The complex cleavage pattern in ascidian embryos can be explained by a simple rule of centrosome attraction mediated by localized PEM activity. PEM is the first gene identified in ascidians that is required for multiple spindle-positioning events.

β-catenin is a transcriptional cofactor mediating the "canonical" Wnt signaling pathway, which activates target genes in a complex with TCF (LEF) transcription factors [1]. In many metazoans, embryos are first subdivided during early cleavage stages into nuclear β-catenin-positive and -negative domains, with β-catenin specifying endoderm or mesendoderm fate. This process has been demonstrated in a wide range of phyla including cnidarians, nemerteans, and invertebrate deuterostomes (echinoderms, hemichordates, and ascidians), implying that β-catenin-dependent (mes)endoderm specification is evolutionarily ancient [2-10]. However, the mechanisms leading to the segregation of mesoderm and endoderm fates from a transient mesendodermal state are less well defined. We show that subdivision of the ascidian embryo into the three germ layers involves differential nuclear β-catenin activity coupled with the first two animal-vegetal (A-V)-oriented cell divisions. We reveal that each of these A-V divisions operates as a binary fate choice: the first between ectoderm and mesendoderm and the second between margin (notochord and neural) and endoderm, such that a β-catenin activation sequence of ON-to-ON specifies endoderm, OFF-to-OFF ectoderm, and ON-to-OFF margin.

Suppression of zygotic transcription in early embryonic germline cells is tightly linked to their separation from the somatic lineage. Many invertebrate embryos utilize localized maternal factors that are successively inherited by the germline cells for silencing the germline. Germline quiescence has also been associated with the underphosphorylation of Ser2 of the C-terminal domain (CTD-Ser2) of RNA polymerase II [1-3]. Here, using the ascidian Halocynthia roretzi, we identified a first deuterostome example of a maternally localized factor, posterior end mark (PEM), which globally represses germline transcription. PEM knockdown resulted in ectopic transcription and ectopic phosphorylation of CTD-Ser2 in the germline. Overexpression of PEM abolished all transcription and led to the underphosphorylation of CTD-Ser2 in the somatic cells. PEM protein was reiteratively detected in the nucleus of the germline cells and coimmunoprecipitated with CDK9, a component of posterior transcription elongation factor b (P-TEFb). These results suggest that nonhomologous proteins, PEM and Pgc of Drosophila [3-5] and PIE-1 of C. elegans [1, 6, 7], repress germline gene expression through analogous functions: by keeping CTD-Ser2 underphosphorylated through binding to the P-TEFb complex. The present study is an interesting example of evolutionary constraint on how a mechanism of germline silencing can evolve in diverse animals.

Targeted protein degradation using the auxin-inducible degron (AID) system is garnering attention in the research field of Caenorhabditis elegans, because of the rapid and efficient target depletion it affords, which can be controlled by treating the animals with the phytohormone auxin. However, the current AID system has drawbacks, i.e., leaky degradation in the absence of auxin and the requirement for high auxin doses. Furthermore, it is challenging to deplete degron-fused proteins in embryos because of their eggshell, which blocks auxin permeability. Here, we apply an improved AID2 system utilizing AtTIR1(F79G) and 5-Ph-IAA to C. elegans and demonstrated that it confers better degradation control vs. the previous system by suppressing leaky degradation and inducing sharp degradation using 1300-fold lower 5-Ph-IAA doses. We successfully degraded the endogenous histone H2A.Z protein fused to an mAID degron and disclosed its requirement in larval growth and reproduction, regardless of the presence of maternally inherited H2A.Z molecules. Moreover, we developed an eggshell-permeable 5-Ph-IAA analogue, 5-Ph-IAA-AM, that affords an enhanced degradation in laid embryos. Our improved system will contribute to the disclosure of the roles of proteins in C. elegans, in particular those that are involved in embryogenesis and development, through temporally controlled protein degradation.