Blastomeres Of Early Embryos Research Articles

USP7 reduction leads to developmental failure of mouse early embryos

As a member of Ubiquitin-specific protease subfamily, ubiquitin specific protease 7 (USP7) has been reported to participate in a variety of cellular processes, including cell cycle, apoptosis, DNA damage response, and epigenetic modification. However, its function in preimplantation embryos is still obscure. To investigate the functions of USP7 during preimplantation embryo development, we used siRNA to degrade endogenous USP7 messenger RNA. We found that USP7 knockdown significantly decreased the development rate of mouse early embryos. Moreover, depletion of USP7 induced the accumulation of the DNA lesions and apoptotic blastomeres in early embryos. In addition, USP7 knockdown caused an abnormal H3K27me3 modification in 2-cell embryos. Overall, our results indicate that USP7 maintains genome stability perhaps via regulating H3K27me3 and DNA damage, consequently controlling the embryo quality.

SETD2 reduction adversely affects the development of mouse early embryos.

SET domain-containing protein 2 (SETD2), the protein of regulating trimethylation status of histone H3 at lysine 36 (H3K36), participates in the maintenance of chromatin architecture, transcription elongation, genome stability, and other biological events. However, its function in preimplantation embryos is still obscure. In this study, specific small interfering RNA was employed to investigate the functions of SETD2. We find that deletion of SETD2 results in the developmental delay of mouse early embryos, indicative of the compromised developmental potential. Remarkably, SETD2 knockdown induces the accumulation of the DNA lesions and apoptotic blastomeres in early embryos. In addition, the methylation level of H3K36 is significantly reduced in two-cell embryos depleted of SETD2. In summary, our data indicate that SETD2 maintains genome stability perhaps via regulating trimethylation status of H3K36, consequently controlling the embryo quality. These findings pave the avenue for understanding the cross-talk between epigenome and SETD2 during early embryo development.

Chemical enhancement in embryo development and stem cell derivation from single blastomeres.

Several chemicals targeting the mitogen-activated protein (MAP) kinase signaling pathway, which play an important role in regulating cell growth and differentiation, have shown enhancing effects on the development of the inner cell mass (ICM) and the derivation of ES cells. However, investigation of such chemicals on early embryonic development and the establishment of ES cell lines has not been elucidated. This study was aimed to determine if ACTH, MAP2K1 inhibitor [MAP2K1 (I)], and MAPK14 inhibitor [MAPK14 (I)] could enhance the development of the ICM in preimplantation mouse embryos and blastocyst outgrowths, and the establishment of ES cell lines from blastomeres of early embryos. We have demonstrated that both MAP2K1 (I) and MAPK14 (I) delay early embryo development and inhibit the development of embryos from early blastomeres. On the other hand, ACTH had a positive effect on embryos derived from early blastomeres. As a result, 17 ES cell lines were established. Among these ES cell lines, nine and five ES cell lines were established from single blastomeres of two-cell embryos with and without the supplement of ACTH, respectively. In addition to two-cell isolated blastomeres, three ES cell lines were established from blastomeres of four-cell embryos only with the supplement of ACTH. Our results suggest that ACTH can enhance the derivation of ES cells from single blastomere-derived embryos.

Global Transcriptional Repression in C. elegans Germline Precursors by Regulated Sequestration of TAF-4

In C. elegans, four asymmetric divisions, beginning with the zygote (P0), generate transcriptionally repressed germline blastomeres (P1-P4) and somatic sisters that become transcriptionally active. The protein PIE-1 represses transcription in the later germline blastomeres but not in the earlier germline blastomeres P0 and P1. We show here that OMA-1 and OMA-2, previously shown to regulate oocyte maturation, repress transcription in P0 and P1 by binding to and sequestering in the cytoplasm TAF-4, a component critical for assembly of TFIID and the pol II preinitiation complex. OMA-1/2 binding to TAF-4 is developmentally regulated, requiring phosphorylation by the DYRK kinase MBK-2, which is activated at meiosis II after fertilization. OMA-1/2 are normally degraded after the first mitosis, but ectopic expression of wild-type OMA-1 is sufficient to repress transcription in both somatic and later germline blastomeres. We propose that phosphorylation by MBK-2 serves as a developmental switch, converting OMA-1/2 from oocyte to embryo regulators.

Mechanisms of cell positioning during C. elegans gastrulation.

Cell rearrangements are crucial during development. In this study, we use C. elegans gastrulation as a simple model to investigate the mechanisms of cell positioning. During C. elegans gastrulation, two endodermal precursor cells move from the ventral surface to the center of the embryo, leaving a gap between these ingressing cells and the eggshell. Six neighboring cells converge under the endodermal precursors, filling this gap. Using an in vitro system, we observed that these movements occurred consistently in the absence of the eggshell and the vitelline envelope. We found that movement of the neighbors towards each other is not dependent on chemotactic signaling between these cells. We further found that C. elegans gastrulation requires intact microfilaments, but not microtubules. The primary mechanism of microfilament-based motility does not appear to be through protrusive structures, such as lamellipodia or filopodia. Instead, our results suggest an alternative mechanism. We found that myosin activity is required for gastrulation, that the apical sides of the ingressing cells contract, and that the ingressing cells determine the direction of movement of their neighboring cells. Based on these results, we propose that ingression is driven by an actomyosin-based contraction of the apical side of the ingressing cells, which pulls neighboring cells underneath. We conclude that apical constriction can function to position blastomeres in early embryos, even before anchoring junctions form between cells.

Characterization of an ascidian DEAD-box gene, Ci-DEAD1: specific expression in the germ cells and its mRNA localization in the posterior-most blastomeres in early embryos.

We isolated DEAD-box genes from three ascidian species (Ciona intestinalis, Ciona savignyi, and Halocynthia roretzi) by polymerase chain reaction methods. We obtained two types from each of C. intestinalis and C. savignyi, and four types from H. roretzi. The first type (DEAD1) belonged to the vasa subfamily, the second type (DEAD2) to the PL10 subfamily, the third type (DEAD3) to the p68 subfamily, and the forth type (DEAD4) did not belong to any of the subfamilies. We further analyzed in detail the expression pattern of C. intestinalis vasa-type gene (Ci-DEAD1) by in situ hybridization. In sections of the ovary and testis, the Ci-DEAD1-specific probe reacted intensely to small germ cells, oogonium, and/or oocyte and spermatogonium and/or spermatocyte, respectively. In whole-mount specimens of juveniles this probe specifically reacted to the primordial germ cells in the gonad rudiment. These gonad-specific expressions were confirmed by reverse transcriptase polymerase chain reaction of RNA from various tissues. The transcript was present in unfertilized eggs and in the central cytoplasm of blastomeres until the two-cell stage. During the second cleavage a part of the transcripts moved to the posterior region of embryos and, during early embryogenesis, was localized in the posterior-most blastomeres. In the tailbud, one or two hybridization signals were detected in the caudal endodermal strand. Based on these observations, we propose precursors of primordial germ cells in ascidians.

Cec-1,a Soma-Specific Chromobox-Containing Gene inC. elegans

The chromo domain is a phylogenetically conserved sequence motif which was identified as a region of homology between the repressor protein Pc and the heterochromatin constitutive protein HP1 ofDrosophila.The specific function of the chromo domain is not yet understood, but it seems to be required for protein–protein interactions in chromatin-associated complexes. Here, we present a new chromobox-containing gene fromCaenorhabditis elegans(cec-1). It encodes a nuclear protein that is present in all somatic cells from the 50- to 80-cell stage on throughout development and in adult animals. Nocec-1protein was detected in the cells of early embryos, in germ cells, and in their precursor cells Z2 and Z3.cec-1mRNA, however, is already present in all the blastomeres of early embryos. Immunolocalization experiments revealed a homogeneous distribution of CEC-1 within interphase nuclei, while during mitosis CEC-1 seems to dissociate from the condensing chromosomes. The expression pattern of thecec-1gene suggests that it may represent a new regulatory gene inC. elegans.

Differentiation Expression in Blastomeres of Cleavage-Arrested Embryos of the Ascidian Halocynthia roretzi: (differentiation without cleavage/monoclonal antibodies/exclusive differentiation/ascidian embryo).

The question whether two or more different genetic programs are expressed in the common cytoplasm of single blastomeres or the expression of one genetic program somehow excludes expression of the other, was analyzed by assessing the occurrence of a muscle-specific and two epidermis-specific antigens in cleavage-arrested blastomeres in early embryos of the ascidian Halocynthia roretzi. Blastomeres which had been arrested in 1- to 4-cell stages expressed only the epidermis markers. Arrested 8-cell to 32-cell embryos produced both epidermis and muscle markers, but each cell expressed only one program of differentiation, even though some possessed the potential to express both. The differentiation expressions followed their cell lineages. These results indicate that at least in this experimental system differentiation markers of the two different cell-types are expressed exclusively. A distinct order was noticed in expression of the two epidermis markers in a single blastomeres; a marker is always superiorly expressed, whereas the other appears only when the superior marker is expressed.

Electrical coupling of blastomeres in early embryos of ascidians and sea urchins

In the early embryos of ascidians and sea urchins, blastomeres are in electrical communication; however, the type and extent of interaction is related to the basic characteristics of the embryo. In the mosaic-like structure of the ascidian embryo, blastomeres have a coupling ratio of about 1 throughout the division cycle. Coupling is facilitated by the extremely low conductance of the non-junctional membrane and possibly mediated via specialized low resistance junctions. Sea urchin embryos do not have specialized low resistance junctions; however, blastomeres are electrically coupled, probably via cytoplasmic bridges, during the first half of the division cycle. The coupling ratio in sea urchins, initially about 0.3, progressively decreases, together with the conductance of the nonjunctional membrane. During the latter half of the division cycle blastomeres are uncoupled; however, a structural junction appears at the periphery, which may play a role in their destiny.

Celestin tubes

<h3>Abstract</h3> The activation of Wnt/β-catenin (cWnt) signaling at the future posterior end of early embryos is a highly conserved mechanism for initiating pattern formation along the anterior-posterior (AP) axis in bilaterians. Moreover, in many bilaterian taxa, in addition, to activation of cWnt signaling at the posterior end, inhibition of cWnt signaling at the anterior end is required for normal development of anterior structures. In most cases, inhibition of cWnt signaling at the anterior end occurs around gastrulation and it is typically mediated by secreted factors that block signal transduction through the cWnt cell surface receptor-ligand complex. This phenomenon has been fairly well characterized, but the emerging role for intracellular inhibition of cWnt signaling in future anterior blastomeres—in cleavage stage embryos—to regulate correct AP patterning is less well understood. To investigate this process in an invertebrate deuterostome embryo we studied the function of Axin, a critical negative regulator of cWnt signaling, during early sea urchin embryogenesis. Sea urchin <i>Axin</i> is ubiquitously expressed in early embryos and by the blastula stage the expression of the gene becomes restricted to the posterior end of the embryo. Strikingly, knockdown of Axin protein levels using antisense Axin morpholinos (MO) led to ectopic nuclearization of β-catenin and activation of endomesoderm gene expression in anterior blastomeres in early embryos. These embryos developed a severely posteriorized phenotype that could be fully rescued by co-injection of Axin MO with wild-type Axin mRNA. Axin is known to negatively regulate cWnt by its role in mediating β-catenin stability within the destruction complex. Consistent with this function overexpression of Axin by mRNA injection led to the downregulation of nuclear β-catenin, inhibition of endomesoderm specification and a strong anteriorization of embryos. Axin has several well-defined domains that regulate its interaction with β-catenin and the key regulators of the destruction complex, Adenomatous Polyposis Coli (APC), Glycogen Synthase Kinase 3β(GSK-3β), and Dishevelled (Dvl). Using Axin constructs with single deletions of the binding sites for these proteins we showed that only the GSK-3βbinding site on Axin is required for its inhibition of cWnt in the sea urchin embryo. Strikingly, overexpression of the GSK-3β-binding domain alone led to embryos with elevated levels of endomesoderm gene expression and a strongly posteriorized phenotype. These results indicated that Axin has a critical global role in inhibiting cWnt signaling in the early sea urchin embryo, and moreover, that the interaction of Axin with GSK-3βis critical for this inhibition. These results also add to the growing body of evidence that Axin plays a global function in suppressing cWnt signaling in early embryos and indicates that modulation of Axin function may be a critical early step during patterning of the AP axis during bilaterian development