Transgenic Techniques for Investigating Cell Biology During Development

Abstract

Ascidians are increasingly being used as a system for investigating cell biology during development. The extreme genetic and cellular simplicity of ascidian embryos in combination with superior experimental tractability make this an ideal system for in vivo analysis of dynamic cellular processes. Transgenic approaches to cellular and sub-cellular analysis of ascidian development have begun to yield new insights into the mechanisms regulating developmental signaling and morphogenesis. This chapter focuses on the targeted expression of fusion proteins in ascidian embryos and how this technique is being deployed to garner new insights into the cell biology of development.

Keywords: Ascidians; Cardiac induction; Cell cycle progression; Ciona intestinalis; Collective cell migration; Fusion proteins; Halocynthia roretzi; Phallusia mammillata; Spindle positioning.

Fig. 14.1.

CLIP, SNAP, and HALO tags for covalent labeling of ascidian protein. Genetically encoded CLIP tag (New England BioLabs, NEB), SNAP tag (NEB) and HALO tag (Promega) schematically depicted in (a) can be used in conjunction with commercially available fluorophore-­conjugated substrates to covalently label and visualize proteins in living or fixed transgenic ascidian embryos. (b) Ventral projection of a pair of mitotic pre-­cardiac founder cells in a Ciona embryo expressing an endosome-associated CLIP tag fusion protein under the control of the cardiopharyngeal lineage specific promoter, Mesp (Mesp>CLIP::Rab4). The embryo was fixed and then incubated with the CLIP-Cell TMR-Star (red; NEB) substrate before imaging using confocal microscopy. Phalloidin was used to detect F-actin and DRAQ5 was used to detect DNA (blue). Anterior is to the right