Par-aPKC-dependent and -independent mechanisms cooperatively control cell polarity, Hippo signaling, and cell positioning in 16-cell stage mouse embryos.

In preimplantation mouse embryos, the Hippo signaling pathway plays a central role in regulating the fates of the trophectoderm (TE) and the inner cell mass (ICM). In early blastocysts with more than 32 cells, the Par-aPKC system controls polarization of the outer cells along the apicobasal axis, and cell polarity suppresses Hippo signaling. Inactivation of Hippo signaling promotes nuclear accumulation of a coactivator protein, Yap, leading to induction of TE-specific genes. However, whether similar mechanisms operate at earlier stages is not known. Here, we show that slightly different mechanisms operate in 16-cell stage embryos. Similar to 32-cell stage embryos, disruption of the Par-aPKC system activated Hippo signaling and suppressed nuclear Yap and Cdx2 expression in the outer cells. However, unlike 32-cell stage embryos, 16-cell stage embryos with a disrupted Par-aPKC system maintained apical localization of phosphorylated Ezrin/Radixin/Moesin (p-ERM), and the effects on Yap and Cdx2 were weak. Furthermore, normal 16-cell stage embryos often contained apolar cells in the outer position. In these cells, the Hippo pathway was strongly activated and Yap was excluded from the nuclei, thus resembling inner cells. Dissociated blastomeres of 8-cell stage embryos form polar-apolar couplets, which exhibit different levels of nuclear Yap, and the polar cell engulfed the apolar cell. These results suggest that cell polarization at the 16-cell stage is regulated by both Par-aPKC-dependent and -independent mechanisms. Asymmetric cell division is involved in cell polarity control, and cell polarity regulates cell positioning and most likely controls Hippo signaling.

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.

Wnt signaling maintains the notochord fate for progenitor cells and supports the posterior extension of the notochord.

The notochord develops from notochord progenitor cells (NPCs) and functions as a major signaling center to regulate trunk and tail development. NPCs are initially specified in the node by Wnt and Nodal signals at the gastrula stage. However, the underlying mechanism that maintains the NPCs throughout embryogenesis to contribute to the posterior extension of the notochord remains unclear. Here, we demonstrate that Wnt signaling in the NPCs is essential for posterior extension of the notochord. Genetic labeling revealed that the Noto-expressing cells in the ventral node contribute the NPCs that reside in the tail bud. Robust Wnt signaling in the NPCs was observed during posterior notochord extension. Genetic attenuation of the Wnt signal via notochord-specific beta-catenin gene ablation resulted in posterior truncation of the notochord. In the NPCs of such mutant embryos, the expression of notochord-specific genes was down-regulated, and an endodermal marker, E-cadherin, was observed. No significant alteration of cell proliferation or apoptosis of the NPCs was detected. Taken together, our data indicate that the NPCs are derived from Noto-positive node cells, and are not fully committed to a notochordal fate. Sustained Wnt signaling is required to maintain the NPCs' notochordal fate.

The Hippo signaling pathway components Lats and Yap pattern Tead4 activity to distinguish mouse trophectoderm from inner cell mass.

Outside cells of the preimplantation mouse embryo form the trophectoderm (TE), a process requiring the transcription factor Tead4. Here, we show that transcriptionally active Tead4 can induce Cdx2 and other trophoblast genes in parallel in embryonic stem cells. In embryos, the Tead4 coactivator protein Yap localizes to nuclei of outside cells, and modulation of Tead4 or Yap activity leads to changes in Cdx2 expression. In inside cells, Yap is phosphorylated and cytoplasmic, and this involves the Hippo signaling pathway component Lats. We propose that active Tead4 promotes TE development in outside cells, whereas Tead4 activity is suppressed in inside cells by cell contact- and Lats-mediated inhibition of nuclear Yap localization. Thus, differential signaling between inside and outside cell populations leads to changes in cell fate specification during TE formation.

Characterization of a sperm factor for egg activation at fertilization of the newt Cynops pyrrhogaster.

Eggs of the newt, Cynops pyrrhogaster, arrested at the second meiotic metaphase are activated by sperm at fertilization and then complete meiosis to initiate development. We highly purified a sperm factor for egg activation from a sperm extract with several chromatographies. The purified fraction containing only a 45 kDa protein induced egg activation accompanied by an intracellular Ca2+ increase when injected into unfertilized eggs. Although injection of mouse phospholipase C (PLC) zeta-mRNA caused a Ca2+ increase and egg activation, partial amino acid sequences of the 45 kDa protein were homologous to those of Xenopus citrate synthase, but not to PLCs. An anti-porcine citrate synthase antibody recognized the 45 kDa protein both in the purified fraction and in the sperm extract. Treatment with the anti-citrate synthase antibody reduced the egg-activation activity in the sperm extract. Injection of porcine citrate synthase or mRNA of Xenopus citrate synthase induced a Ca2+ increase and caused egg activation. A large amount of the 45 kDa protein was localized in two lines elongated from the neck to the middle piece of sperm. These results indicate that the 45 kDa protein is a major component of the sperm factor for egg activation at newt fertilization.

Characterization of Xenopus egg membrane microdomains containing uroplakin Ib/III complex: roles of their molecular interactions for subcellular localization and signal transduction.

A single-transmembrane protein uroplakin III (UPIII) and its tetraspanin binding-partner uroplakin Ib (UPIb) are members of the UP proteins that were originally identified in mammalian urothelium. In Xenopus laevis eggs, these proteins: xUPIII and xUPIb, are components of the cholesterol-enriched membrane microdomains or "rafts" and involved in the sperm-egg membrane interaction and subsequent egg activation signaling via Src tyrosine kinase at fertilization. Here, we investigate whether the xUPIII-xUPIb complex is in close proximity to CD9, a tetraspanin that has been implicated in the sperm-egg fusion in the mouse and GM1, a ganglioside typically enriched in egg rafts. Preparation of the egg membrane microdomains using different non-ionic detergents (Brij 98 and Triton X-100), chemical cross-linking, co-immunoprecipitation, in vitro kinase assay and in vitro fertilization experiments demonstrated that GM1, but not CD9, is in association with the xUPIII-xUPIb complex and contributes to the sperm-dependent egg activation. Transfection experiments using HEK293 cells demonstrated that xUPIII and xUPIb localized efficiently to the cholesterol-dependent membrane microdomains when they were co-expressed, whereas co-expression of xUPIII and CD9, instead of xUPIb, did not show this effect. Furthermore, xUPIII and xUPIb were shown to suppress kinase activity of the wild type, but not a constitutively active form of, Xenopus Src protein co-expressed in HEK293 cells. These results provide novel insight into the molecular architecture of the egg membrane microdomains containing xUPIII, xUPIb and Src, which may contribute to the understanding of sperm-egg interaction and signaling during Xenopus fertilization.

Molecular identification and characterization of Xenopus egg uroplakin III, an egg raft-associated transmembrane protein that is tyrosine-phosphorylated upon fertilization.

Here we describe mass spectrometric identification, molecular cloning, and biochemical characterization of a lipid/membrane raft-associated protein that is tyrosine-phosphorylated upon Xenopus egg fertilization. This protein is homologous to mammalian uroplakin III, a member of the uroplakin family proteins (UPs) that constitute asymmetric unit membranes in the mammalian urothelial tissues, thus termed Xenopus uroplakin III (xUPIII). xUPIII contains N-linked sugars and is highly expressed in Xenopus eggs, ovary, urinary tract, and kidney. In unfertilized eggs, xUPIII is predominantly localized to the lipid/membrane rafts and exposed on the cell surface, as judged by surface biotinylation experiments and indirect immunofluorescent studies. After fertilization or hydrogen peroxide-induced egg activation, xUPIII becomes rapidly phosphorylated on tyrosine residue-249, which locates in the carboxyl-terminal cytoplasmic tail of the molecule. Raft localization and tyrosine phosphorylation of xUPIII can be reconstituted in HEK293 cells by coexpression of xUPIII, and Xenopus c-Src, a tyrosine kinase whose fertilization-induced activation in egg rafts is required for initiation of development. In mammals, UPIII is forming a complex with a tetraspanin molecule uroplakin Ib. As another tetraspanin, CD9, is known to be a critical component for sperm-egg fusion in the mouse, we have assumed that xUPIII is involved in sperm-egg interaction. An antibody against the extracellular domain of xUPIII blocks sperm-egg interaction, as judged by the occurrence of egg activation and first cell cleavage. Thus, xUPIII represents an egg raft-associated protein that is likely involved in sperm-egg interaction as well as subsequent Src-dependent intracellular events of egg activation in Xenopus.

Molecular dissection of egg fertilization signaling with the aid of tyrosine kinase-specific inhibitor and activator strategies.

Fertilization is triggered by sperm-egg interaction and fusion that initiate a transient rise(s) in the free intracellular calcium ([Ca(2+)](i)) that is responsible for a series of biochemical and cell biological events, so-called "egg activation". Calcium-dependent egg activation leads to the initiation of developmental program that culminates in the birth of individuals. A growing body of knowledge has uncovered the molecular mechanisms underlying sperm-induced transient [Ca(2+)](i) increase(s) to some extent; namely, in most animals so far studied, a second messenger inositol 1,4,5-trisphosphate (IP(3)) seems to play a pivotal role in inducing [Ca(2+)](i) transient(s) at fertilization. However, signaling mechanisms used by sperm to initiate IP(3)-[Ca(2+)](i) transient pathway have not been elucidated. To approach this problem, we have employed African clawed frog, Xenopus laevis, as a model animal and conducted experiments designed specifically to determine the role of the Src family protein-tyrosine kinases (SFKs or Src family PTKs) in the sperm-induced egg activation. This review compiles information about the use of PTK-specific inhibitors and activators for analyzing signal transduction events in egg fertilization. Specifically, we focus on molecular identification of Xenopus Src and the signaling mechanism of the Src-dependent egg activation that has been established recently. We also summarize recent advances in understanding the role of the Src family kinases in egg fertilization of other model organisms, and discuss future directions of the field.