Search

Zebrafish germ plasm ribonucleoparticles (RNPs) become recruited to furrows of early zebrafish embryos through their association with astral microtubules ends. During the initiation of cytokinesis, microtubules are remodeled into a furrow microtubule array (FMA), which is thought to be analogous to the mammalian midbody involved in membrane abscission. During furrow maturation, RNPs and FMA tubules transition from their original distribution along the furrow to enrichments at the furrow distal ends, which facilitates germ plasm mass compaction. We show that nebel mutants exhibit reduced furrow-associated slow calcium waves (SCWs), caused at least in part by defective enrichment of calcium stores. RNP and FMA distal enrichment mirrors the medial-to-distal polarity of SCWs, and inhibition of calcium release or downstream mediators such as Calmodulin affects RNP and FMA distal enrichment. Blastomeres with reduced or lacking SCWs, such as early blastomeres in nebel mutants and wild-type blastomeres at later stages, exhibit medially bundling microtubules similar to midbodies in other cell types. Our data indicate that SCWs provide medial-to-distal directionality along the furrow to facilitate germ plasm RNP enrichment at the furrow ends., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

To understand how undifferentiated pluripotent cells cope with cell corpses, we examined the clearance of polar bodies born during female meiosis. We found that polar bodies lose membrane integrity and expose phosphatidylserine in Caenorhabditis elegans. Polar body signaling recruits engulfment receptors to the plasma membrane of embryonic blastomeres using the PI3K VPS-34, RAB-5 GTPase and the sorting nexin SNX-6. The second polar body is then phagocytosed using receptor-mediated engulfment pathways dependent on the Rac1 ortholog CED-10 but undergoes non-apoptotic programmed cell death independent of engulfment. RAB-7 GTPase is required for lysosome recruitment to the polar body phagosome, while LC3 lipidation is required for degradation of the corpse membrane after lysosome fusion. The polar body phagolysosome vesiculates in an mTOR- and ARL-8-dependent manner, which assists its timely degradation. Thus, we established a genetic model to study clearance by LC3-associated phagocytosis and reveal insights into the mechanisms of phagosome maturation and degradation., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Derepression of chromatin-mediated transcriptional repression of paternal and maternal genomes is considered the first major step that initiates zygotic gene expression after fertilization. The histone variant H3.3 is present in both male and female gametes and is thought to be important for remodeling the paternal and maternal genomes for activation during both fertilization and embryogenesis. However, the underlying mechanisms remain poorly understood. Using our H3.3B-HA-tagged mouse model, engineered to report H3.3 expression in live animals and to distinguish different sources of H3.3 protein in embryos, we show here that sperm-derived H3.3 (sH3.3) protein is removed from the sperm genome shortly after fertilization and extruded from the zygotes via the second polar bodies (PBII) during embryogenesis. We also found that the maternal H3.3 (mH3.3) protein is incorporated into the paternal genome as early as 2 h postfertilization and is detectable in the paternal genome until the morula stage. Knockdown of maternal H3.3 resulted in compromised embryonic development both of fertilized embryos and of androgenetic haploid embryos. Furthermore, we report that mH3.3 depletion in oocytes impairs both activation of the Oct4 pluripotency marker gene and global de novo transcription from the paternal genome important for early embryonic development. Our results suggest that H3.3-mediated paternal chromatin remodeling is essential for the development of preimplantation embryos and the activation of the paternal genome during embryogenesis., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB -28 (A>G) mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor (BE) system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB -28 (A>G) mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB -28 (A>G) homozygous mutation. Data showed that base editor could precisely correct HBB -28 (A>G) mutation in the patient's primary cells. To model homozygous mutation disease embryos, we constructed nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured (IVM) oocytes. Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB -28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.

The maternal-zygotic transition (MZT) describes the developmental reprogramming of gene expression marked by the degradation of maternally supplied gene products and activation of the zygotic genome. While the timing and duration of the MZT vary among taxa, little is known about early-stage transcriptional dynamics in the non-bilaterian phylum Ctenophora. We sought to better understand the extent of maternal mRNA loading and subsequent differential transcript abundance during the earliest stages of development by performing comprehensive RNA-sequencing-based analyses of mRNA abundance in single- and eight-cell stage embryos in the lobate ctenophore Mnemiopsis leidyi. We found 1,908 contigs with significant differential abundance between single- and eight-cell stages, of which 1,208 contigs were more abundant at the single-cell stage and 700 contigs were more abundant at the eight-cell stage. Of the differentially abundant contigs, 267 were exclusively present in the eight-cell samples, providing strong evidence that both the MZT and zygotic genome activation (ZGA) have commenced by the eight-cell stage. Many highly abundant transcripts encode genes involved in molecular mechanisms critical to the MZT, such as maternal transcript degradation, serine/threonine kinase activity, and chromatin remodeling. Our results suggest that chromosomal restructuring, which is critical to ZGA and the initiation of transcriptional regulation necessary for normal development, begins by the third cleavage within 1.5 hr post-fertilization in M. leidyi., (© 2017 Wiley Periodicals, Inc.)

Mouse embryonic stem cells derived from the epiblast contribute to the somatic lineages and the germline but are excluded from the extra-embryonic tissues that are derived from the trophectoderm and the primitive endoderm upon reintroduction to the blastocyst. Here we report that cultures of expanded potential stem cells can be established from individual eight-cell blastomeres, and by direct conversion of mouse embryonic stem cells and induced pluripotent stem cells. Remarkably, a single expanded potential stem cell can contribute both to the embryo proper and to the trophectoderm lineages in a chimaera assay. Bona fide trophoblast stem cell lines and extra-embryonic endoderm stem cells can be directly derived from expanded potential stem cells in vitro. Molecular analyses of the epigenome and single-cell transcriptome reveal enrichment for blastomere-specific signature and a dynamic DNA methylome in expanded potential stem cells. The generation of mouse expanded potential stem cells highlights the feasibility of establishing expanded potential stem cells for other mammalian species.

Intracellular Ca 2+ signaling regulates cellular activities during embryogenesis and in adult organisms. We generated stable Tg[βactin2:GCaMP6s] stl351 and Tg[ubi:GCaMP6s] stl352 transgenic lines that combine the ubiquitously-expressed Ca 2+ indicator GCaMP6s with the transparent characteristics of zebrafish embryos to achieve superior in vivo Ca 2+ imaging. Using the Tg[βactin2:GCaMP6s] stl351 line featuring strong GCaMP6s expression from cleavage through gastrula stages, we detected higher frequency of Ca 2+ transients in the superficial blastomeres during the blastula stages preceding the midblastula transition. Additionally, GCaMP6s also revealed that dorsal-biased Ca 2+ signaling that follows the midblastula transition persisted longer during gastrulation, compared with earlier studies. We observed that dorsal-biased Ca 2+ signaling is diminished in ventralized ichabod/β-catenin2 mutant embryos and ectopically induced in embryos dorsalized by excess β-catenin. During gastrulation, we directly visualized Ca 2+ signaling in the dorsal forerunner cells, which form in a Nodal signaling dependent manner and later give rise to the laterality organ. We found that excess Nodal increases the number and the duration of Ca 2+ transients specifically in the dorsal forerunner cells. The GCaMP6s transgenic lines described here enable unprecedented visualization of dynamic Ca 2+ events from embryogenesis through adulthood, augmenting the zebrafish toolbox., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Our recent perplexing findings that polyploid giant cancer cells (PGCCs) acquired embryonic-like stemness and were capable of tumor initiation raised two important unanswered questions: how do PGCCs acquire such stemness, and to which stage of normal development do PGCCs correspond. Intriguingly, formation of giant cells due to failed mitosis/cytokinesis is common in the blastomere stage of the preimplantation embryo. However, the relationship between PGCCs and giant blastomeres has never been studied. Here, we tracked the fate of single PGCCs following paclitaxel-induced mitotic failure. Morphologically, early spheroids derived from PGCCs were indistinguishable from human embryos at the blastomere, polyploid blastomere, compaction, morula and blastocyst-like stages by light, scanning electron or three-dimensional confocal scanning microscopy. Formation of PGCCs was associated with activation of senescence, while budding of daughter cells was associated with senescence escape. PGCCs showed time- and space-dependent activation of expression of the embryonic stem cell markers OCT4, NANOG, SOX2 and SSEA1 and lacked expression of Xist. PGCCs acquired mesenchymal phenotype and were capable of differentiation into all three germ layers in vitro. The embryonic-like stemness of PGCCs was associated with nuclear accumulation of YAP, a key mediator of the Hippo pathway. Spheroids derived from single PGCCs grew into a wide spectrum of human neoplasms, including germ cell tumors, high-grade and low-grade carcinomas and benign tissues. Daughter cells derived from PGCCs showed attenuated capacity for invasion and increased resistance to paclitaxel. We also observed formation of PGCCs and dedifferentiation in ovarian cancer specimens from patients treated with chemotherapy. Taken together, our findings demonstrate that PGCCs represent somatic equivalents of blastomeres, the most primitive cancer stem cells reported to date. Thus, our studies reveal an evolutionarily conserved archaic embryonic program in somatic cells that can be de-repressed for oncogenesis. Our work offers a new paradigm for cancer origin and disease relapse.

Following fertilization in mammals, it is generally accepted that totipotent cells are exclusive to the zygote and to each of the two blastomeres originating from the first mitotic division. This model of totipotency was inferred from a minority of cases in which blastomeres produced monozygotic twins in mice. Was this due to experimental limitation or biological constraint? Here we removed experimental obstacles and achieved reliable quantification of the prevalence of dual totipotency among mouse two-cell stage blastomeres. We separated the blastomeres of 1,252 two-cell embryos, preserving 1,210 of the pairs. Two classes of monozygotic twins became apparent at the blastocyst stage: 27% formed a functional epiblast in both members (concordant), and 73% did so in only one member of the pair (discordant) - a partition that proved insensitive to oocyte quality, sperm-entry point, culture environment and pattern of cleavage. In intact two-cell embryos, the ability of sister blastomeres to generate epiblast was also skewed. Class discovery clustering of the individual blastomeres' and blastocysts' transcriptomes points to an innate origin of concordance and discordance rather than developmental acquisition. Our data place constraints on the commonly accepted idea that totipotency is allocated equally between the two-cell stage blastomeres in mice.

Myosin-1 (Myo1) represents a mechanical link between the membrane and actin-cytoskeleton in animal cells. We have studied the effect of Myo1 inhibitor PClP in 1-8 cell Zebrafish embryos. Our results indicate a unique involvement of Myo1 in early development of Zebrafish embryos. Inhibition of Myo1 (by PClP) and Myo2 (by Blebbistatin) lead to arrest in cell division. While Myo1 isoforms appears to be important for both the formation and the maintenance of cleavage furrows, Myo2 is required only for the formation of furrows. We found that the blastodisc of the embryo, which contains a thick actin cortex (~13 μm), is loaded with cortical Myo1. Myo1 appears to be crucial for maintaining the blastodisc morphology and the actin cortex thickness. In addition to cell division and furrow formation, inhibition of Myo1 has a drastic effect on the dynamics and distribution of lipid droplets (LDs) in the blastodisc near the cleavage furrow. All these results above are effects of Myo1 inhibition exclusively; Myo2 inhibition by blebbistatin does not show such phenotypes. Therefore, our results demonstrate a potential role for Myo1 in the maintenance and formation of furrow, blastodisc morphology, cell-division and LD organization within the blastodisc during early embryogenesis.

The canonical Wnt/β-catenin signaling pathway plays multiple roles during Xenopus gastrulation, including posteriorization of the neural plate, patterning of the mesoderm, and induction of the neural crest. Wnt signaling stabilizes β-catenin, which then activates target genes. However, few targets of this signaling pathway that mediate early developmental processes are known. Here we sought to identify transcriptional targets of the Wnt/β-catenin signaling pathway using a genome-wide approach. We selected putative targets using the criteria of reduced expression upon zygotic Wnt knockdown, β-catenin binding within 50kb of the gene, and expression in tissues that receive Wnt signaling. Using these criteria, we found 21 novel direct transcriptional targets of Wnt/β-catenin signaling during gastrulation and in addition have identified putative regulatory elements for further characterization in future studies., (Copyright © 2017. Published by Elsevier Inc.)

Our study examined the in vivo chimeric and survival capacities of chimeras created by injecting tetraploid embryonic stem cells (ESCs) expressing green fluorescent protein (GFP) into diploid embryos. At 3.5 days post-coitum (dpc) and 4.5 dpc, the tetraploid ESCs were able to contribute to the inner cell mass (ICM) just as diploid ESCs tagged with GFP. At 6.5 dpc, 8.0 dpc and 10.5 dpc, the tetraploid ESCs manifested in the same location as the diploid ESCs. The GFP cells in the extraembryonic tissues and fetuses of tetraploid ESC chimeras were tetraploid as determined by fluorescence activated cell sorting (FACS). Furthermore, tetraploid ESCs contributed to the development of the placenta, embryolemma and umbilical cord at 13.5 dpc and 16.5 dpc; however, very less GFP cells were found in the fetuses of tetraploid ESC chimeras. We further found that the proliferation of tetraploid ESCs was slower than that of diploid ESCs. In addition, the relative mRNA expression in the three germ layers and the trophoblast was abnormal in the EBs of tetraploid ESCs compared with diploid ESCs. In short, slower proliferation and abnormal differentiation potential of tetraploid ESCs might be two of the reasons for their poor survival and chimeric capacities.

Studying the early function of essential genes is an important and challenging problem in developmental biology. Here, we established a method for rapidly inducing CRISPR-Cas9-mediated mutations in one blastomere of two-cell stage embryos, termed 2-cell embryo-CRISPR-Cas9 injection (2CC), to study the in vivo function of essential (or unknown) genes in founder chimeric mice. By injecting both Cre mRNA and CRISPR-Cas9 targeting the gene of interest into fluorescent reporter mice, the 2CC method can trace both wild-type and mutant cells at different developmental stages, offering internal control for phenotypic analyses of mutant cells. Using this method, we identified novel functions of the essential gene Tet3 in regulating excitatory and inhibitory synaptic transmission in the developing mouse cerebral cortex. By generating chimeric mutant mice, the 2CC method allows for the rapid screening of gene function in multiple tissues and cell types in founder chimeric mice, significantly expanding the current armamentarium of genetic tools.

The oocyte-to-embryo transition (OET) transforms a differentiated gamete into pluripotent blastomeres. The accompanying maternal-zygotic RNA exchange involves remodeling of the long non-coding RNA (lncRNA) pool. Here, we used next generation sequencing and de novo transcript assembly to define the core population of 1,600 lncRNAs expressed during the OET (lncRNAs). Relative to mRNAs, OET lncRNAs were less expressed and had shorter transcripts, mainly due to fewer exons and shorter 5' terminal exons. Approximately half of OET lncRNA promoters originated in retrotransposons suggesting their recent emergence. Except for a small group of ubiquitous lncRNAs, maternal and zygotic lncRNAs formed two distinct populations. The bulk of maternal lncRNAs was degraded before the zygotic genome activation. Interestingly, maternal lncRNAs seemed to undergo cytoplasmic polyadenylation observed for dormant mRNAs. We also identified lncRNAs giving rise to trans-acting short interfering RNAs, which represent a novel lncRNA category. Altogether, we defined the core OET lncRNA transcriptome and characterized its remodeling during early development. Our results are consistent with the notion that rapidly evolving lncRNAs constitute signatures of cells-of-origin while a minority plays an active role in control of gene expression across OET. Our data presented here provide an excellent source for further OET lncRNA studies., (© The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

The role of CDX2 in trophectoderm (TE) cells has been extensively studied, yet the results are contradictory and species specific. Here, CDX2 expression and function were explored in early porcine embryos. Notably, siRNA-mediated gene knockdown and lentivirus-mediated TE-specific gene regulation demonstrated that CDX2 is essential for the maintenance of blastocyst integrity by regulating the BMP4-mediated blastocyst niche and classic protein kinase C (PKC)-mediated TE polarity in mammalian embryos. Mechanistically, CDX2 -depleted porcine embryos stalled at the blastocyst stage and exhibited apoptosis and inactive cell proliferation, possibly resulting from BMP4 downregulation. Moreover, TE cells in CDX2 -depleted blastocysts displayed defective F-actin apical organization associated with downregulation of PKCα ( PRKCA ). Collectively, these results provide further insight into the functional diversity of CDX2 in early mammalian embryos., (© 2017. Published by The Company of Biologists Ltd.)

The mouse preimplantation embryo generates the precursors of trophectoderm (TE) and inner cell mass (ICM) during the 8- to 16-cell stage transition, when the apico-basal polarized blastomeres undergo divisions that give rise to cells with different fate. Asymmetric segregation of polar domain at 8-16 cell division generate two cell types, polar cells which adopt an outer position and develop in TE and apolar cells which are allocated to inner position as the precursors of ICM. It is still not know when the blastomeres of 8-cell stage start to be determined to undergo asymmetric division. Here, we analyze the frequency of symmetric and asymmetric divisions of blastomeres isolated from 8-cell stage embryo before and after compaction. Using p-Ezrin as the polarity marker we found that size of blastomeres in 2/16 pairs cannot be used as a criterion for distinguishing symmetric and asymmetric divisions. Our results showed that at early 8-cell stage, before any visible signs of cortical polarity, a subset of blastomeres had been already predestined to divide asymmetrically. We also showed that almost all of 8-cell stage blastomeres isolated from compacted embryo divide asymmetrically, whereas in intact embryos, the frequency of asymmetric divisions is significantly lower. Therefore we conclude that in intact embryo the frequency of symmetric and asymmetric division is regulated by cell-cell interactions.

Insulin-like growth factors (IGFs) regulate diverse processes including energy metabolism, cell proliferation and embryonic development. They activate the IGF signaling pathway via binding to cell surface receptors. Here we report an essential role of IGF2 in maintaining the pluripotency of embryonic stem (ES) cell from medaka (Oryzias latipes). The medaka igf2 gene was cloned for prokaryotically expression of IGF2 ligand and green fluorescent protein-tagged IGF2 namely IGF2:GFP. With flow cytometry analysis, we demonstrated that the IGF2:GFP can bind to the cultured ES cells from medaka and zebrafish respectively. We also verified that IGF2 is able to activate the phosphorylation of Erk1/2 and Akt, and sustain the viability and pluripotency of medaka ES cells in culture. Furthermore, we characterized the binding of IGF2:GFP to freshly isolated blastomeres by fluorescence microscopy and electron microscopy. Most importantly, we revealed the important role of IGF2 in supporting the derivation of blastomeres in short-term culture. Therefore, Medaka IGF2 is essential for the self-renewal of cultured ES cells and blastomeres from fish embryos. This finding underscores a conserved role of the IGF signaling pathway in stem cells from fish to mammals.

To understand mammalian active DNA demethylation, various methods have been developed to map the genomic distribution of the demethylation intermediates 5-formylcysotine (5fC) and 5-carboxylcytosine (5caC). However, the majority of these methods requires a large number of cells to begin with. In this study, we describe low-input methylase-assisted bisulfite sequencing (liMAB-seq ) and single-cell MAB-seq (scMAB-seq), capable of profiling 5fC and 5caC at genome scale using ∼100 cells and single cells, respectively. liMAB-seq analysis of preimplantation embryos reveals the oxidation of 5mC to 5fC/5caC and the positive correlation between chromatin accessibility and processivity of ten-eleven translocation (TET) enzymes. scMAB-seq captures the cell-to-cell heterogeneity of 5fC and 5caC and reveals the strand-biased distribution of 5fC and 5caC. scMAB-seq also allows the simultaneous high-resolution mapping of sister chromatid exchange (SCE), facilitating the study of this type of genomic rearrangement. Therefore, our study not only establishes new methods for the genomic mapping of active DNA demethylation using limited numbers of cells or single cells but also demonstrates the utilities of the methods in different biological contexts., (© 2017 Wu et al.; Published by Cold Spring Harbor Laboratory Press.)

Objective: To identify the quantitative profiling of adiponectin and its receptors (AdipoR1, AdipoR2, and T-cadherin) in cumulus cells (CCs) and to evaluate their roles in the early embryo development of polycystic ovary syndrome (PCOS) patients, in part, with obesity., Methods: Fifty-five subjects were divided into two groups according to the body mass index. Oocytes were further inseminated and only mature and normal fertilized oocytes (2PN) were included in this research. Real-time PCR and western blot were performed to identify adiponectin and its receptors in CCs., Results: Adiponectin and receptors were ubiquitously expressed in CCs of PCOS and non-PCOS patients. The level of AdipoR2 in CCs from the oocytes yielding blastocyst after 5/6 days in vitro culture was markedly higher than in those from oocytes could not develop to blastocyst stage after Day 6, for non-obese or obese PCOS patients (0.1647 ± 0.0161 versus 0.0783 ± 0.0385, 0.1948 ± 0.0307 versus 0.1057 ± 0.0236, respectively, p < 0.05). In addition, only in patients with PCOS and concurrent obesity the AdipoR1 in CCs was considerably increased in CC-B + compared with CC-B - subgroup (0.5162 ± 0.0371 versus 0.2448 ± 0.0333, p < 0.01)., Conclusion: The development of early embryo was associated with the up-regulation of AdipoR1 and AdipoR2 in PCOS patients. Our results suggested that adiponectin could positively modulate embryo development in humans. Further investigations should be carried out to unlock the crucial role that adiponectin plays in embryo development.

During development from oocyte to embryo, genetic programs in mouse germ cells are reshaped by chromatin remodeling to orchestrate the onset of development. Epigenetic modifications of specific amino acid residues of core histones and their isoforms can dramatically alter activation and suppression of gene expression. H3.3 is a histone H3 variant that plays essential roles in mouse oocytes and early embryos, but the functional role of individual amino acid residues has been unclear because of technical hurdles. Here, we describe two strategies that successfully investigated the functions of three individual H3.3 residues in oogenesis, cleavage-stage embryogenesis and early development. We first generated genetic mosaic ovaries and blastocysts with stochastic expression of wild-type or mutant H3.3 alleles and showed dominant negative effects of H3.3R26 and H3.3K27 in modulating oogenesis and partitioning cells to the inner cell mass of the early embryo. Time-lapse imaging assays also revealed the essential roles of H3.3K56 in efficient H2B incorporation and paternal pronuclei formation. Application of these strategies can be extended to investigate roles of additional H3.3 residues and has implications for use in other developmental systems., (© 2017. Published by The Company of Biologists Ltd.)

The MS blastomere produces one-third of the body wall muscles (BWMs) in the C. elegans embryo. MS-derived BWMs require two distinct cell-cell interactions, the first inhibitory and the second, two cell cycles later, required to overcome this inhibition. The inductive interaction is not required if the inhibitory signal is absent. Although the Notch receptor GLP-1 was implicated in both interactions, the molecular nature of the two signals was unknown. We now show that zygotically expressed MOM-2 (Wnt) is responsible for both interactions. Both the inhibitory and the activating interactions require precise spatiotemporal expression of zygotic MOM-2, which is dependent upon two distinct Notch signals. In a Notch mutant defective only in the inductive interaction, MS-derived BWMs can be restored by preventing zygotic MOM-2 expression, which removes the inhibitory signal. Our results suggest that the inhibitory interaction ensures the differential lineage specification of MS and its sister blastomere, whereas the inductive interaction promotes the expression of muscle-specifying genes by modulating TCF and β-catenin levels. These results highlight the complexity of cell fate specification by cell-cell interactions in a rapidly dividing embryo., (© 2017. Published by The Company of Biologists Ltd.)

The assembly of the first centrosome occurs upon fertilisation when male centrioles recruit pericentriolar material (PCM) from the egg cytoplasm. The mechanisms underlying the proper assembly of centrosomes during early embryogenesis remain obscure. We identify Wdr8 as a novel maternally essential protein that is required for centrosome assembly during embryonic mitoses of medaka (Oryzias latipes). By CRISPR-Cas9-mediated knockout, maternal/zygotic Wdr8-null (m/zWdr8 -/- ) blastomeres exhibit severe defects in centrosome structure that lead to asymmetric division, multipolar mitotic spindles and chromosome alignment errors. Via its WD40 domains, Wdr8 interacts with the centriolar satellite protein SSX2IP. Combining targeted gene knockout and in vivo reconstitution of the maternally essential Wdr8-SSX2IP complex reveals an essential link between maternal centrosome proteins and the stability of the zygotic genome for accurate vertebrate embryogenesis. Our approach provides a way of distinguishing maternal from paternal effects in early embryos and should contribute to understanding molecular defects in human infertility.

The proper positioning of microtubule (MT) asters underlies fundamental processes such as nuclear centration, cell polarity, division positioning, and embryogenesis. In large eggs and early blastomeres, MT asters may exhibit long range motions with atypical speed and precision to target their functional position. The biophysical mechanisms regulating such motions remain however largely unknown. The centration of sperm asters in sea urchin embryos is a stereotypical example of such aster long range motion. In this chapter, we describe methods developed in this system to (1) quantify sperm aster 3-D motion with confocal microscopy and automated image analysis and (2) severe a portion of astral MTs with a UV laser. These methods may serve as a template to dissect the generic mechanisms of aster motion and force production in other embryos and cell types., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The nucleus of mouse two-cell embryos houses the coilin-containing bodies of two types: 1) 1—3 large spherical structures of 1 mm and 2) small foci, which vary in number in different blastomeres. The largest coilin-containing structures, unlike the smallest ones, contain RNA polymerase I, nucleic acid chaperon YB-1, and also actin. Neither large nor small coilin-positive domains contain symplekin, one of the signature components of histone locus bodies. In the nuclei of late two-cell embryos, symplekin localizes to 1—2 well-formed roundish bodies that are observed both in close proximity to the coilin-positive structures and far away from them. Large coilin-containing bodies were not observed in embryos at the morula stage as well as in the nuclei of late two-cell embryos after artificial suppression of transcription activity. Thus, a population of coilin-containing bodies in the nuclei of late two-cell embryos of mice is heterogeneous in morphology and molecular composition. It could be assumed that the largest coilin-containing bodies are provisional nuclear domains that are formed at the background of significant changes of nuclear metabolism at the final stages of embryonic genome activation and the initial stages of reactivation of nucleolar transcription.

Study Question: Does fetuin-B inhibit premature zona pellucida (ZP) hardening in mouse oocytes in vitro and thus increase IVF rate?, Summary Answer: Supplementation of oocyte in vitro maturation (IVM) media with recombinant mouse fetuin-B (rmFetuB) increased fertilization rate without affecting mouse embryo development into blastocysts., What Is Known Already: Mice deficient in fetuin-B are infertile owing to premature ZP hardening. Premature ZP hardening also occurs during oocyte IVM leading to decreased fertilization rate., Study Design, Size, Duration: We fertilized batches of 20-30 mouse metaphase II (Mll) stage oocytes from C57BL/6 mice with fresh sperm, and studied early embryo development until blastocyst hatching., Participants/materials, Setting, Methods: Oocytes were maintained with or without rmFetuB during IVM and IVF. Exogenous rmFetuB was added to media prior to oocyte isolation. ZP hardening was quantified by chymotrypsin digestion timing and by counting attached sperm., Main Results and the Role of Chance: In the absence of cumulus cells, rmFetuB dose-dependently inhibited ZP hardening and increased IVF rate (P = 0.039). Fetuin-B at ≥0.03 mg/ml also inhibited physiological, fertilization-triggered ZP hardening (indicated by increased sperm binding, P = 0.0002), without increasing embryo death. Exogenous rmFetuB increased IVF rate for up to 5 hours of IVM (P = 0.02 at 1 hour, P = 0.01 at 5 hours of IVM)., Limitations, Reasons for Caution: Mll stage oocytes in this study were isolated from the ampullae of fetuin-B expressing mice. Thus, oocytes were protected against premature ZP hardening by endogenous fetuin-B. In humans and livestock, oocytes are usually isolated by follicle puncture before ovulation. In this situation, the deprivation of endogenous fetuin-B would occur earlier and the effect of exogenous fetuin-B in the IVF medium may be even more pronounced. Fertilization-triggered ZP hardening is essential for embryo development but in this study the effect of fetuin-B supplementation was only studied to blastocyst stage. Any influence of added fetuin-B on later embryo development after transplantation remains to be determined., Wider Implications of the Findings: The astacin-type protease ovastacin triggers definitive ZP hardening by cleaving the zona pellucida protein 2. Animal sera are known to inhibit premature ZP hardening. The addition of rFetuB to the culture medium of oocytes could increase IVF rates by the inhibition of premature ZP hardening. In this regard, the results could be useful for clinical activity., Large Scale Data: None., Study Funding/competing Interests: The research was supported by a grant from Deutsche Forschungsgemeinschaft and by the START program of the Medical Faculty of RWTH Aachen University. The authors ED, JF and WJD are named inventors on a patent application of RWTH Aachen University covering the use of fetuin-B in ovary and oocyte culture., (© The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.)

Life for all animals starts with a precise 3D choreography of reductive divisions of the fertilized egg, known as cleavage patterns. These patterns exhibit conserved geometrical features and striking interspecies invariance within certain animal classes. To identify the generic rules that may govern these morphogenetic events, we developed a 3D-modeling framework that iteratively infers blastomere division positions and orientations, and consequent multicellular arrangements. From a minimal set of parameters, our model predicts detailed features of cleavage patterns in the embryos of fishes, amphibians, echinoderms, and ascidians, as well as the genetic and physical perturbations that alter these patterns. This framework demonstrates that a geometrical system based on length-dependent microtubule forces that probe blastomere shape and yolk gradients, biased by cortical polarity domains, may dictate division patterns and overall embryo morphogenesis. These studies thus unravel the default self-organization rules governing early embryogenesis and how they are altered by deterministic regulatory layers., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: To investigate the prevalence, developmental potential, chromosomal constitution and clinical outcome of embryos with direct unequal cleavages (DUC)., Design: A retrospective observational study., Setting: Academic Institution., Participant: 21,261 embryos from 3,155 cycles cultured in EmbryoScope®., Results: The total incidence of DUCs per embryo occupying the first three cleavages were 26.1%. Depending of the cell stage, DUC rate was 9.8% at first cleavage (DUC-1), 9.1% at second cleavage (DUC-2), and 3.7% at third cleavage (DUC-3) with 3.6% of embryos exhibiting multiple DUCs (DUC-Plus). The occurrence of DUCs was not correlated with female gamete age or source. The incidence of DUC-1 was significantly higher in embryos fertilized by epididymal and testicular sperm (13.6% and 11.4%, respectively) compared to ejaculated sperm (9.1%, all p<0.05). The total incidences of DUCs were strongly correlated with the onset of blastomere multinucleation (MNB) during the first three divisions. In MNB embryos, DUCs incidence are two to three times more likely to develop when compared to non-MNB embryos (OR = 3.11, 95% CI [2.64, 3.67] at 1-cell stage, OR = 2.64, 95% CI [2.39, 2.91] at 2-cell stage and OR = 2.51, 95% CI [1.84, 3.43] at 4-cell stage). The blastocyst formation rates gradually decreased from 61.0% in non-DUC to 40.2% in DUC-3, 18.8% in DUC-2, 8.2% in DUC-1 and 5.6% in multiple DUC embryos (DUC-Plus). The known implantation rates (FH) for day 3 (D3) transfers were 12.42% (n = 3172) in Non-DUC embryos, 6.3% (n = 127) in DUC-3, and 2.7% (n = 260) in DUC-2 embryos. No live births resulted from either DUC-1 (n = 225) or DUC-Plus (n = 100) embryo transfers. For blastocyst transfers, lower implantation rates (33.3%) but similar live birth (LB) rates (40%) were observed if DUC blastocysts were transferred. Comparatively rates in Non-DUC blastocyst were 45.2% and 34.8%, respectively. The euploid rate gradually increased from DUC-1, -2, -3 to Non-DUC (13.3%, 19.5%, 33.3%, 45.6%, p<0.001) for D3 biopsied embryos. Interestingly, the trend of decreased euploidy disappeared in DUC D5/6 biopsied embryos and similar rates were exemplified in DUC (D5 56.3%, D6 35.6%) vs. non-DUC (D5 51.4%, D6 33.8%) embryos., Conclusion: Blastocyst formation, implantation potential and euploid rate were significantly reduced in DUC embryos. DUC embryos should be deselected for D3 transfers, but should be culture to blastocyst stage for possible ET., Competing Interests: The authors have declared that no competing interests exist.

The first lineage specification during mammalian embryo development can be visually distinguished at the blastocyst stage. Two cell lineages are observed on the embryonic-abembryonic axis of the blastocyst: the inner cell mass and the trophectoderm. The timing and mechanisms driving this process are still not fully understood. In mouse embryos, cells seem prepatterned to become certain cell lineage because the first cleavage plane has been related with further embryonic-abembryonic axis at the blastocyst stage. Nevertheless, this possibility has been very debatable. Our objective was to determine whether this would be the case in another mammalian species, the bovine. To achieve this, cells of in vitro produced bovine embryos were traced from the 2-cell stage to the blastocyst stage. Blastocysts were then classified according to the allocation of the labeled cells in the embryonic and/or abembryonic part of the blastocyst. Surprisingly, we found that there is a significant percentage of the embryos (∼60%) with labeled and nonlabeled cells randomly distributed and intermingled. Using time-lapse microscopy, we have identified the emergence of this random pattern at the third to fourth cell cycle, when cells started to intermingle. Even though no differences were found on morphokinetics among different embryos, these random blastocysts and those with labeled cells separated by the embryonic-abembryonic axis (deviant pattern) are significantly bigger; moreover deviant embryos have a significantly higher number of cells. Interestingly, we observed that daughter cells allocation at the blastocyst stage is not affected by biopsies performed at an earlier stage., (© 2016 by the Society for the Study of Reproduction, Inc.)

CDC25B has been demonstrated to activate the complex of CDK1/Cyclin B and trigger mitosis. We have recently demonstrated that p-CDC25B-Ser351 is located at the centrosomes of mouse oocytes and contributes to the release of mouse oocytes from prophase I arrest. But much less is known about CDC25B function at the centrosome in two-cell stage mouse embryos. Here we investigate the effect of CDC25B regulating the microtubules nucleation. Microinjection of anti-CDC25B antibody caused aberrant microtubule nucleation. In addition, embryos injected with anti-CDC25B antibody showed the marked absence of microtubule repolymerization and Nek2 foci after nocodazole washout. CDC25B overexpression caused microtubule-organizing center (MTOC) overduplication. Moreover, overexpression of CDC25B-▵65 mutant resulted in the loss of CDC25B localization in the perinuclear region and made CDC25B less efficient in inducing mitosis. We additionally identified that CDC25B is responsible for the pericentrin localization to the MTOC. Our data suggest an important role of CDC25B for microtubule nucleation and organization. N-terminal of CDC25B is required for regulating the microtubule dynamics and mitotic function., (© 2016 Japanese Society of Developmental Biologists.)

The initiation of embryonic gene expression in ascidian embryos appears to be tightly regulated by the number of DNA replication cycles. DNA methylation is thought to contribute to the clock mechanism that counts the rounds of DNA replication. We used mass spectrometry and whole genome bisulfite sequencing to characterize DNA methylation changes that occur in early developmental stages of the ascidian, Ciona intestinalis. We found that global DNA methylation in early Ciona development was static, and a base-wise comparison between the genomes of consecutive developmental stages found no DNA demethylation that was related to zygotic gene activation. Additionally, 5hmC was hardly detected by mass spectrometry in the developmental samples, suggesting a lack of demethylation mediated by ten eleven translocation (TET) methylcytosine dioxygenase in C. intestinalis. We conclude that DNA methylation is not involved in regulating DNA replication-dependent transcriptional activation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Quantification of protein expression in single cells promises to advance a systems-level understanding of normal development. Using a bottom-up proteomic workflow and multiplexing quantification by tandem mass tags, we recently demonstrated relative quantification between single embryonic cells (blastomeres) in the frog (Xenopus laevis) embryo. In this study, we minimize derivatization steps to enhance analytical sensitivity and use label-free quantification (LFQ) for single Xenopus cells. The technology builds on a custom-designed capillary electrophoresis microflow-electrospray ionization high-resolution mass spectrometry platform and LFQ by MaxLFQ (MaxQuant). By judiciously tailoring performance to peptide separation, ionization, and data-dependent acquisition, we demonstrate an ∼75-amol (∼11 nm) lower limit of detection and quantification for proteins in complex cell digests. The platform enabled the identification of 438 nonredundant protein groups by measuring 16 ng of protein digest, or <0.2% of the total protein contained in a blastomere in the 16-cell embryo. LFQ intensity was validated as a quantitative proxy for protein abundance. Correlation analysis was performed to compare protein quantities between the embryo and n = 3 different single D11 blastomeres, which are fated to develop into the nervous system. A total of 335 nonredundant protein groups were quantified in union between the single D11 cells spanning a 4 log-order concentration range. LFQ and correlation analysis detected expected proteomic differences between the whole embryo and blastomeres, and also found translational differences between individual D11 cells. LFQ on single cells raises exciting possibilities to study gene expression in other cells and models to help better understand cell processes on a systems biology level., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Artificial collapse of the blastocoel cavity before vitrification can improve the quality of warmed embryos, yet how reduction of blastocoel fluid impacts formation of the blastocyst cell lineages is not clear. The present study assessed the effect of pre-vitrification blastocoel fluid reduction on the survival, hatching rate, and the expression of genes related to apoptosis (Tp53), pluripotency (Pou5f1, Nanog), and differentiation (Cdx2, Eomes, Gata6) in mouse blastocysts. In vivo-produced blastocysts were randomly divided into three groups: The first group was vitrified and warmed; the second group underwent artificial collapse of the blastocoel cavity prior to vitrification and warming; the third group served as the control, in which neither vitrification or artificial collapse was performed. The survival rate of treatment groups was similar to the control group, whereas the hatching rate of artificial collapse/vitrified blastocysts was significantly higher than vitrified blastocysts. Quantitative reverse-transcription PCR analysis revealed a considerable reduction in the expression of Cdx2, Eomes, Gata6, Grb2, and Tp53 transcripts following artificial collapse/vitrification in comparison to the vitrification-alone group; the abundance of Pou5f1 and Nanog, however, did not change. These results suggest that artificial collapse of the blastocoel cavity before vitrification leads to relatively normal expression of apoptosis and development-related genes plus higher hatching rates. Mol. Reprod. Dev. 83: 735-742, 2016 © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Recent advances in genome editing have facilitated the generation of nonhuman primate (NHP) models, with potential to unmask the complex biology of human disease not revealed by rodent models. However, their broader use is hindered by the challenges associated with generation of adult NHP models as well as the cost of their production. Here, we describe the generation of a marmoset model of severe combined immunodeficiency (SCID). This study optimized zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) to target interleukin-2 receptor subunit gamma (IL2RG) in pronuclear stage marmoset embryos. Nine of 21 neonates exhibited mutations in the IL2RG gene, concomitant with immunodeficiency, and three neonates have currently survived from 240 days to 1.8 years. Our approach demonstrates highly efficient production of founder NHP with SCID phenotypes, with promises of multiple pre-clinical and translational applications., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Ovarian xenotransplantation is a promising alternative to preserve fertility of oncologic patients. However, several functional aspects of this procedure remained to be addressed. The aim of this study was evaluate the feasibility of xenotransplantation as a strategy to maintain bovine ovarian grafts and produce oocytes. Adult ovarian cortical pieces were xenotransplanted to the dorsal subcutaneous of female NOD-SCID mice (n = 62). Grafts were recovered ten days after xenotransplantation. Host and graft weights; folliculogenesis progression; blood perfusion, relative gene expression and number of macrophage and neutrophil of xenografts; in vitro developmental competence of graft-derived oocytes were evaluated. Folliculogenesis was supported in the grafts, as indicated by the presence of primordial, primary, secondary, antral, and atretic follicles. The xenografts showed a greater volumetric density of atretic follicles and higher hyperemia and number of host-derived macrophage and neutrophil (P<0.05), when compared to non-grafted fragments. There was a higher blood perfusion under the back skin in the transplantation sites of host animals than in control and non-grafted (P<0.01). BAX and PRDX1 genes were up-regulated, while BCL2, FSHR, IGF1R and IGF2R were down-regulated, when compared to the control (P<0.01). Twenty seven oocytes were successfully harvested from grafts, and some of these oocytes were able to give rise to blastocysts after in vitro fertilization. However, cleavage and blastocyst rates of xenograft derived oocytes were lower than in control (P<0.01). Despite showing some functional modifications, the ovarian xenografts were able to support folliculogenesis and produce functional oocytes.

Protamines are expressed in the spermatid nucleus and allow denser packaging of DNA compared with histones. Disruption of the coding sequence of one allele of either protamine 1 (Prm1) or Prm2 results in failure to produce offspring, although sperm with disrupted Prm1 or Prm2 alleles are produced. Here, we produced Prm1-deficient female chimeric mice carrying Prm1-deficient oocytes. These mice successfully produced Prm1(+/-) male mice. Healthy Prm1(+/-) offspring were then produced by transferring blastocysts obtained via in vitro fertilization using zona-free oocytes and sperm from Prm1(+/-) mice. This result suggests that sperm lacking Prm1 can generate offspring despite being abnormally shaped and having destabilised DNA, decondensed chromatin and a reduction in mitochondrial membrane potential. Nevertheless, these mice showed little derangement of expression profiles.

The decision by embryonic ectoderm to give rise to epidermal versus neural derivatives is the result of signaling events during blastula and gastrula stages. However, there also is evidence in Xenopus that cleavage stage blastomeres contain maternally derived molecules that bias them toward a neural fate. We used a blastomere explant culture assay to test whether maternally deposited transcription factors bias 16-cell blastomere precursors of epidermal or neural ectoderm to express early zygotic neural genes in the absence of gastrulation interactions or exogenously supplied signaling factors. We found that Foxd4l1, Zic2, Gmnn, and Sox11 each induced explants made from ventral, epidermis-producing blastomeres to express early neural genes, and that at least some of the Foxd4l1 and Zic2 activities are required at cleavage stages. Similarly, providing extra Foxd4l1 or Zic2 to explants made from dorsal, neural plate-producing blastomeres significantly increased the expression of early neural genes, whereas knocking down either significantly reduced them. These results show that maternally delivered transcription factors bias cleavage stage blastomeres to a neural fate. We demonstrate that mouse and human homologs of Foxd4l1 have similar functional domains compared to the frog protein, as well as conserved transcriptional activities when expressed in Xenopus embryos and blastomere explants. genesis 54:334-349, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The aim of this study was to explore whether the morphology of polar bodies (PBs) estimated at 16-18 h after insemination can be used as an additional marker for predicting human embryo quality or pregnancy outcome. The data from 355 patients who received standard in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) treatment after controlled ovarian hyperstimulation were recruited. Normal fertilized 3048 zygotes from 382 cycles were divided into two groups, PBs intact or fragmented, according to the morphology of PBs assessed at 16-18 h after insemination. Embryo quality and pregnancy outcome were compared between the two groups. It was shown that the day 3 (D3) good embryo rate, good quality blastocyst rate and available embryo rate of the PBs intact group were all significantly higher than that of the corresponding fragmented groups. However, no significant differences in pregnancy rate (PR) or implantation rate (IR) were observed between the intact and fragmented groups. Although PBs morphology estimated at 16-18 h after insemination had little effect on PR or IR in fresh embryo transfer cycles, a better embryo quality can be achieved in the PB-intact group, which is valuable for embryo selection.

Maternal factors initiate the zygotic developmental program in animal embryos. In embryos of the chordate, Ciona intestinalis, three maternal factors-Gata.a, β-catenin, and Zic-r.a-are required to establish three domains of gene expression at the 16-cell stage; the animal hemisphere, vegetal hemisphere, and posterior vegetal domains. Here, we show how the maternal factors establish these domains. First, only β-catenin and its effector transcription factor, Tcf7, are required to establish the vegetal hemisphere domain. Second, genes specifically expressed in the posterior vegetal domain have additional repressive cis-elements that antagonize the activity of β-catenin/Tcf7. This antagonizing activity is suppressed by Zic-r.a, which is specifically localized in the posterior vegetal domain and binds to DNA indirectly through the interaction with Tcf7. Third, Gata.a directs specific gene expression in the animal hemisphere domain, because β-catenin/Tcf7 weakens the Gata.a-binding activity for target sites through a physical interaction in the vegetal cells. Thus, repressive regulation through protein-protein interactions among the maternal transcription factors is essential to establish the first distinct domains of gene expression in the chordate embryo.

Dramatic genome dynamics, such as chromosome instability, contribute to the remarkable genomic heterogeneity among the blastomeres comprising a single embryo during human preimplantation development. This heterogeneity, when compatible with life, manifests as constitutional mosaicism, chimerism, and mixoploidy in live-born individuals. Chimerism and mixoploidy are defined by the presence of cell lineages with different parental genomes or different ploidy states in a single individual, respectively. Our knowledge of their mechanistic origin results from indirect observations, often when the cell lineages have been subject to rigorous selective pressure during development. Here, we applied haplarithmisis to infer the haplotypes and the copy number of parental genomes in 116 single blastomeres comprising entire preimplantation bovine embryos (n = 23) following in vitro fertilization. We not only demonstrate that chromosome instability is conserved between bovine and human cleavage embryos, but we also discovered that zygotes can spontaneously segregate entire parental genomes into different cell lineages during the first post-zygotic cleavage division. Parental genome segregation was not exclusively triggered by abnormal fertilizations leading to triploid zygotes, but also normally fertilized zygotes can spontaneously segregate entire parental genomes into different cell lineages during cleavage of the zygote. We coin the term "heterogoneic division" to indicate the events leading to noncanonical zygotic cytokinesis, segregating the parental genomes into distinct cell lineages. Persistence of those cell lines during development is a likely cause of chimerism and mixoploidy in mammals., (© 2016 Destouni et al.; Published by Cold Spring Harbor Laboratory Press.)

Chromosomal abnormalities are common in human embryos. Previous studies have suggested links between centrosome number and chromosome abnormalities, but information regarding abnormalities in centrosome number in human embryos is limited. We analyzed abnormalities in centrosome number in human embryos and embryonic stem cells (hESCs). Following normal fertilization, supernumerary centrosomes were present at rates of 7.3% in two-pronucleus (2PN)-stage zygotes and 6.5% in first-cleavage zygotes. Supernumerary centrosomes were also detected in 24.4% of blastomeres from 60% of embryos derived from 2PN zygotes. Conversely, in mono- (1PN) and tri-pronucleus (3PN) zygotes, the frequency of abnormal centrosome number increased substantially at first cleavage. Rates in blastomeres of Day-3 embryos, however, were about the same between embryos derived from 1PN and 2PN zygotes, whereas abnormalities in centrosome number were higher in those from 3PN zygotes. By comparison, the rate of abnormal centrosome numbers in hESCs was 1.5-11.2%. Thus, abnormalities in centrosome number existed in human zygotes and cleaved embryos-especially those resulting from aberrant fertilization-but the frequency of such abnormalities was lower in hESCs derived from these embryos. These findings identify a source of the chromosomal instability in human embryos and hESCs, and highlight new safety issues for human assisted reproductive technology. Mol. Reprod. Dev. 83: 392-404, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The adult striped pattern of zebrafish is composed of melanophores, iridophores and xanthophores arranged in superimposed layers in the skin. Previous studies have revealed that the assembly of pigment cells into stripes involves heterotypic interactions between all three chromatophore types. Here we investigate the role of homotypic interactions between cells of the same chromatophore type. Introduction of labelled progenitors into mutants lacking the corresponding cell type allowed us to define the impact of competitive interactions via long-term in vivo imaging. In the absence of endogenous cells, transplanted iridophores and xanthophores show an increased rate of proliferation and spread as a coherent net into vacant space. By contrast, melanophores have a limited capacity to spread in the skin even in the absence of competing endogenous cells. Our study reveals a key role for homotypic competitive interactions in determining number, direction of migration and individual spacing of cells within chromatophore populations.

This study investigated re-expansion dynamics during culture of bovine blastocysts cryopreserved either by slow-freezing or vitrification. Also, the extent and localization of membrane damage and DNA fragmentation in re-expanded embryos were studied. Frozen-thawed embryos showed a significantly lower re-expansion rate during 24 h of post-thawing culture compared to vitrified embryos. Vitrified embryos reached the maximum level of re-expansion rate by 12 h of culture whereas frozen embryos showed a gradual increase in re-expansion rate by 24 h of culture. When assayed by Hoechst/propidium iodide staining there was no difference in the numbers and ratio of membrane damaged cells between re-expanded frozen and vitrified embryos; however, the extent of membrane damage in blastomeres was significantly higher in both groups compared with non-cryopreserved embryos (control). TUNEL assay combined with differential ICM and TE staining revealed a significantly higher number and ratio of TE cells showing DNA-fragmentation in frozen-thawed re-expanded blastocysts compared to vitrified ones; however, vitrification also resulted in an increased extent of DNA fragmentation in TE cells compared with control blastocysts. In frozen-thawed blastocysts increased extent of DNA fragmentation was associated with reduced numbers and proportion of TE cells compared with vitrified and control embryos. The number and ratio of ICM cells and the extent of DNA fragmentation in ICM did not differ among control, frozen and vitrified groups. In conclusion, compared with vitrified embryos, blastocysts preserved by slow-freezing showed a delayed timing of re-expansion which was associated with an increased frequency of DNA fragmentation in TE cells., (Copyright © 2016 Elsevier Inc. All rights reserved.)

During mouse early embryogenesis, blastomeres increase in number by the morula stage. Among them, the outer cells are polarized and differentiated into trophectoderm (TE), while the inner cells remain unpolarized and give rise to inner cell mass (ICM). TE provides an important liquid environment for ICM development. In spite of extensive research, the molecular mechanisms underlying TE formation are still obscure. In order to investigate the roles of estrogen receptor α (ERα) in this course, mouse 8-cell embryos were collected and cultured in media containing ERα specific antagonist MPP and/or agonist PPT. The results indicated that MPP treatment inhibits blastocyst formation in a dose-dependent manner, while PPT, at proper concentration, promotes the cavitation ratio of mouse embryos. Immunofluorescence staining results showed that MPP significantly decreased the nuclear expression of CDX2 in morula, but no significant changes of OCT4 were observed. Moreover, after MPP treatment, the expression levels of the genes related to TE specification, Tead4, Gata3 and Cdx2, were significantly reduced. Overall, these results indicated that ERα might affect mouse embryo cavitation by regulating TE lineage differentiation., (© 2016 Japanese Society of Developmental Biologists.)

Cell lineage determination during early embryogenesis has profound effects on adult animal development. Pre-patterning of embryos, such as that of Drosophila and Caenorhabditis elegans, is driven by asymmetrically localized maternal or zygotic factors, including mRNA species and RNA binding proteins. However, it is not clear how mammalian early embryogenesis is regulated and what the early cell fate determinants are. Here we show that, in mouse, mitochondrial ribosomal RNAs (mtrRNAs) are differentially distributed between 2-cell sister blastomeres. This distribution pattern is not related to the overall quantity or activity of mitochondria which appears equal between 2-cell sister blastomeres. Like in lower species, 16S mtrRNA is found to localize in the cytoplasm outside of mitochondria in mouse 2-cell embryos. Alterations of 16S mtrRNA levels in one of the 2-cell sister blastomere via microinjection of either sense or anti-sense RNAs drive its progeny into different cell lineages in blastocyst. These results indicate that mtrRNAs are differentially distributed among embryonic cells at the beginning of embryogenesis in mouse and they are functionally involved in the regulation of cell lineage allocations in blastocyst, suggesting an underlying molecular mechanism that regulates pre-implantation embryogenesis in mouse., (© 2016 Society for Reproduction and Fertility.)

Mouse parthenogenetic embryonic stem cells (PgESCs) could be applied to study imprinting genes and are used in cell therapy. Our previous study found that stem cells established by aggregation of two parthenogenetic embryos at 8-cell stage (named as a2 PgESCs) had a higher efficiency than that of PgESCs, and the paternal expressed imprinting genes were observably upregulated. Therefore, we propose that increasing the number of parthenogenetic embryos in aggregation may improve the development of parthenogenetic mouse and imprinting gene expression of PgESCs. To verify this hypothesis, we aggregated four embryos together at the 4-cell stage and cultured to the blastocyst stage (named as 4aPgB). qPCR detection showed that the expression of imprinting genes Igf2, Mest, Snrpn, Igf2r, H19, Gtl2 in 4aPgB were more similar to that of fertilized blastocyst (named as fB) compared to 2aPgB (derived from two 4-cell stage parthenogenetic embryos aggregation) or PgB (single parthenogenetic blastocyst). Post-implantation development of 4aPgB extended to 11 days of gestation. The establishment efficiency of GFP-a4 PgESCs which derived from GFP-4aPgB is 62.5%. Moreover, expression of imprinting genes Igf2, Mest, Snrpn, notably downregulated and approached the level of that in fertilized embryonic stem cells (fESCs). In addition, we acquired a 13.5-day fetus totally derived from GFP-a4 PgESCs with germline contribution by 8-cell under zona pellucida (ZP) injection. In conclusion, four embryos aggregation improves parthenogenetic development, and compensates imprinting genes expression in PgESCs. It implied that a4 PgESCs could serve as a better scientific model applied in translational medicine and imprinting gene study., (© 2016 Japanese Society of Developmental Biologists.)

By evolving bilaterally separated shell plates, bivalves acquired a unique body plan in which their soft tissues are completely protected by hard shell plates. In this unique body plan, mobility between the separated shell plates is provided by novel structures such as a ligament and adductor muscles. As a first step towards understanding how the bivalve body plan was established, we investigated the development of the separated shell plates and ligament. Over 100 years ago, it was hypothesized that the development of separated shell plates is tightly linked with the unique cell cleavage (division) pattern of bivalves during development, wherein each bilateral daughter cell of the 2d descendant 2d(1121) develops into one of the bilateral shell fields. In this study, we tested this hypothesis by tracing the cell lineages of the Japanese purple mussel Septifer virgatus. Although the shell fields were found to be exclusively derived from the bilateral descendant cells of 2d: 2d(11211) and 2d(11212), the descendants of these cells were not restricted to shell fields alone, nor were they confined to the left or right side of the shell field based on their lineage. Our study demonstrated that ligament cells are also derived from 2d(11211) and 2d(11212), indicating that the ligament cells emerged as a subpopulation of shell field cells. This also suggests that the establishment of the novel developmental system for the ligament cells was critical for the evolution of the unique body plan of bivalves., (© 2016 The Author(s).)

As one of the key nutrient sensors, insulin signaling plays an important role in integrating environmental energy cues with organism growth. In adult organisms, relative insufficiency of insulin signaling induces compensatory expansion of insulin-secreting pancreatic beta (β) cells. However, little is known about how insulin signaling feedback might influence neogenesis of β cells during embryonic development. Using genetic approaches and a unique cell transplantation system in developing zebrafish, we have uncovered a novel role for insulin signaling in the negative regulation of pancreatic progenitor cell differentiation. Blocking insulin signaling in the pancreatic progenitors hastened the expression of the essential β cell genes insulin and pdx1, and promoted β cell fate at the expense of alpha cell fate. In addition, loss of insulin signaling promoted β cell regeneration and destabilization of alpha cell character. These data indicate that insulin signaling constitutes a tunable mechanism for β cell compensatory plasticity during early development. Moreover, using a novel blastomere-to-larva transplantation strategy, we found that loss of insulin signaling in endoderm-committed blastomeres drove their differentiation into β cells. Furthermore, the extent of this differentiation was dependent on the function of the β cell mass in the host. Altogether, our results indicate that modulation of insulin signaling will be crucial for the development of β cell restoration therapies for diabetics; further clarification of the mechanisms of insulin signaling in β cell progenitors will reveal therapeutic targets for both in vivo and in vitro β cell generation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The parental genomes are initially spatially separated in each pronucleus after fertilization. Here we have used green-to-red photoconversion of Dendra2-H2B-labeled pronuclei to distinguish maternal and paternal chromatin domains and to track their spatial distribution in living Caenorhabditis elegans embryos starting shortly after fertilization. Intermingling of the parental chromatin did not occur until after the division of the AB and P1 blastomeres, at the 4-cell stage. Unexpectedly, we observed that the intermingling of chromatin did not take place during mitosis or during chromatin decondensation, but rather ∼ 3-5 minutes into the cell cycle. Furthermore, unlike what has been observed in mammalian cells, the relative spatial positioning of chromatin domains remained largely unchanged during prometaphase in the early C. elegans embryo. Live imaging of photoconverted chromatin also allowed us to detect a reproducible 180° rotation of the nuclei during cytokinesis of the one-cell embryo. Imaging of fluorescently-labeled P granules and polar bodies showed that the entire embryo rotates during the first cell division. To our knowledge, we report here the first live observation of the initial separation and subsequent mixing of parental chromatin domains during embryogenesis.

Bovine embryos are now routinely used in agricultural systems as a means of disseminating superior genetics worldwide, ultimately with the aim of feeding an ever-growing population. Further investigations, common for human IVF embryos, thus have priority to improve cattle IVF, as has screening for aneuploidy (abnormal chromosome number). Although the incidence and consequences of aneuploidy are well documented in human preimplantation embryos, they are less well known for the embryos of other animals. To address this, we assessed aneuploidy levels in thirty-one 2-cell bovine embryos derived from early- and late-cleaving zygotes. Contemporary approaches ( Whole Genome Amplification and next-generation sequencing) allowed aneuploidy assessment for all chromosomes in oocytes from donors aged 4-7 years. We also quantified mitochondrial DNA (mtDNA) levels in all blastomeres assessed, thereby testing the hypothesis that they are related to levels of aneuploidy. The overall incidence of aneuploidy in this cohort of bovine embryos was 41.1% and correlated significantly with the timing of cleavage (77.8% in late-cleaving vs. 31.8% in early-cleaving embryos). Moreover, based on mtDNA sequence read counts, we observed that the median mtDNA quantity is significantly lower in late-cleaving embryos. These findings further reinforce the use of the bovine system as a model for human IVF studies., (© 2016 S. Karger AG, Basel.)