Ascidian notochord morphogenesis

Abstract

The development of the notochord involves a complex set of cellular behaviors. While these morphogenic behaviors are common to all chordates, the ascidian provides a particularly attractive experimental model because of its relative simplicity. In particular, all notochord morphogenesis in ascidians takes place with only 40 cells, as opposed to the hundreds of cells in vertebrate model systems. Initial steps in ascidian notochord development convert a monolayer of epithelial-like cells in the pregastrula embryo to a cylindrical rod of single-cell diameter. Convergent extension is responsible for the intercalation of notochord cells and some degree of notochord elongation, while a second phase of elongation is observed as the notochord narrows medially and increases in volume. The mechanism by which the volume of the notochord increases differs between ascidian species. Some ascidians produce extracellular pockets that will eventually coalesce to form a lumen running the length of the notochord; whereas others do not. By either mechanism, the resulting notochord serves as a hydrostatic skeleton allowing for the locomotion of the swimming larva. Several basic cell behaviors, such as cell shape changes, cell rearrangement, establishment of cell polarity, and alteration of extracellular environment, are displayed in the process of notochord morphogenesis. Modern analysis of ascidian notochord morphogenesis promises to contribute to our understanding of these fundamental biological processes.

Figure 1

Ascidian notochord development from the onset of gastrulation to the completion of convergent extension. (A) Ten presumptive notochord cells (red) forms a semicircular arc anterior to the blastopore (*). Two rounds of cell division generates 20 (B) and finally 40 notochord cells that form a monolayer epithelia (C). Infolding (not shown here) and convergent extension transform the notochord precursor into a column of 40 stacked cells (D-F). To collect these images Ciona savignyi embryos were stained with bodipy-phalloidin and imaged with a laser scanning confocal microscope. hpf: hours post fertilization at 18°C. All images are dorsal view, with anterior to the left.

Figure 2

Model of mediolateral polarity establishment in notochord. (A) Disheveled (red) is localized at notochord cell membrane (arrowhead) but is excluded from the portion that borders lateral muscle cells (arrow). This polarized Disheveled localization may be the result of signaling from the muscle tissue to initiate notochord intercalation along mediolateral axis. In the absence of normal Prickle activity, Disheveled membrane localization is lost and becomes cytoplasmic (red shading) (B). N: notochord; M: muscle. Dorsal view; anterior to the left.

Figure 3

Post convergent extension notochord morphogenesis. Normaski (A-H) and fluorescent (A’-H’) images of notochord in stable transgenic Ciona savignyi embryos expressing GFP under the Ciona intestinalis brachyury promoter. In these cells the GFP tends to accumulate in nuclei. Arrows indicate emergence of intercellular pockets (D and D’). Approximately 10 cells from the second quarter of notochord column are imaged. hpf: hours post fertilization at 16°C. Dorsal view, anterior to the left.

Figure 4

Notochord morphology at post convergent extension elongation stage (A-H) and swimming larva stage (A’-H’) for various ascidian species. Images collected by Normaski microscopy.

Figure 5

Extracellular pockets in notochord column. A membrane-targeted GFP is expressed in notochord cells in mosaic pattern by electroporation. (A) A string of notochord cells expressing membrane-bound GFP at different levels. (B) An isolated GFP-positive cell. * indicates extracellular pockets. Arrowheads indicate intracellular yolk granules. Anterior to the left.

Figure 6

A Ciona savignyi swimming tadpole imaged by Normarski microscopy. At this stage, notochord is a tubular lumen is at the center (*) encircled by endothelial-like notochord cells (arrows) in the periphery. The notochord at this stage plays an important role in locomotion by serving as a hydrostatic skeleton in the larval tail. Lateral view; anterior to the left.