A Nodal/Eph signalling relay drives the transition from apical constriction to apico-basal shortening in ascidian endoderm invagination.

Gastrulation is the first major morphogenetic event during animal embryogenesis. Ascidian gastrulation starts with the invagination of 10 endodermal precursor cells between the 64- and late 112-cell stages. This process occurs in the absence of endodermal cell division and in two steps, driven by myosin-dependent contractions of the acto-myosin network. First, endoderm precursors constrict their apex. Second, they shorten apico-basally, while retaining small apical surfaces, thereby causing invagination. The mechanisms that prevent endoderm cell division, trigger the transition between step 1 and step 2, and drive apico-basal shortening have remained elusive. Here, we demonstrate a conserved role for Nodal and Eph signalling during invagination in two distantly related ascidian species, Phallusia mammillata and Ciona intestinalis Specifically, we show that the transition to step 2 is triggered by Nodal relayed by Eph signalling. In addition, our results indicate that Eph signalling lengthens the endodermal cell cycle, independently of Nodal. Finally, we find that both Nodal and Eph signals are dispensable for endoderm fate specification. These results illustrate commonalities as well as differences in the action of Nodal during ascidian and vertebrate gastrulation.

Distinct modes of mitotic spindle orientation align cells in the dorsal midline of ascidian embryos.

The orientation of cell division can have important consequences on the choice of cell fates adopted by each daughter cell as well as on the architecture of the tissue within which the dividing cell resides. We have studied in detail the oriented cell divisions that take place in the dorsal midline of the ascidian embryo. The dorsal midline cells of the ascidian embryo emerge following an asymmetric cell division oriented along the animal-vegetal (A-V) axis. This division generates the NN (Notochord-Neural) cell at the margin and the E (Endoderm) cell more vegetally. Deviating from the default mode of cell division, these sister cells divide again along the A-V axis to generate a column of four cells. We describe these cell divisions in detail. We show that the NN cell mitotic spindle rotates 90° to align along the A-V axis while the E cell spindle forms directly along the axis following the asymmetric migration of its centrosomes. We combine live imaging, embryo manipulations and pharmacological modulation of cytoskeletal elements to address the mechanisms underlying these distinct subcellular behaviours. Our evidence suggests that, in E cells, aster asymmetry together with the E cell shape contribute to the asymmetric centrosome migration. In NN cells, an intrinsic cytoplasmic polarisation of the cell results in the accumulation of dynein to the animal pole side. Our data support a model in which a dynein-dependent directional cytoplasmic pulling force may be responsible for the NN cell spindle rotation.

G protein-coupled receptors Flop1 and Flop2 inhibit Wnt/ß-catenin signaling and are essential for head formation in Xenopus.

Patterning of the vertebrate anterior-posterior axis is regulated by the coordinated action of growth factors whose effects can be further modulated by upstream and downstream mediators and the cross-talk of different intracellular pathways. In particular, the inhibition of the Wnt/ß-catenin signaling pathway by various factors is critically required for anterior specification. Here, we report that Flop1 and Flop2 (Flop1/2), G protein-coupled receptors related to Gpr4, contribute to the regulation of head formation by inhibiting Wnt/ß-catenin signaling in Xenopus embryos. Using whole-mount in situ hybridization, we showed that flop1 and flop2 mRNAs were expressed in the neural ectoderm during early gastrulation. Both the overexpression and knockdown of Flop1/2 resulted in altered embryonic head phenotypes, while the overexpression of either Flop1/2 or the small GTPase RhoA in the absence of bone morphogenetic protein (BMP) signaling resulted in ectopic head induction. Examination of the Flops' function in Xenopus embryo animal cap cells showed that they inhibited Wnt/ß-catenin signaling by promoting ß-catenin degradation through both RhoA-dependent and -independent pathways in a cell-autonomous manner. These results suggest that Flop1 and Flop2 are essential regulators of Xenopus head formation that act as novel inhibitory components of the Wnt/ß-catenin signaling pathway.

Practical tips for imaging ascidian embryos.

Decades of studies on ascidian embryogenesis have culminated in deciphering the first gene regulatory "blueprint" for the generation of all major larval tissue types in chordates. However, the current gene regulatory network (GRN) is not well integrated with the morphogenetic and cellular processes that are also taking place during embryogenesis. Describing these processes represents a major on-going challenge, aided by recent advances in imaging and fluorescent protein (FP) technologies. In this report, we describe the application of these technologies to the developmental biology of ascidians and provide a detailed practical guide on the preparation of ascidian embryos for imaging.

The auxin-inducible degron 2 (AID2) system enables controlled protein knockdown during embryogenesis and development in Caenorhabditis elegans.

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-phenyl-indole-3-acetic acid (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 1,300-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 analog, 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.

Polo-like kinase 1 is required for localization of Posterior End Mark protein to the centrosome-attracting body and unequal cleavages in ascidian embryos.

In ascidian embryos, the posterior-localized maternal factor Posterior End Mark (PEM) is responsible for patterning embryos along the anterior-posterior axis with regard to both cleavage pattern involving unequal cell divisions and gene expression. Although PEM plays important roles in embryogenesis, its mechanism of action is still unclear because PEM has no known functional domain. In the present study, we explored the candidate of PEM partner proteins in Halocynthia roretzi using yeast two-hybrid screening. We isolated a homologue of Polo-like kinase 1 (Plk1), a key regulator of cell division and highly conserved in eukaryotes, as the first potential binding partner of PEM. We biochemically confirmed that interaction occurred between the Plk1 and PEM proteins. Immunostaining showed that Plk1 protein concentrates in the centrosome-attracting body (CAB) at the posterior pole, where PEM protein is also localized. The CAB is a subcellular structure that plays an important role in generating the posterior cleavage pattern. Plk1 localization to the CAB was dependent on the cell cycle phases during unequal cleavage. Inhibition of Plk1 with specific drugs resulted in failure of the nucleus to migrate towards the posterior pole and formation of a microtubule bundle between the CAB and a centrosome, similarly to inhibition of PEM function, suggesting that both proteins are involved in the same process of unequal cleavages. This interrupted nuclear migration was rescued by overexpression of PEM. In Plk1-inhibited embryos, the localization of PEM protein to the CAB was impaired, indicating that Plk1 is required for appropriate localization of PEM.

Localized PEM mRNA and protein are involved in cleavage-plane orientation and unequal cell divisions in ascidians.

BACKGROUND: Orientation and positioning of the cell division plane are essential for generation of invariant cleavage patterns and for unequal cell divisions during development. Precise control of the division plane is important for appropriate partitioning of localized factors, spatial arrangement of cells for proper intercellular interactions, and size control of daughter cells. Ascidian embryos show complex but invariant cleavage patterns mainly due to three rounds of unequal cleavage at the posterior pole. RESULTS: The ascidian embryo is an emerging model for studies of developmental and cellular processes. The maternal Posterior End Mark (PEM) mRNA is localized within the egg and embryo to the posterior region. PEM is a novel protein that has no known domain. Immunostaining showed that the protein is also present in the posterior cortex and the in centrosome-attracting body (CAB) and that the localization is extraction-resistant. Here we show that PEM of Halocynthia roretzi is required for correct orientation of early-cleavage planes and subsequent unequal cell divisions because it repeatedly pulls a centrosome toward the posterior cortex and the CAB, respectively, where PEM mRNA and protein are localized. When PEM activity is suppressed, formation of the microtubule bundle linking the centrosome and the posterior cortex did not occur. PEM possibly plays a role in anchoring microtubule ends to the cortex. In our model of orientation of the early-cleavage planes, we also amend the allocation of the conventional animal-vegetal axis in ascidian embryos, and discuss how the newly proposed A-V axis provides the rationale for various developmental events and the fate map of this animal. CONCLUSIONS: 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.

Asymmetric and Unequal Cell Divisions in Ascidian Embryos.

Asymmetric cell division during embryogenesis contributes to cell diversity by generating daughter cells that adopt distinct developmental fates. In this chapter, we summarize current knowledge of three examples of asymmetric cell division occurring in ascidian early embryos: (1) Three successive cell divisions that are asymmetric in terms of cell fate and unequal in cell size in the germline lineage at the embryo posterior pole. A subcellular structure, the centrosome-attracting body (CAB), and maternal PEM mRNAs localized within it control both the positioning of the cell division planes and segregation of the germ cell fates. (2) Asymmetric cell divisions involving endoderm and mesoderm germ layer separation. Asymmetric partitioning of zygotically expressed mRNA for Not, a homeodomain transcription factor, promotes the mesoderm fate and suppresses the endoderm fate. This asymmetric partitioning is mediated by transient nuclear migration toward the mesodermal pole of the mother cell, where the mRNA is delivered. In this case, there is no special regulation of cleavage plane orientation. (3) Asymmetric cell divisions in the marginal region of the vegetal hemisphere. The directed extracellular FGF and ephrin signals polarize the mother cells, inducing distinct fates in a pair of daughter cells (nerve versus notochord and mesenchyme versus muscle). The directions of cell division are regulated and oriented but independently of FGF and ephrin signaling. In these examples, polarization of the mother cells is facilitated by localized maternal factors, by delivery of transcripts from the nucleus to one pole of each cell, and by directed extracellular signals. Two cellular processes-asymmetric fate allocation and orientation of the cell division plane-are coupled by a single factor in the first example, but these processes are regulated independently in the third example. Thus, various modes of asymmetric cell division operate even at the early developmental stages in this single type of organism.

Physical association between a novel plasma-membrane structure and centrosome orients cell division.

In the last mitotic division of the epidermal lineage in the ascidian embryo, the cells divide stereotypically along the anterior-posterior axis. During interphase, we found that a unique membrane structure invaginates from the posterior to the centre of the cell, in a microtubule-dependent manner. The invagination projects toward centrioles on the apical side of the nucleus and associates with one of them. Further, a cilium forms on the posterior side of the cell and its basal body remains associated with the invagination. A laser ablation experiment suggests that the invagination is under tensile force and promotes the posterior positioning of the centrosome. Finally, we showed that the orientation of the invaginations is coupled with the polarized dynamics of centrosome movements and the orientation of cell division. Based on these findings, we propose a model whereby this novel membrane structure orchestrates centrosome positioning and thus the orientation of cell division axis.

ß-Catenin-driven binary fate specification segregates germ layers in ascidian embryos.

ß-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 ß-catenin-positive and -negative domains, with ß-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 ß-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 ß-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 ß-catenin activation sequence of ON-to-ON specifies endoderm, OFF-to-OFF ectoderm, and ON-to-OFF margin.

A maternal factor unique to ascidians silences the germline via binding to P-TEFb and RNAP II regulation.

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.