Vernadeth B. Alarcon

Polarity-dependent distribution of angiomotin localizes Hippo signaling in preimplantation embryos.

BACKGROUND In preimplantation mouse embryos, the first cell fate specification to the trophectoderm or inner cell mass occurs by the early blastocyst stage. The cell fate is controlled by cell position-dependent Hippo signaling, although the mechanisms underlying position-dependent Hippo signaling are unknown. RESULTS We show that a combination of cell polarity and cell-cell adhesion establishes position-dependent Hippo signaling, where the outer and inner cells are polar and nonpolar, respectively. The junction-associated proteins angiomotin (Amot) and angiomotin-like 2 (Amotl2) are essential for Hippo pathway activation and appropriate cell fate specification. In the nonpolar inner cells, Amot localizes to adherens junctions (AJs), and cell-cell adhesion activates the Hippo pathway. In the outer cells, the cell polarity sequesters Amot from basolateral AJs to apical domains, thereby suppressing Hippo signaling. The N-terminal domain of Amot is required for actin binding, Nf2/Merlin-mediated association with the E-cadherin complex, and interaction with Lats protein kinase. In AJs, S176 in the N-terminal domain of Amot is phosphorylated by Lats, which inhibits the actin-binding activity, thereby stabilizing the Amot-Lats interaction to activate the Hippo pathway. CONCLUSIONS We propose that the phosphorylation of S176 in Amot is a critical step for activation of the Hippo pathway in AJs and that cell polarity disconnects the Hippo pathway from cell-cell adhesion by sequestering Amot from AJs. This mechanism converts positional information into differential Hippo signaling, thereby leading to differential cell fates.

Cell Polarity Regulator PARD6B Is Essential for Trophectoderm Formation in the Preimplantation Mouse Embryo

In preimplantation mouse development, the first cell lineages to be established are the trophectoderm (TE) and inner cell mass. TE possesses epithelial features, including apical-basal cell polarity and intercellular junctions, which are crucial to generate a fluid-filled cavity in the blastocyst. Homologs of the partitioning defective (par) genes in Caenorhabditis elegans are critical regulators of cell polarity. However, their roles in regulating TE differentiation and blastocyst formation remain unclear. Here, the role of mouse Pard6b, a homolog of par-6 gene and a component of the PAR-atypical protein kinase C (aPKC) complex, was investigated. Pard6b expression was knocked down by microinjecting RNA interference construct into zygotes. Pard6b-knockdown embryos cleaved and compacted normally but failed to form the blastocyst cavity. The cavitation failure is likely the result of defective intercellular junctions, because Pard6b knockdown caused abnormal distribution of actin filaments and TJP1 (ZO-1) tight junction (TJ) protein and interfered with cavitation in chimeras containing cells from normal embryos. Defective TJ formation may be caused by abnormal cell polarization, because the apical localization of PRKCZ (aPKCzeta) was absent in Pard6b-knockdown embryos. Pard6b knockdown also diminished the expression of CDX2, a TE-lineage transcription factor, in the outer cells. TEAD4, a transcriptional activator that is required for Cdx2 expression and cavity formation, was not essential for the transcription of Pard6b. Taken together, Pard6b is necessary for blastocyst morphogenesis, particularly the development of TE-specific features—namely, the apical-basal cell polarity, formation of TJ, paracellular permeability sealing, and up-regulated expression of Cdx2.

Deviation of the Blastocyst Axis from the First Cleavage Plane Does Not Affect the Quality of Mouse Postimplantation Development

Abstract 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 × 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 (1,1′-dioctadecyl-3,3,3′,3′-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.

Establishment of trophectoderm and inner cell mass lineages in the mouse embryo

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, that is, 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. Mol. Reprod. Dev. 76: 1019–1032, 2009.

Inhibition of RHO-ROCK signaling enhances ICM and suppresses TE characteristics through activation of Hippo signaling in the mouse blastocyst.

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.

Aggregated P19 Mouse Embryonal Carcinoma Cells as a Simple In Vitro Model to Study the Molecular Regulations of Mesoderm Formation and Axial Elongation Morphogenesis

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 β‐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. genesis 47:93–106, 2009.

Identification of new localized RNAs in the Xenopus oocyte by differential display PCR

We have identified localized transcripts in full-grown Xenopus oocytes by differential display PCR. One clone, An4a, has two transcripts, which localize to the animal half of the stage VI oocyte. The transcripts are expressed throughout early development, with embryonic expression primarily in anterior neural tissues. An4a has a high degree of sequence identity to a human cDNA clone of unknown function. Another clone, the previously identified beta-transducin repeat containing protein (beta-TrCP), has three transcripts with a unique pattern of localization, one localized to the animal half and two localized primarily to the vegetal cortex. This cDNA has previously been shown to rescue a yeast cell division cycle mutant, raising the possibility that the different Xenopus transcripts are involved in animal and vegetal cell cycles. Embryonic expression is primarily in the cement gland. These new localized transcripts contribute to the general observation that the vegetal cortex, but not the animal cortex, is a specific site for RNA localization.

Misexpression of Six2 is associated with heritable frontonasal dysplasia and renal hypoplasia in 3H1 Br mice

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. Developmental Dynamics 237:1767–1779, 2008.

Rho-Associated Kinase Activity Is Required for Proper Morphogenesis of the Inner Cell Mass in the Mouse Blastocyst

ABSTRACT 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.

Spatial alignment of the mouse blastocyst axis across the first cleavage plane is caused by mechanical constraint rather than developmental bias among blastomeres

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. Mol. Reprod. Dev. 75: 1143–1153, 2008.