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In brief Trophectoderm is the first tissue to differentiate in the early mammalian embryo and is essential for hatching, implantation, and placentation. This review article discusses the roles of Ras homolog family members (RHO) and RHO-associated coiled-coil containing protein kinases (ROCK) in the molecular and cellular regulation of trophectoderm formation. Abstract The trophectoderm (TE) is the first tissue to differentiate during the preimplantation development of placental mammals. It constitutes the outer epithelial layer of the blastocyst and is responsible for hatching, uterine attachment, and placentation. Thus, its formation is the key initial step that enables the viviparity of mammals. Here, we first describe the general features of TE formation at the morphological and molecular levels. Prospective TE cells form an epithelial layer enclosing an expanding fluid-filled cavity by establishing the apical-basal cell polarity, intercellular junctions, microlumen, and osmotic gradient. A unique set of genes is expressed in TE that encode the transcription factors essential for the development of trophoblasts of the placenta upon implantation. TE-specific gene expressions are driven by the inhibition of HIPPO signaling, which is dependent on the prior establishment of the apical-basal polarity. We then discuss the specific roles of RHO and ROCK as essential regulators of TE formation. RHO and ROCK modulate the actomyosin cytoskeleton, apical-basal polarity, intercellular junctions, and HIPPO signaling, thereby orchestrating the epithelialization and gene expressions in TE. Knowledge of the molecular mechanisms underlying TE formation is crucial for assisted reproductive technologies in human and farm animals, as it provides foundation to help improve procedures for embryo handling and selection to achieve better reproductive outcomes.

Remdesivir (RDV) is the first antiviral drug to be approved by the US Food and Drug Administration for the treatment of COVID-19. While the general safety of RDV has been studied, its reproductive risk, including embryotoxicity, is largely unknown. Here, to gain insights into its embryotoxic potential, we investigated the effects of RDV on mouse preimplantation embryos cultured in vitro at the concentrations comparable to the therapeutic plasma levels. Exposure to RDV (2-8 µM) did not affect the initiation of blastocyst formation, although the maintenance of the cavity failed at 8 µM due to increased cell death. While exposure to 2-4 µM permitted the cavity maintenance, expressions of developmental regulator genes associated with the inner cell mass (ICM) lineage were significantly diminished. Adverse effects of RDV depended on the duration and timing of exposure, as treatment between the 8-cell to early blastocyst stage most sensitively affected cavity expansion, gene expressions, and cell proliferation, particularly of the ICM than the trophectoderm lineage. GS-441524, a major metabolite of RDV, did not impair blastocyst formation or cavity expansion, although it altered gene expressions in a manner differently from RDV. Additionally, RDV reduced the viability of human embryonic stem cells, which were used as a model for the human ICM lineage, more potently than GS-441524. These findings suggest that RDV is potentially embryotoxic to impair the pluripotent lineage, and will be useful for designing and interpreting further in vitro and in vivo studies on the reproductive toxicity of RDV.

Pluripotent stem cells recapitulate many aspects of embryogenesis in vitro. Here, we established a novel culture system to differentiate human embryonic stem cell aggregates (HESCA), and evaluated its utility for teratogenicity assessment. Culture of HESCA with modulators of developmental signals induced morphogenetic and molecular changes associated with differentiation of the paraxial mesoderm and neuroectoderm. To examine impact of teratogenic exposures on HESCA differentiation, 18 compounds were tested, for which adequate information on in vivo plasma concentrations is available. HESCA treated with each compound were examined for gross morphology and transcript levels of 15 embryogenesis regulator genes. Significant alterations in the transcript levels were observed for 94% (15/16) of the teratogenic exposures within 5-fold margin, whereas no alteration was observed for 92% (11/12) of the non-teratogenic exposures. Our study demonstrates that transcriptional changes in HESCA serve as predictive indicator of teratogenicity in a manner comparable to in vivo exposure levels.

Early embryos are vulnerable to environmental insults, such as medications taken by the mother. Due to increasing prevalence of hypercholesterolemia, more women of childbearing potential are taking cholesterol-lowering medications called statins. Previously, we showed that inhibition of the mevalonate pathway by statins impaired mouse preimplantation development, by modulating HIPPO signaling, a key regulator for trophectoderm (TE) lineage specification. Here, we further evaluated molecular events that are altered by mevalonate pathway inhibition during the timeframe of morphogenesis and cell lineage specification. Whole transcriptome analysis revealed that statin treatment dysregulated gene expression underlying multiple processes, including cholesterol biosynthesis, HIPPO signaling, cell lineage specification and endoplasmic reticulum (ER) stress response. We explored mechanisms that link the mevalonate pathway to ER stress, because of its potential impact on embryonic health and development. Upregulation of ER stress-responsive genes was inhibited when statin-treated embryos were supplemented with the mevalonate pathway product, geranylgeranyl pyrophosphate (GGPP). Inhibition of geranylgeranylation was sufficient to upregulate ER stress-responsive genes. However, ER stress-responsive genes were not upregulated by inhibition of ras homolog family member A (RHOA), a geranylgeranylation target, although it interfered with TE specification and blastocyst cavity formation. In contrast, inhibition of Rac family small GTPase 1 (RAC1), another geranylgeranylation target, upregulated ER stress-responsive genes, while it did not impair TE specification or cavity formation. Thus, our study suggests that the mevalonate pathway regulates cellular homeostasis (ER stress repression) and differentiation (TE lineage specification) in preimplantation embryos through GGPP-dependent activation of two distinct small GTPases, RAC1 and RHOA, respectively. Translation of the findings to human embryos and clinical settings requires further investigations.

STUDY QUESTION: What molecular signals are required to maintain the functional trophectoderm (TE) during blastocyst expansion of the late stage of preimplantation development? SUMMARY ANSWER: The activity of ras homology family member A (RHOA) GTPases is necessary to retain the expanded blastocyst cavity and also to sustain the gene expression program specific to TE. WHAT IS KNOWN ALREADY: At the early stages of preimplantation development, the precursor of the TE lineage is generated through the molecular signals that integrate RHOA, RHO-associated coiled-coil containing protein kinase (ROCK), the apicobasal cell polarity, and the HIPPO-Yes-associated protein (YAP) signaling pathway. By contrast, molecular mechanisms regulating the maintenance of the TE characteristics at the later stage, which is crucial for blastocyst hatching and implantation, are scarcely understood. STUDY DESIGN, SIZE, DURATION: Expanding mouse blastocysts, obtained from crosses of the F1 (C57BL6 × DBA/2) strain, were exposed to chemical agents that interfere with RHOA, ROCK, or the actin cytoskeleton for up to 8 h, and effects on the blastocyst cavity, HIPPO-YAP signaling, and cell lineage-specific gene expression profiles were examined. PARTICIPANTS/MATERIALS, SETTING, METHODS: Mouse embryos at the embryonic stage E3.5 (expanding blastocysts) and E4.5 (fully expanded blastocysts) were treated with RHOA inhibitor (C3 exoenzyme), ROCK inhibitor (Y27632), or actin filament disruptors (cytochalasin B and latrunculin A). The integrity of the blastocyst cavity was evaluated based on the gross morphology. Effects on HIPPO-YAP signaling were assessed based on the presence of nuclearized YAP protein by immunofluorescence staining and the expression of YAP/TEA domain family member (TEAD) target genes by quantitative RT-PCR (qRT-PCR). The impact of these disruptors on cell lineages was evaluated based on expression of the TE-specific and inner cell mass-specific marker genes by qRT-PCR. The integrity of the apicobasal cell polarity was assessed by localization of protein kinase C zeta (PRKCZ; apical) and scribbled planar cell polarity (SCRIB; basal) proteins by immunofluorescence staining. For comparisons, cultured cell lines, NIH/3T3 (mouse fibroblast) and P19C5 (mouse embryonal carcinoma), were also treated with RHOA inhibitor, ROCK inhibitor, and actin filament disruptors for up to 8 h, and effects on HIPPO-YAP signaling were assessed based on expression of YAP/TEAD target genes by qRT-PCR. Each experiment was repeated using three independent batches of embryos (n = 40–80 per batch) or cell collections. Statistical analyses of data were performed, using one-way ANOVA and two-sample t-test. MAIN RESULTS AND THE ROLE OF CHANCE: Inhibition of RHOA deflated the cavity, diminished nuclear YAP (P < 0.01), and down-regulated the YAP/TEAD target and TE-specific marker genes in both E3.5 and E4.5 blastocysts (P < 0.05), indicating that the maintenance of the key TE characteristics is dependent on RHOA activity. However, inhibition of ROCK or disruption of actin filament only deflated the blastocyst cavity, but did not alter HIPPO-YAP signaling or lineage-specific gene expressions, suggesting that the action of RHOA to sustain the TE-specific gene expression program is not mediated by ROCK or the actomyosin cytoskeleton. By contrast, ROCK inhibitor and actin filament disruptors diminished YAP/TEAD target gene expressions in cultured cells to a greater extent than RHOA inhibitor, implicating that the regulation of HIPPO-YAP signaling in expanding blastocysts is distinctly different from that in the cell lines. Furthermore, the apicobasal cell polarity proteins in the expanding blastocyst were mislocalized by ROCK inhibition but not by RHOA inhibition, indicating that cell polarity is not linked to regulation of HIPPO-YAP signaling. Taken together, our study suggests that RHOA activity is essential to maintain the TE lineage in the expanding blastocyst and it regulates HIPPO-YAP signaling and the lineage-specific gene expression program through mechanisms that are independent of ROCK or actomyosin cytoskeleton. LARGE-SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: This study was conducted using one species, the mouse. Direct translation of the experiments and findings to human fertility preservation and ART requires further investigations. WIDER IMPLICATIONS OF THE FINDINGS: The elucidation of the mechanisms of TE formation is highly pertinent to fertility preservation in women. Our findings may raise awareness among providers of ART that the TE is sensitive to disturbance even in the late stage of blastocyst expansion and that rational approaches should be devised to avoid conditions that may impair the TE and its function. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by grants from the Ingeborg v.F. McKee Fund of the Hawaii Community Foundation (16ADVC-78882 to V.B.A.), and the National Institutes of Health (P20 GM103457 and R03 HD088839 to V.B.A.). The authors have no conflict of interest to declare.

In placental mammalian development, the first cell differentiation produces two distinct lineages that emerge according to their position within the embryo: the trophectoderm (TE, placenta precursor) differentiates in the surface, while the inner cell mass (ICM, fetal body precursor) forms inside. Here, we discuss how such position-dependent lineage specifications are regulated by the RHOA subfamily of small GTPases and RHO-associated coiled-coil kinases (ROCK). Recent studies in mouse show that activities of RHO/ROCK are required to promote TE differentiation and to concomitantly suppress ICM formation. RHO/ROCK operate through the HIPPO signaling pathway, whose cell position-specific modulation is central to establishing unique gene expression profiles that confer cell fate. In particular, activities of RHO/ROCK are essential in outside cells to promote nuclear localization of transcriptional co-activators YAP/TAZ, the downstream effectors of HIPPO signaling. Nuclear localization of YAP/TAZ depends on the formation of apicobasal polarity in outside cells, which requires activities of RHO/ROCK. We propose models of how RHO/ROCK regulate lineage specification and lay out challenges for future investigations to deepen our understanding of the roles of RHO/ROCK in preimplantation development. Finally, as RHO/ROCK may be inhibited by certain pharmacological agents, we discuss their potential impact on human preimplantation development in relation to fertility preservation in women.

Specification of the trophectoderm (TE) and inner cell mass (ICM) lineages in the mouse blastocyst correlates with cell position, as TE derives from outer cells whereas ICM from inner cells. Differences in position are reflected by cell polarization and Hippo signaling. Only in outer cells, the apical–basal cell polarity is established, and Hippo signaling is inhibited in such a manner that LATS1 and 2 (LATS1/2) kinases are prevented from phosphorylating YAP, a key transcriptional co-activator of the TE-specifying gene Cdx2. However, the molecular mechanisms that regulate these events are not fully understood. Here, we showed that inhibition of RHO–ROCK signaling enhances ICM and suppresses TE characteristics through activation of Hippo signaling and disruption of apical–basal polarity. Embryos treated with ROCK inhibitor Y-27632 exhibited elevated expression of ICM marker NANOG and reduced expression of CDX2 at the blastocyst stage. Y-27632-treated embryos failed to accumulate YAP in the nucleus, although it was rescued by concomitant inhibition of LATS1/2. Segregation between apical and basal polarity regulators, namely PARD6B, PRKCZ, SCRIB, and LLGL1, was dampened by Y-27632 treatment, whereas some of the polarization events at the late 8-cell stage such as compaction and apical localization of p-ERM and tyrosinated tubulin occurred normally. Similar abnormalities of Hippo signaling and apical–basal polarization were also observed in embryos that were treated with RHO GTPases inhibitor. These results suggest that RHO–ROCK signaling plays an essential role in regulating Hippo signaling and cell polarization to enable proper specification of the ICM and TE lineages.

Activation of Wnt/β-catenin signaling is crucial for the differentiation of pluripotent stem cells, namely the epiblast, embryonic stem, and embryonal carcinoma cells, into mesendoderm. However, downstream events of Wnt/β-catenin signaling that control the formation of mesendoderm are still unclear. In the present study, we used mouse P19 embryonal carcinoma cells as a model, and identified a homeodomain protein Nkx1-2 as a key regulator of mesendoderm formation. In the mouse embryo, Nkx1-2 was expressed in the primitive streak, in which the nascent mesendoderm emerges. In P19 cells, the expression of Nkx1-2 was activated by Wnt/β-catenin signaling independently of Brachyury, an evolutionary conserved early mesendoderm gene. In contrast, the expression of Nkx1-2 was both necessary and sufficient for the activation of Brachyury. Nkx1-2 acted as a transcriptional repressor to mediate the action of Wnt/β-catenin signaling to activate the Brachyury expression. We found Tcf3 as a potential target of gene repression by Nkx1-2, and the down-regulation of Tcf3 was partly required for effective activation of Brachyury by Wnt/β-catenin signaling. These results suggest that Nkx1-2 is a critical component of the gene regulatory network that operates downstream of Wnt/β-catenin signaling to regulate the formation of mesendoderm.

To assess the impact of embryonic stem cell culture medium (ESCM) on the pre- and post-implantation development of the mouse embryo, as a mammalian model, in comparison with the conventional culture medium, a potassium simplex optimized medium (KSOM).Development in ESCM versus KSOM was compared in terms of embryo morphology, cleavage, cavitation, hatching, cell number, expression of TE and ICM transcription factors (Cdx2 and Oct4, respectively), implantation, and development in utero.An enriched medium like ESCM can be beneficial for in vitro embryo development when cultured from the 8-cell stage, as evidenced by promotion of blastocyst development with respect to cavity expansion, hatching, and cell division. Such benefits were not observed when embryos were cultured from the 2-cell stage.ESCM may augment in vitro embryo development from the 8-cell stage. Using different culture media at different stages may be beneficial to achieve more effective human in vitro fertilization.

Transcription factor Oct4 is expressed in pluripotent cell lineages during mouse development, namely, in inner cell mass (ICM), primitive ectoderm, and primordial germ cells. Functional studies have revealed that Oct4 is essential for the maintenance of pluripotency in inner cell mass and for the survival of primordial germ cells. However, the function of Oct4 in the primitive ectoderm has not been fully explored. In this study, we investigated the role of Oct4 in mouse P19 embryonal carcinoma (EC) cells, which exhibit molecular and developmental properties similar to the primitive ectoderm, as an in vitro model. Knockdown of Oct4 in P19 EC cells upregulated several early mesoderm-specific genes, such as Wnt3, Sp5, and Fgf8, by activating Wnt/β-catenin signaling. Overexpression of Oct4 was sufficient to suppress Wnt/β-catenin signaling through its action as a transcriptional activator. However, Brachyury, a key regulator of early mesoderm development and a known direct target of Wnt/β-catenin signaling, was unable to be upregulated in the absence of Oct4, even with additional activation of Wnt/β-catenin signaling. Microarray analysis revealed that Oct4 positively regulated the expression of Tdgf1, a critical component of Nodal signaling, which was required for the upregulation of Brachyury in response to Wnt/β-catenin signaling in P19 EC cells. We propose a model that Oct4 maintains pluripotency of P19 EC cells through 2 counteracting actions: one is to suppress mesoderm-inducing Wnt/β-catenin signaling, and the other is to provide competence to Brachyury gene to respond to Wnt/β-catenin signaling.

The first cell lineage specification in mouse embryo development is the formation of trophectoderm (TE) and inner cell mass (ICM) of the blastocyst. This article is to review and discuss the current knowledge on the cellular and molecular mechanisms of this particular event. Several transcription factors have been identified as the critical regulators of the formation or maintenance of the two cell lineages. The establishment of TE manifests as the formation of epithelium, and is dependent on many structural and regulatory components that are commonly found and that function in many epithelial tissues. Distinct epithelial features start to emerge at the late 8-cell stage, but the fates of blastomeres are not fixed as TE or ICM until around 32-cell stage. The location of blastomeres at this stage, i.e., external or internal of the embryo, in effect defines the commitment towards the TE or ICM lineage, respectively. Some studies implicate the presence of a developmental bias among blastomeres at 2- or 4-cell stage, although it is unlikely to play a decisive role in the establishment of TE and ICM. The unique mode of cell lineage specification in the mouse embryo is further discussed in comparison with the formation of initial cell lineages, namely the three germ layers, in non-mammalian embryos.

Because of their capacity to give rise to various types of cells in vitro, embryonic stem and embryonal carcinoma (EC) cells have been used as convenient models to study the mechanisms of cell differentiation in mammalian embryos. In this study, we explored the mouse P19 EC cell line as an effective tool to investigate the factors that may play essential roles in mesoderm formation and axial elongation morphogenesis. We first demonstrated that aggregated P19 cells not only exhibited gene expression patterns characteristic of mesoderm formation but also displayed elongation morphogenesis with a distinct anterior-posterior body axis as in the embryo. We then showed by RNA interference that these processes were controlled by various regulators of Wnt signaling pathways, namely beta-catenin, Wnt3, Wnt3a, and Wnt5a, in a manner similar to normal embryo development. We further showed by inhibitor treatments that the axial elongation morphogenesis was dependent on the activity of Rho-associated kinase. Because of the convenience of these experimental manipulations, we propose that P19 cells can be used as a simple and efficient screening tool to assess the potential functions of specific molecules in mesoderm formation and axial elongation morphogenesis.

In mammals, Sry is the master regulator of male sex determination, although how it functions is still unclear. By contrast, female sex determination depends on the action of Rspo1 and Wnt4, the regulators of Wnt/beta-catenin signaling. To seek a possible interaction between male and female sex determination mechanisms, we examined whether Sry affects Wnt/beta-catenin signaling. Using the TOPFLASH reporter system to measure Lef/Tcf-dependent transcriptional activity, we showed that ectopic expression of mouse Sry strongly suppressed Wnt/beta-catenin signaling in mouse embryonal carcinoma and human embryonic kidney cell lines. This inhibition occurred downstream of beta-catenin but upstream of Lef/Tcf, and depended on both the HMG-box and the C-terminal transcriptional activation domain. By contrast, TOPFLASH was not inhibited by human SRY, which apparently lacks a transcriptional activation domain. However, a fusion construct consisting of human SRY attached to the C-terminal domain of mouse Sry was able to inhibit TOPFLASH effectively. Furthermore, Sry constructs carrying point mutations equivalent to those in human sex reversal mutations were less effective in inhibiting Wnt/beta-catenin signaling. Also, we showed that the action of Sry as a transcriptional activator was both necessary and sufficient to inhibit Wnt/beta-catenin signaling, suggesting that the transcriptional targets of Sry are responsible for the inhibition of signaling. Sox9 is a potential transcriptional target of Sry, although quantitative RT-PCR analysis indicates that the expression of Sox9 was not up-regulated by the ectopic expression of mouse Sry in mouse embryonal carcinoma cells. While the present study demonstrates an impact of mouse Sry on Wnt/beta-catenin signaling at an in vitro level, it requires further investigations to assess whether such action also takes place in vivo to regulate male sex determination.

A radiation-induced mouse mutant, Brachyrrhine (Br), exhibits frontonasal dysplasia and renal hypoplasia, two malformations associated with deficiencies in mesenchymal condensation. The purpose of this study was to resolve the Br locus, evaluate possible candidate genes, and identify developmental defects in the mutant chondrocranium. Linkage analysis mapped the Br mutation to a critical region distal to D17Mit76, which contains only one gene, the transcription factor Six2. Sequence analysis of the Six2 gene, including 1.5 kb of the promoter, failed to reveal the Br mutation. However, homozygous Br/Br embryos showed almost complete absence of Six2 mRNA and protein in craniofacial and renal tissues while heterozygous Br/+ embryos displayed intermediate Six2 levels. Mutant embryos displayed malformations of neural crest-derived structures of the anterior cranium where Six2 is normally expressed. These data suggest a mutation in a novel cis-acting regulatory region inhibits Six2 expression and is associated with frontonasal dysplasia and renal hypoplasia.

STUDY HYPOTHESIS Statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase of the mevalonate pathway and prescription drugs that treat hypercholesterolemia, compromise preimplantation mouse development via modulation of HIPPO signaling. STUDY FINDING HMG-CoA reductase activity is required for trophectoderm specification, namely blastocyst cavity formation and Yes-associated protein (YAP) nuclear localization, through the production of isoprenoid geranylgeranyl pyrophosphate (GGPP) and the action of geranylgeranyl transferase. WHAT IS KNOWN ALREADY Previous studies have shown that treatment of mouse embryos with mevastatin prevents blastocyst formation, but how HMG-CoA reductase is involved in preimplantation development is unknown. HIPPO signaling regulates specification of the trophectoderm lineage of the mouse blastocyst by controlling the nuclear localization of YAP. In human cell lines, the mevalonate pathway regulates YAP to mediate self-renewal and survival through geranylgeranylation of RHO proteins. These studies suggest that in preimplantation development, statins may act through HIPPO pathway to interfere with trophectoderm specification and thereby inhibit blastocyst formation. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Eight-cell stage (E2.5) mouse embryos were treated in hanging drop culture with chemical agents, namely statins (lovastatin, atorvastatin, cerivastatin and pravastatin), mevalonic acid (MVA), cholesterol, squalene, farnesyl pyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP), geranylgeranyltransferase inhibitor GGTI-298, RHO inhibitor I, and squalene synthase inhibitor YM-53601, up to the late blastocyst stage (E4.5). Efficiency of blastocyst formation was assessed based on gross morphology and the measurement of the cavity size using an image analysis software. Effects on cell lineages and HIPPO signaling were analyzed using immunohistochemistry with confocal microscopy based on the expression patterns of the lineage-specific markers and the nuclear accumulation of YAP. Effects on cell lineages were also examined by quantitative RT-PCR based on the transcript levels of the lineage-specific marker genes. Data were analyzed using one-way ANOVA and two-sample t-test. MAIN RESULTS AND THE ROLE OF CHANCE All four statins examined inhibited blastocyst formation. The adverse impact of statins was rescued by supplementation of MVA (P < 0.01) or GGPP (P < 0.01) but not squalene nor cholesterol. Blastocyst formation was also prevented by GGTI-298 (P < 0.01). These results indicate that HMG-CoA reductase activity is required for blastocyst formation mainly through the production of GGPP but not cholesterol. Inhibition of RHO proteins, known targets of geranylgeranylation, impaired blastocyst formation, which was not reversed by GGPP supplementation. Nuclear localization of YAP was diminished by statin treatment but fully restored by supplementation of MVA (P < 0.01) or GGPP (P < 0.01). This suggests that HIPPO signaling is regulated by GGPP-dependent mechanisms, possibly geranylgeranylation of RHO, to enable trophectoderm formation. YM-53601 prevented blastocyst formation (P < 0.01), but its adverse impact was not rescued by supplementation of squalene or cholesterol, suggesting that squalene synthesis inhibition was not the cause of blastocyst defects. LIMITATIONS, REASONS FOR CAUTION Analyses were conducted on embryos cultured ex vivo, but they enable the determination of specific concentrations that impair embryo development which can be compared with drug concentrations in the reproductive tract when testing in vivo impact of statins through animal experimentations. Also, analyses were conducted in only one species, the mouse. Epidemiological studies on the effects of various types of statins on the fertility of women are necessary. WIDER IMPLICATIONS OF THE FINDINGS Our study reveals how the mevalonate pathway is required for blastocyst formation and intersects with HIPPO pathway to provide a mechanistic basis for the embryotoxic effect of statins. This bears relevance for women who are taking statins while trying to conceive, since statins have potential to prevent the conceptus from reaching the blastocyst stage and to cause early conceptus demise. LARGE SCALE DATA Not applicable. STUDY FUNDING AND COMPETING INTERESTS This study was supported by grants from the George F. Straub Trust of the Hawaii Community Foundation (13ADVC-60315 to V.B.A.) and the National Institutes of Health, USA (P20GM103457 to V.B.A.). The authors have no conflict of interest to declare.

The enhancer-trap system is a useful tool to uncover genes that exhibit a unique tissue-specific expression. Here, we established a transgenic mouse line in which the reporter gene LacZ was specifically expressed in the developing limbs and face in the embryo. To identify the endogenous genes that are controlled by the limb- and face-specific enhancers, we pinpointed the integration site of the transgene, and analyzed the expression pattern of the genes that were located near the integration site. We found that the gene encoding KIF26B, a member of the kinesin superfamily, was preferentially expressed in the limb buds, face, and somite derivatives. Moreover, while a 7.5-kb mRNA was the major Kif26B transcript in the embryo, it was absent in many adult tissues. These results imply that KIF26B may play a role in embryogenesis, specifically in the development of limbs, face, and somites.

To study the mechanisms of mouse peri-implantation development, we explored the in vitro culture of the isolated inner cell mass (ICM) of the blastocyst. As previously reported, individually cultured ICM recapitulated several early embryological events, such as the formation of primitive endoderm, epiblast, and proamniotic cavity. However, we found that the timing and efficiency of these morphogenetic processes significantly varied among the ICM. Due to this unpredictability in developmental potential, in- dividually cultured ICM may be unsuitable for further analysis. By contrast, we found that when five ICM were fused into a single mass, such aggregates (5� ICM) underwent efficient and synchronous mor- phogenesis. The synchronous nature of 5� ICM development was also demonstrated by the temporal and spatial pattern of apoptotic cell death. TUNEL assay showed that a number of the epiblast cells committed apoptosis in 48 hr of culture, which took place after primitive endoderm differentiation but prior to proamniotic cavity formation. In situ hybridization analysis showed that Oct4 was downregulated and a-fetoprotein was upregulated in the primitive en- doderm of the cultured 5� ICM. In addition, RT-PCR analysis revealed the expression of various primitive endodermal genes, but not of extraembryonic ectoder- mal markers in the cultured 5� ICM. Taken together, we propose that the 5� ICM is a useful in vitro tool to study the mechanisms of peri-implantation develop- ment of the mouse embryo. Mol. Reprod. Dev. 67: 83-90, 2004. 2004 Wiley-Liss, Inc.

Cytokeratins of the liver were examined during the lamprey life cycle. This organ transforms, at metamorphosis, from a ductal larval structure to an aductal adult organ lacking the biliary passageways. Specifically, the relationship between liver morphology and cytokeratins 7, 18, and 19, and several unidentified cytokeratins was compared in larvae, transformers, and adults. Immunofluorescence staining showed that spatiotemporal cytokeratin distributions are cell-type-specific among hepatocytes, bile-duct cells, and gallbladder cells. In the larva, cytokeratins 7 and 19, and other unknown cytokeratins occur diffusely in the hepatocyte cytoplasm and prominently in the apices of hepatocytes near a bile canaliculus, so that collectively these cytokeratins form a pericanalicular ring. At mid-metamorphosis, the ring disappears, and cytokeratins are located predominantly in the periphery of bile-duct cells. In the adult, cytokeratin 19 has a sexually dimorphic distribution. Electrophoretic separations of cytoskeletal extracts of larval and adult livers have revealed larval cytokeratins 46, 49, 58, and 63.5, and adult male cytokeratins 46, 48, 49, 52.5, 58, 63.5, 64.5, 65, and 66. Immunoblotting has shown that certain cytokeratins are each related to at least two mammalian cytokeratins. We suggest that lamprey-specific liver cytokeratins exist, and that the metamorphosing liver can be used as a model for studying the relationship between cytokeratin organization and cell function.

The blastocyst consists of the outer layer of trophectoderm and pluripotent inner cell mass (ICM), the precursor of the placenta and fetus, respectively. During blastocyst expansion, the ICM adopts a compact, ovoidal shape, whose proper morphology is crucial for normal embryogenesis. Rho-associated kinase (ROCK), an effector of small GTPase RHO signaling, mediates the diverse cellular processes of morphogenesis, but its role in ICM morphogenesis is unclear. Here, we demonstrate that ROCK is required for cohesion of ICM cells and formation of segregated tissues called primitive endoderm (PrE) and epiblast (Epi) in the ICM of the mouse blastocyst. Blastocyst treatment with ROCK inhibitors Y-27632 and Fasudil caused widening or spreading of the ICM, and intermingling of PrE and Epi. Widening of ICM was independent of trophectoderm because isolated ICMs as well as colonies of mouse embryonic stem cells (mESC) also spread upon Y-27632 treatment. PrE, Epi, and trophectoderm cell numbers were similar between control and treated blastocysts, suggesting that ROCK inhibition affected ICM morphology but not lineage differentiation. Rock1 and Rock2 knockdown via RNA interference in mESC also induced spreading, supporting the conclusion that morphological defects caused by the pharmacological inhibitors were due to ROCK inactivation. When blastocysts were transferred into surrogates, implantation efficiencies were unaffected by ROCK inhibition, but treated blastocysts yielded greater fetal loss. These results show that proper ICM morphology is dependent on ROCK activity and is crucial for fetal development. Our studies have wider implication for improving efficiencies of human assisted reproductive technologies that diminish pregnancy loss and promote successful births.

In eukaryotes, DNA synthesis is preceded by licensing of replication origins. We examined the subcellular localization of two licensing proteins, ORC2 and MCM7, in the mouse zygotes and two-cell embryos. In somatic cells ORC2 remains bound to DNA replication origins throughout the cell cycle, while MCM7 is one of the last proteins to bind to the licensing complex. We found that MCM7 but not ORC2 was bound to DNA in metaphase II oocytes and remained associated with the DNA until S-phase. Shortly after fertilization, ORC2 was detectable at the metaphase II spindle poles and then between the separating chromosomes. Neither protein was present in the sperm cell at fertilization. As the sperm head decondensed, MCM7 was bound to DNA, but no ORC2 was seen. By 4 h after fertilization, both pronuclei contained DNA bound ORC2 and MCM7. As expected, during S-phase of the first zygotic cell cycle, MCM7 was released from the DNA, but ORC2 remained bound. During zygotic mitosis, ORC2 again localized first to the spindle poles, then to the area between the separating chromosomes. ORC2 then formed a ring around the developing two-cell nuclei before entering the nucleus. Only soluble MCM7 was present in the G2 pronuclei, but by zygotic metaphase it was bound to DNA, again apparently before ORC2. In G1 of the two-cell stage, both nuclei had salt-resistant ORC2 and MCM7. These data suggest that licensing follows a unique pattern in the early zygote that differs from what has been described for other mammalian cells that have been studied.

Trophectoderm (TE) is the first cell type that emerges during development and plays pivotal roles in the viviparous mode of reproduction in placental mammals. TE adopts typical epithelium morphology to surround a fluid-filled cavity, whose expansion is critical for hatching and efficient interaction with the uterine endometrium for implantation. TE also differentiates into trophoblast cells to construct the placenta. This chapter is an overview of the cellular and molecular mechanisms that control the critical aspects of TE formation, namely, the formation of the blastocyst cavity, the expression of key transcription factors, and the roles of cell polarity in the specification of the TE lineage. Current gaps in our knowledge and challenging issues are also discussed that should be addressed in future investigations in order to further advance our understanding of the mechanisms of TE formation.

The embryonic – abembryonic (Em–Ab) axis of the mouse blastocyst has been found in several studies to align orthogonal to the first cleavage plane, raising the possibility that a developmental prepattern already exists at the two-cell stage. However, it is also possible that such alignment is not due to any developmental disparity between the two-cell stage blastomeres, but rather is caused by an extrinsic mechanical constraint that is conferred by an irregular shape of the zona pellucida (ZP). Here, we conducted a series of experiments to distinguish between these possibilities. We showed that the shape of the ZP at the two-cell stage varied among embryos, ranging from near spherical to ellipsoidal, and that the ZP shape did not change until the blastocyst stage. In those embryos with an ellipsoidal ZP, the Em–Ab axis tended to lie orthogonal to the first cleavage plane, while in those embryos with a near spherical ZP, there was no such relationship. The clonal boundary between the descendants of the two-cell stage blastomeres tended to lie orthogonal to the Em–Ab axis when the rotation of the embryo within the ZP was experimentally prevented, while the control embryos did not exhibit such tendency. These results support the possibility that an apparent correlation between the first cleavage plane and the blastocyst axis can be generated by the mechanical constraint from the ZP but not by a developmental prepattern. Moreover, recent reports indicate that the vegetal blastomere of the four-cell stage embryo that had undergone a specific type of second cleavages is destined to contribute to the abembryonic side of the blastocyst. However, our present study shows that in spite of such specific second cleavages, the vegetal blastomere did not preferentially give rise to the abembryonic side. This result implicates that the lineage of the four-cell stage blastomere is not restricted even when embryos undergo a specific type of second cleavages.

During differentiation, somatic cell nuclei acquire unique patterns of epigenetic modifications, such as DNA methylation, which affect the transcriptional activity of specific genes. Upon transfer into oocytes, however, the somatic nucleus undergoes reprogramming of these epigenetic modifications to achieve pluripotency. Oct4 is one of the critical pluripotency regulators, and is expressed in the germ line, including the pluripotent early embryonic cells. Previous studies showed that the upstream regulatory sequences of the Oct4 gene are distinctly methylated in somatic cells, and the DNA methylation of the regulatory sequences suppresses the transcriptional activity. Thus, successful reprogramming of the somatic cell nucleus to gain pluripotency must be accompanied by the demethylation of the Oct4 regulatory sequences. Here, we investigated the methylation pattern of the Oct4 promoter during early development of cloned mouse embryos. We found that the Oct4 promoter was only gradually demethylated during the early cleavage stages and that the ineffective demethylation of the promoter was associated with developmental retardation. We also found that the upstream sequences of the other pluripotency regulators, namely Nanog, Sox2, and Foxd3, were considerably under-methylated in cumulus cells. These results suggest that the Oct4 gene, as compared to the other pluripotency regulators, needs to undergo extensive demethylation during nuclear reprogramming, and that the failure of such demethylation is associated with inefficient development of cloned somatic cell embryos.

Several recent studies have proposed a model that the organization of the mouse blastocyst is determined by the pattern of early cleavages: the plane of first cleavage divides the two-cell embryo into embryonic (Em) and abembryonic (Ab) halves, while the timing of the second cleavages specifies which blastomere becomes the Em half. This model is still controversial because of conflicting observations in various studies. Here, we investigated the possibility that the difference between mouse strains contributed to the discrepancy of the findings of different experiments regarding the relationship between the first two cleavages and the blastocyst axial pattern. First, we showed by using a lipophilic, fluorescent tracer that the plane of the first cleavage bears no consistent spatial relationship to the Em-Ab axis of the blastocyst regardless of the genotypic background. Secondly, the order of the second cleavage does not correlate with the Em-Ab polarity of the blastocyst. This was demonstrated by tracing the lineage of the early- and later-dividing two-cell stage blastomeres in the whole embryo as well as by comparing the developmental potential of isolated early- and later-dividing blastomeres and chimeras made entirely of early- or later-dividing blastomeres. These results suggest that contrary to recent studies, the differences between the early- and later-dividing blastomeres of the two-cell embryo are not functionally evident and do not define the Em-Ab polarity of the blastocyst. The significance of these findings is discussed in relation to human assisted reproduction and preimplantation genetic diagnosis.

The transcription factor Oct4 is specifically expressed in the germ line and pluripotent stem cells, and is indispensable for normal mouse development. To understand the epigenetic control of Oct4 expression, we examined the DNA methylation pattern of the Oct4 regulatory element in various types of cells. Bisulfite analysis showed that the regulatory element was unmethylated in P19 embryonal carcinoma cells, which robustly express Oct4. By contrast, the regulatory element was distinctly methylated in somatic cells, including cell lines, such as NIH3T3 embryonic fibroblast and Hepa1-6 hepatoma, as well as tissues from the adult body, such as liver, spleen, and cumulus cells. However, we found that the extent of methylation was considerably heterogeneous among the alleles in the adult somatic cells. Using a luciferase reporter construct, we demonstrated that the extent of methylation directly affects the efficiency of gene expression driven by the Oct4 regulatory element in P19 cells. These results raise the possibility that the epigenetic status of Oct4 is heterogeneous among a population of somatic cells, which may affect the efficiency of Oct4 reactivation after somatic cell nuclear transfer.

Several researchers have suggested recently that the embryonic-abembryonic (Em-Ab) axis of the mouse blastocyst is orthogonal to the first cleavage plane of the two-cell embryo. To determine the universality of this relationship, we used embryos of two different genotypes, F1 (C57BL/6 3 DBA/2) and CD-1. The position of the first cleavage plane in the early blastocyst was determined by labeling a blastomere with the fluorescent lineage tracer DiI (1,19-dioctadecyl-3,3,39,39-tetramethylindocarbocyanine perchlorate) at the two-cell stage. Approximately one quarter of the blastocysts from both genotypes possessed an Em-Ab axis that respected the orthogonal relationship with the first cleavage plane. However, the remainder of the blastocysts deviated from the orthogonal relationship. This result indicates that the orthogonal orientation of the Em-Ab axis to the first cleavage plane is not a universal phenomenon. We also tested whether the angular relationship between the Em-Ab axis and first cleavage plane influences postimplantation embryo development. We sorted the blastocysts that had the Em-Ab axis orthogonal to the first cleavage plane from the ones that did not. These two types of blastocysts were transferred separately into surrogates, and fetal development was examined in late gestation. The results revealed that both types of blastocysts produced normal fetuses at a similar frequency. Thus, the relationship of the blastocyst axis to the first cleavage plane does not significantly influence later development. assisted reproductive technology, conceptus, developmental biology, early development, embryo

The body plan of the embryo is established by a polarized source of developmental information in the oocyte. The Xenopus laevis oocyte creates polarity by anchoring mRNAs in the vegetal cortex, including Vg1 and Xwnt-11, which might function in body plan specification, and Xcat-2, which might function in germ cell development. To identify components of the RNA anchoring mechanism, we used the manually isolated vegetal cortex (IVC) to assay loss or change in spatial arrangement of mRNAs caused by disruption of cortical elements. The role of cytoskeleton in mRNA anchoring was tested by treating oocytes with inhibitors that selectively disrupted actin microfilaments and cytokeratin filaments. Treatment of oocytes with cytochalasin B caused clumping of Vg1 and Xwnt-11 as revealed by in situ hybridization of the IVC, but did not cause their release, as confirmed by RT-PCR analysis. These mRNA clumps did not match the distribution of actin microfilament clumps, but were distributed similarly to the remnant cytokeratin filaments. Treatment of oocytes with monoclonal anti-cytokeratin antibody C11 released these mRNAs from the cortex. C11 altered the texture of the cytokeratin network, but did not affect the actin meshwork. These results show that Vg1 and Xwnt-11 are retained by a cytokeratin filament-dependent mechanism, and that organization of the cytokeratin network depend on an intact actin meshwork. Colcemid did not disrupt Vg1 and Xwnt-11 retention in the IVC, so anchoring of these mRNAs are independent of microtubules. Membrane disruption in the IVC by Triton X-100 decreased Vg1 and Xwnt-11. Loss of these mRNAs was due mainly to ribonuclease activity released from membrane components. However, when ribonuclease activity was suppressed under cold temperature, a higher amount of Vg1 and Xwnt-11 was recovered in the supernatant. This result suggested that a fraction of these mRNAs required membranes to be retained in the cortex. By contrast, Xcat-2 mRNA was neither released nor degraded following treatments with cytochalasin B, C11, colcemid and Triton X-100 under cold temperature, so no cortical element could be implicated in its anchoring.