Exploration of the differentiation of vegetal cilia using heavy metal exposure and microscopy

Abstract

Echinoderms have been used for over 100 years as model organisms for studying early development. In particular, they have been used for studying cilia, the long whip-like appendages that stick out of cells. The purpose of the present study was to visualize the growth and differentiation of the vegetal cilia during the early development of echinoid embryos. The technique of embryo vegetalization was used to better visualize the cilia of the vegetal hemisphere. DIC microscopy was used on normal and vegetalized embryos to explore the differentiation of cilia along the vegetal pole, and to characterize the lengths of cilia around the vegetal hemisphere. Our data suggest that cilia at the tip of the gut are longer than those that project from the sides of the gut. Cilia lengths on one, vegetalized embryo were quantified for apical tuft cilia, lateral cilia, and vegetal (or gut) cilia and compared with measurements taken from the literature. Additionally, confocal microscopy was then employed on normal and vegetalized embryos to characterize the type of cilia that were present on the vegetal hemisphere. Our results suggest that the cilia of the gut have an abrupt change in length from the lateral cilia to the gut cilia. This implies that there is some gene regulation which causes retraction and regrowth of cells fated to be gut to differentiate from the lateral cilia. Finally, in order to orient the embryos later in development when it was difficult to tell which part of the embryo was animal or vegetal, a dye called Calcein-AM (CAM) was exposed to the embryos to attempt to label the micromeres.