success rates of metabolically compromised oocytes but this may increase the risk of reduced embryo quality and may thus have other long-term consequences.

Effect of Rhodiola sachalinensis Aqueous Extract on In Vitro Maturation of Porcine Oocytes and Subsequent In Vitro Embryonic Development.

Oxidative stress can impede maturation of the nucleus and cytoplasm of oocytes during in vitro maturation (IVM). Rhodiola sachalinensis, an herb commonly used in traditional Chinese medicine, conveys antioxidative effects to cryopreserved bovine sperm. Therefore, the aims of this study were to evaluate the effects of different concentrations of R. sachalinensis aqueous extract (RSAE) on IVM and subsequent in vitro embryonic development after parthenogenetic activation (PA), in vitro fertilization (IVF), and somatic cell nuclear transfer (SCNT). The results showed that RSAE supplementation (6 and 60 mg/L) significantly increased intracellular glutathione levels, but had no effect on maturation rates or reactive oxygen species. After in vitro culture, greater blastocyst formation was observed in PA embryos (6 mg/L RSAE), as well as in IVF and SCNT embryos (60 mg/L) matured in RSAE-supplemented IVM media. In conclusion, although there was no significant improvement in the maturation rate, RSAE supplementation conveyed an antioxidative effect during IVM, and improved subsequent embryonic development in vitro. Further studies are needed to explore gene expression pattern in oocytes and embryos treated with RSAE.

Characterization of DNA Methylation Patterns and Mining of Epigenetic Markers During Genomic Reprogramming in SCNT Embryos.

Somatic cell nuclear transfer (SCNT), also known as somatic cell cloning, is a commonly used technique to study epigenetic reprogramming. Although SCNT has the advantages of being safe and able to obtain pluripotent cells, early developmental arrest happens in most SCNT embryos. Overcoming epigenetic barriers is currently the primary strategy for improving reprogramming efficiency and improving developmental rate in SCNT embryos. In this study, we analyzed DNA methylation profiles of in vivo fertilized embryos and SCNT embryos with different developmental fates. Overall DNA methylation level was higher in SCNT embryos during global de-methylation process compared to in vivo fertilized embryos. In addition, promoter region, first intron and 3′UTR were found to be the major genomic regions that were hyper-methylated in SCNT embryos. Surprisingly, we found the length of re-methylated region was directly related to the change of methylation level. Furthermore, a number of genes including Dppa2 and Dppa4 which are important for early zygotic genome activation (ZGA) were not properly activated in SCNT embryos. This study comprehensively analyzed genome-wide DNA methylation patterns in SCNT embryos and provided candidate target genes for improving efficiency of genomic reprogramming in SCNT embryos. Since SCNT technology has been widely used in agricultural and pastoral production, protection of endangered animals, and therapeutic cloning, the findings of this study have significant importance for all these fields.

SARS-CoV-2 Entry Factors: ACE2 and TMPRSS2 Are Expressed in Peri-Implantation Embryos and the Maternal-Fetal Interface.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread throughout the world, leading to large-scale population infection. Angiotensin-converting enzyme 2 (ACE2) is the receptor of both severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. However, it is still controversial whether vertical transmission exists. In order to investigate the potential risk of SARS-CoV-2 vertical transmission, we explored ACE2 and TMPRSS2 (encoding transmembrane protease serine 2) expression patterns in peri-implantation embryos and the maternal–fetal interface using previously published single-cell transcriptome data. The results showed that day 6 (D6) trophectoderm (TE) cells in peri-implantation embryos, as well as syncytiotrophoblast (STB) at 8 weeks of gestation (STB_8W) and extravillous trophoblast (EVT) cells at 24 weeks of gestation (EVT_24W) in the maternal–fetal interface, strongly co-expressed ACE2 and TMPRSS2, indicating a SARS-CoV-2 infection susceptibility. The ACE2 positive-expressing cells in the three cell types mentioned above were found to share common characteristics, which were involved in autophagy and immune-related processes. ACE2 showed no gender bias in post-implantation embryos but showed a significant gender difference in D6_TE, D6 primitive endoderm (PE) cells, and ACE2 positive-expressing STBs. These findings suggest that there may be different SARS-CoV-2 infection susceptibilities of D6 embryos of different genders and during the gestation of different genders. Our results reveal potential SARS-CoV-2 infection risks during embryo transfer, peri-implantation embryo development, and gestation.

Optogenetic Rescue of a Patterning Mutant

Animal embryos are patterned by a handful of highly conserved inductive signals. Yet, in most cases, it is unknown which pattern features (i.e., spatial gradients or temporal dynamics) are required to support normal development. An ideal experiment to address this question would be to "paint" arbitrary synthetic signaling patterns on "blank canvas" embryos to dissect their requirements. Here, we demonstrate exactly this capability by combining optogenetic control of Ras/extracellular signal-related kinase (ERK) signaling with the genetic loss of the receptor tyrosine-kinase-driven terminal signaling patterning in early Drosophila embryos. Blue-light illumination at the embryonic termini for 90min was sufficient to rescue normal development, generating viable larvae and fertile adults from an otherwise lethal terminal signaling mutant. Optogenetic rescue was possible even using a simple, all-or-none light input that reduced the gradient of Erk activity and eliminated spatiotemporal differences in terminal gap gene expression. Systematically varying illumination parameters further revealed that at least three distinct developmental programs are triggered at different signaling thresholds and that the morphogenetic movements of gastrulation are robust to a 3-fold variation in the posterior pattern width. These results open the door to controlling tissue organization with simple optical stimuli, providing new tools to probe natural developmental processes, create synthetic tissues with defined organization, or directly correct the patterning errors that underlie developmental defects.

Analysis of accessible chromatin landscape in the inner cell mass and trophectoderm of human blastocysts

Early embryonic development is characterized by drastic changes in chromatin structure that affects the accessibility of the chromatin. In human, the chromosome reorganization and its involvement in the first linage segregation are poorly characterized due to the difficulties in obtaining human embryonic material and limitation on low input technologies. In this study, we aimed to explore the chromatin remodeling pattern in human preimplantation embryos and gain insight into the epigenetic regulation of inner cell mass (ICM) and trophectoderm (TE) differentiation. We optimized ATAC-seq (an assay for transposase-accessible chromatin using sequencing) to analyze the chromatin accessibility landscape for low DNA input. Sixteen preimplantation human blastocysts frozen on Day 6 were used. Our data showed that ATAC peak distributions of the promoter regions (<1 kb) and distal regions versus other regions were significantly different between ICM versus TE samples (P < 0.01). We detected that a higher percentage of accessible binding loci were located within 1 kb of the transcription start site in ICM compared to TE (P < 0.01). However, a higher percentage of accessible regions was detected in the distal region of TE compared to ICM (P < 0.01). In addition, eight differential peaks with a false discovery rate <0.05 between ICM and TE were detected. This is the first study to compare the landscape of the accessible chromatin between ICM and TE of human preimplantation embryos, which unveiled chromatin-level epigenetic regulation of cell lineage specification in early embryo development.

Transcriptomes shed light on transgenerational and developmental effects of ocean warming on embryos of the sea urchin Strongylocentrotus intermedius

Ocean warming increasingly endangers the fitness of marine invertebrates. Transgenerational effects (TE) potentially mitigate the impacts of environmental stress on the embryos of marine invertebrates. The molecular mechanisms, however, remain largely unknown. Using high-throughput RNA sequencing technology, we investigated the gene expression patterns of embryos (the gastrula stage) of the sea urchin Strongylocentrotus intermedius at different developmental temperatures, whose parents were exposed to long-term (15 months) elevated temperature (A) or not (B). The temperatures at which adults were held for ~4 weeks prior to the start of the experiment (21 °C for A and 18 °C for B) were also used for the development of offspring (high: 21 °C and ambient (laboratory): 18 °C) resulting in four experimental groups (HA and HB at 21 °C, and LA and LB at 18 °C). The embryos were sampled ~24 h after fertilization. All samples were in the gastrula stage. Twelve mRNA libraries (groups HA, HB, LA, LB, 3 replicates for each group) were established for the following sequencing. Embryos whose parents were exposed to elevated temperatures or not showed 1891 significantly different DEGs (differentially expressed genes) at the ambient developmental temperature (LB vs LA, LB as control) and 2203 significantly different DEGs at the high developmental temperature (HB vs HA, HB as control), respectively. This result indicates complex molecular mechanisms of transgenerational effects of ocean warming, in which a large number of genes are involved. With the TE, we found 904 shared DEGs in both LB vs LA (LB as control) and HB vs HA (HB as control) changed in the same direction of expression (i.e., up- or down-regulated), indicating that parental exposed temperatures affect the expression of these genes in the same manner regardless of the development temperature. With developmental exposure, we found 198 shared DEGs in both HB vs LB (HB as control) and HA vs LA (HA as control) changed in the same direction of expression. Of the 198 DEGs, more genes were up-regulated at high developmental temperature. Interestingly, embryos whose parents were exposed to high temperature showed fewer differently expressed DEGs between high and low developmental temperatures than the individuals whose parents were exposed to ambient temperature. The results indicate that gene expressions are probably depressed by the transgenerational effect of ocean warming. The roles of hsp70 and hnf6 in thermal acclimation are highlighted for future studies. The present study provides new insights into the molecular mechanisms of the transgenerational and developmental effects of ocean warming on the embryos of sea urchins.

Mitochondrial ribosomal protein S9M is involved in male gametogenesis and seed development in Arabidopsis.

Mitochondrial function is critical for cell vitality in all eukaryotes including plants. Although plant mitochondria contain many proteins, few have been studied in the context of plant development and physiology. We used knock-down mutant RPS9M to study its important role in male gametogenesis and seed development in Arabidopsis thaliana. Knock-down of RPS9M in the rps9m-3 mutant led to abnormal pollen development and impaired pollen tube growth. In addition, both embryo and endosperm development were affected. Phenotype analysis revealed that the rps9m-3 mutant contained a lower amount of endosperm and nuclear proteins, and both embryo cell division and embryo pattern were affected, resulting in an abnormal and defective embryo. Lowering the level of RPS9M in rps9m-3 affects mitochondrial ribosome biogenesis, energy metabolism and production of ROS. Our data revealed that RPS9M plays important roles in normal gametophyte development and seed formation, possibly by sustaining mitochondrial function.

Intracellular Communication among Morphogen Signaling Pathways during Vertebrate Body Plan Formation.

During embryonic development in vertebrates, morphogens play an important role in cell fate determination and morphogenesis. Bone morphogenetic proteins (BMPs) belonging to the transforming growth factor-β (TGF-β) family control the dorsal–ventral (DV) patterning of embryos, whereas other morphogens such as fibroblast growth factor (FGF), Wnt family members, and retinoic acid (RA) regulate the formation of the anterior–posterior (AP) axis. Activation of morphogen signaling results in changes in the expression of target genes including transcription factors that direct cell fate along the body axes. To ensure the correct establishment of the body plan, the processes of DV and AP axis formation must be linked and coordinately regulated by a fine-tuning of morphogen signaling. In this review, we focus on the interplay of various intracellular regulatory mechanisms and discuss how communication among morphogen signaling pathways modulates body axis formation in vertebrate embryos.

Finding cell-specific expression patterns in the early Ciona embryo with single-cell RNA-seq

Single-cell RNA-seq has been established as a reliable and accessible technique enabling new types of analyses, such as identifying cell types and studying spatial and temporal gene expression variation and change at single-cell resolution. Recently, single-cell RNA-seq has been applied to developing embryos, which offers great potential for finding and characterising genes controlling the course of development along with their expression patterns. In this study, we applied single-cell RNA-seq to the 16-cell stage of the Ciona embryo, a marine chordate and performed a computational search for cell-specific gene expression patterns. We recovered many known expression patterns from our single-cell RNA-seq data and despite extensive previous screens, we succeeded in finding new cell-specific patterns, which we validated by in situ and single-cell qPCR.

Caenorhabditis elegans Gastrulation: A Model for Understanding How Cells Polarize, Change Shape, and Journey Toward the Center of an Embryo.

Gastrulation is fundamental to the development of multicellular animals. Along with neurulation, gastrulation is one of the major processes of morphogenesis in which cells or whole tissues move from the surface of an embryo to its interior. Cell internalization mechanisms that have been discovered to date in Caenorhabditis elegans gastrulation bear some similarity to internalization mechanisms of other systems including Drosophila, Xenopus, and mouse, suggesting that ancient and conserved mechanisms internalize cells in diverse organisms. C. elegans gastrulation occurs at an early stage, beginning when the embryo is composed of just 26 cells, suggesting some promise for connecting the rich array of developmental mechanisms that establish polarity and pattern in embryos to the force-producing mechanisms that change cell shapes and move cells interiorly. Here, we review our current understanding of C. elegans gastrulation mechanisms. We address how cells determine which direction is the interior and polarize with respect to that direction, how cells change shape by apical constriction and internalize, and how the embryo specifies which cells will internalize and when. We summarize future prospects for using this system to discover some of the general principles by which animal cells change shape and internalize during development.

The short and intricate life of the suspensor.

The suspensor is a short-lived tissue critical for proper embryonic development in many higher plants. While the tissue was initially thought to simply suspend the embryo in the endosperm, it has been found through decades of research that it serves multiple important purposes. The suspensor has been found to be vital for proper embryo patterning and numerous studies have been undertaken into the complex transcriptional cross-talk between the suspensor and the embryo proper. Indeed, many suspensor mutants also display abnormalities in the embryo. The suspensor's role as a nutrient conduit has been shown using ultrastructural and histochemical techniques. Biochemical approaches have found that the suspensor is a centre of early embryonic hormone production in several species. The suspensor has also been frequently used as a model for programmed cell death as it shows signs of termination almost immediately upon developing. This review covers the essential functions of the suspensor throughout its short existence from multiple disciplines including structural, genetic and biochemical perspectives.

The notochord gene regulatory network in chordate evolution: Conservation and divergence from Ciona to vertebrates.

The notochord is a structure required for support and patterning of all chordate embryos, from sea squirts to humans. An increasing amount of information on notochord development and on the molecular strategies that ensure its proper morphogenesis has been gleaned through studies in the sea squirt Ciona. This invertebrate chordate offers a fortunate combination of experimental advantages, ranging from translucent, fast-developing embryos to a compact genome and impressive biomolecular resources. These assets have enabled the rapid identification of numerous notochord genes and cis-regulatory regions, and provide a rather unique opportunity to reconstruct the gene regulatory network that controls the formation of this developmental and evolutionary chordate landmark. This chapter summarizes the morphogenetic milestones that punctuate notochord formation in Ciona, their molecular effectors, and the current knowledge of the gene regulatory network that ensures the accurate spatial and temporal orchestration of these processes.

Genetic Screens to Target Embryo and Endosperm Pathways in Arabidopsis and Maize.

The major tissue types and stem-cell niches of plants are established during embryogenesis, and thus knowledge of embryo development is essential for a full understanding of plant development. Studies of seed development are also important for human health, because the nutrients stored in both the embryo and endosperm of plant seeds provide an essential part of our diet. Arabidopsis and maize have evolved different types of seeds, opening a range of experimental opportunities. Development of the Arabidopsis embryo follows an almost invariant pattern, while cell division patterns of maize embryos are variable. Embryo-endosperm interactions are also different between the two species: in Arabidopsis, the endosperm is consumed during seed development, while mature maize seeds contain an enormous endosperm. Genetic screens have provided important insights into seed development in both species. In the genomic era, genetic analysis will continue to provide important tools for understanding embryo and endosperm biology in plants, because single gene functional studies can now be integrated with genome-wide information. Here, we lay out important factors to consider when designing genetic screens to identify new genes or to probe known pathways in seed development. We then highlight the technical details of two previous genetic screens that may serve as useful examples for future experiments.

Effect of Maternal Age on Hippo Pathway Related Gene Expressions and Protein Localization Pattern in Human Embryos.

Objective The Hippo pathway plays an important role in embryo development, and separation of trophectoderm (TE) and inner cell mass (ICM) cell lines. Therefore, this study investigated effect of maternal age on activity of Hippo pathway in human embryos. Materials and Methods In this experimental study, the developed up embryos to the blastocyst stage and the embryos whose growth stopped at the morula stage were collected from women aged 20-30 years old (young group, 94 embryos) and >37 years (old group, 89 embryos). Expression of OCT4, SOX2, CDX2, GATA3, YAP genes and the relevant proteins, in the both groups were evaluated using respectively quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunofluorescence methods. Results There was no significant difference in the expression level of OCT4, SOX2, CDX2, GATA3 and YAP genes in blastocyst and morula stages, between the two groups. However, SOX2 and CDX2 gene expressions in morula stage embryos of the old group was statistically lower than that of the young group (P=0.007 and P=0.008, respectively). Additionally, in the embryos collected from women with >37 years of age, at the blastocyst stage, phospho-YAP (p-YAP) protein was found to be accumulated in the TE, but it was almost disappeared from the ICM. Additionally, in the old group, contrary to the expectation, YAP protein was expressed in the ICM, rather than TE. ConclusionThe results of this study showed that YAP and P-YAP among the Hippo signalling pathway may be altered by increasing age.

Rapid early development and feeding benefits an invasive population of lake trout

Lake trout (Salvelinus namaycush) were discovered in Yellowstone Lake in 1994 and their population expanded dramatically despite intensive suppression. The lake is species-depauperate, with no major lake trout embryo predators. We hypothesized that without this predation threat, lake trout free embryo feeding and growth may be greater than in their native range, leading to increased survival of age-0 individuals and rapid population growth. We compared length, developmental rate, and feeding patterns of lake trout free embryos captured at a spawning site in Yellowstone Lake with free embryos captured in their native range in Lake Champlain, Vermont. More embryos were feeding, contained more food, and were significantly longer at the same developmental stages in Yellowstone Lake. With an abundance of available food and minimal threat of predation, free embryos remained on the spawning site in Yellowstone Lake later into the summer than in Lake Champlain and achieved a greater maximum length before they dispersed. Greater food consumption and associated growth likely leads to high survival of lake trout free embryos in Yellowstone Lake, contributing to rapid population growth.

Setting up for gastrulation: D. melanogaster.

Drosophila melanogaster embryos develop initially as a syncytium of totipotent nuclei and subsequently, once cellularized, undergo morphogenetic movements associated with gastrulation to generate the three somatic germ layers of the embryo: mesoderm, ectoderm, and endoderm. In this chapter, we focus on the first phase of gastrulation in Drosophila involving patterning of early embryos when cells differentiate their gene expression programs. This patterning process requires coordination of multiple developmental processes including genome reprogramming at the maternal-to-zygotic transition, combinatorial action of transcription factors to support distinct gene expression, and dynamic feedback between this genetic patterning by transcription factors and changes in cell morphology. We discuss the gene regulatory programs acting during patterning to specify the three germ layers, which involve the regulation of spatiotemporal gene expression coupled to physical tissue morphogenesis.

A comparative transcriptomic analysis in late embryogenesis of the red claw crayfish Cherax quadricarinatus.

The red claw crayfish (Cherax quadricarinatus) is an emerging and important commercial species in several countries, and is also a potential biological model in crustacean biology. However, its molecular embryonic development mechanism remains largely unknown because of a lack of genomic resources and systematic research. A comprehensive and integrated transcriptomic analysis is necessary to reveal the cell biological function, gene expression profiles, and embryo patterning that occur during embryogenesis. In the present study, transcriptomic profiles of C. quadricarinatus embryos during three developmental stages were investigated by high-throughput Illumina sequencing technology, and the genes related to development were further analyzed. In total, 49,436 unigenes were assembled and clustered, in which 13,727 were annotated in the Nonredundant database, 5087 were classified based on Gene Ontology annotations, and 2735 were associated with 189 Kyoto Encyclopedia of Genes and Genomes pathways. Furthermore, gene expression differences among the embryos stages were analyzed, and 6658 differentially expressed genes (DEGs) were identified. In total, 3300, 5211, and 1262 DEGs were identified between the eye pigments forming stage (EP) and prepare-hatching stage (PH), EP and larvae (L), as well as PH and L; meanwhile, 1595, 2540 and 680 DEGs were annotated, respectively. The fundamental developmental genes related to apoptosis, neurogenesis, and segmentation, as well as signaling pathways related to Hedgehog, MAPK, Wnt, TGF-β and Notch, showed higher expression during the EP stage than in other two stages, indicating that the EP stage has more active biological processes than the latter stages. This transcriptome studies gene expression at different stages of embryonic development and the datasets provide a basis for understanding crustacean developmental biology and guiding seedling production.

Two tonoplast proton pumps function in Arabidopsis embryo development.

Two types of tonoplast proton pumps, H+ -pyrophosphatase (V-PPase) and the H+ -ATPase (V-ATPase), establish the proton gradient that powers molecular traffic across the tonoplast thereby facilitating turgor regulation and nutrient homeostasis. However, how proton pumps regulate development remains unclear. In this study, we investigated the function of two types of proton pumps in Arabidopsis embryo development and pattern formation. While disruption of either V-PPase or V-ATPase had no obvious effect on plant embryo development, knocking out both resulted in severe defects in embryo pattern formation from the early stage. While the first division in wild-type zygote was asymmetrical, a nearly symmetrical division occurred in the mutant, followed by abnormal pattern formation at all stages of embryo development. The embryonic defects were accompanied by dramatic differences in vacuole morphology and distribution, as well as disturbed localisation of PIN1. The development of mutant cotyledons and root, and the auxin response of mutant seedlings supported the hypothesis that mutants lacking tonoplast proton pumps were defective in auxin transport and distribution. Taking together, we proposed that two tonoplast proton pumps are required for vacuole morphology and PIN1 localisation, thereby controlling vacuole and auxin-related developmental processes in Arabidopsis embryos and seedlings.

Insights into the Functions of LncRNAs in Drosophila.

Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs longer than 200 nucleotides (nt). LncRNAs have high spatiotemporal specificity, and secondary structures have been preserved throughout evolution. They have been implicated in a range of biological processes and diseases and are emerging as key regulators of gene expression at the epigenetic, transcriptional, and post-transcriptional levels. Comparative analyses of lncRNA functions among multiple organisms have suggested that some of their mechanisms seem to be conserved. Transcriptome studies have found that some Drosophila lncRNAs have highly specific expression patterns in embryos, nerves, and gonads. In vivo studies of lncRNAs have revealed that dysregulated expression of lncRNAs in Drosophila may result in impaired embryo development, impaired neurological and gonadal functions, and poor stress resistance. In this review, we summarize the epigenetic, transcriptional, and post-transcriptional mechanisms of lncRNAs and mainly focus on recent insights into the transcriptome studies and biological functions of lncRNAs in Drosophila.