SummaryThe aPKC-PAR-6-PAR-3 cell polarity complex localizes to the centrosome attracting body, a macroscopic cortical structure responsible for asymmetric divisions in the early ascidian embryo

Ascidians (marine invertebrates: urochordates) are thought to be the closest sister groups of vertebrates. They are particularly attractive models because of their non-duplicated genome and the fast and synchronous development of large populations of eggs into simple tadpoles made of about 3,000 cells. As a result of stereotyped asymmetric cleavage patterns all blastomeres become fate restricted between the 16- and 110 cell stage through inheritance of maternal determinants and/or cellular interactions. These advantageous features have allowed advances in our understanding of the nature and role of maternal determinants, inductive interactions, and gene networks that are involved in cell lineage specification and differentiation of embryonic tissues. Ascidians have also contributed to our understanding of fertilization, cell cycle control, self-recognition, metamorphosis, and regeneration. In this chapter we provide basic protocols routinely used at the marine station in Villefranche-sur-Mer using the cosmopolitan species of reference Ciona intestinalis and the European species Phallusia mammillata. These two models present complementary advantages with regard to molecular, functional, and imaging approaches. We describe techniques for basic culture of embryos, micro-injection, in vivo labelling, micro-manipulations, fixation, and immuno-labelling. These methods allow analysis of calcium signals, reorganizations of cytoplasmic and cortical domains, meiotic and mitotic cell cycle and cleavages as well as the roles of specific genes and cellular interactions. Ascidians eggs and embryos are also an ideal material to isolate cortical fragments and to isolate and re-associate individual blastomeres. We detail the experimental manipulations which we have used to understand the structure and role of the egg cortex and of specific blastomeres during development.

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Ascidian postplasmic/PEM RNAs constitute a large class of cortical maternal RNAs which include developmental determinants (macho-1 and pem-1). We have analyzed the localization, cortical anchorage and cell type segregation of postplasmic/PEM RNAs in Ciona intestinalis and Phallusia mammillata using very high-resolution fluorescent in situ hybridization. We also compared RNAs extracted from whole oocytes and from isolated cortices using microarrays and localized RNAs possessing clusters of xCACx motifs in their 3'UTRs. Based on these combined approaches we conclude that: (1) the vast majority of the 39 postplasmic/PEM RNAs (including vasa) are localized in the egg cortex. (2) Many postplasmic/PEM RNAs 3'UTR are enriched in xCACx motifs, allowing us to identify 2 novel postplasmic/PEM RNAs (PSD and MnK). (3) Postplasmic/PEM RNAs anchored to cortical Endoplasmic Reticulum (cER) and those associated with granules have different cell destinations. We propose that there are 2 distinct categories of postplasmic/PEM RNAs on the basis of their cortical anchorages and cell destinations: (1) macho-1-like postplasmic/PEM RNAs anchored to cER which segregate into somatic B8.11 cells. (2) vasa-like postplasmic/PEM RNAs associated with granules which in addition to B8.11 cells segregate into B8.12 germ cells.