We are interested in analyzing the dynamic behavior of epithelial cells undergoing common morphogenetic movements such as invagination and cell rearrangement. Such an analysis will allow us to examine the mechanisms underlying these cell movements. We use the sea urchin embryo as our model system for several reasons. It is relatively simply organized consisting of an epithelial monolayer and is transparent which makes it highly amenable to n-dcroscopy. Gastrulation in the sea urchin embryo involves a series of morphogenetic movements whose underlying mechanisms are not understood. At the onset of gastrulation the vegetal plate of the embryo buckles into the blastocoel. This invagination ultimately forms a stout, flat-topped cylinder called the archefiteron. Ultimately, through cell rearrangement, the archenteron spans the entire blastocoel contacting the animal pole. In order to understand the mechanisms underlying gastrulation we are examining the individual cell movements and behaviors which occur during this process in living embryos. To perform this dynamic analysis, we have utilized the 2 photon system in conjunction with two dye labeling techniques. The first dye we have used is the lipophilic dye Dil. Dil randomly labels single cells in the embryo. By panning through a field of labeled embryos we chose the ones with cells labeled in the archenteron and then do time lapse filming of the labeled cell as gastrulation proceeds. Thus we have a dynamic account of a single cell's behavior. The second dye we have used is FM464 which labels the membranes of all the cells in the embryo. Therefore, we are able to analyze the movements of all the cells in the archenteron as they move relative to one another during gastrulation. 2 photon microscopy has allowed us to film for longer periods of time due to a decreased amount of photobleaching of the dyes and poisoning of the embryos. The technique has been invaluable in providing us with the dynamic cell behavioral information we need to analyze the mechanisms of epithelial morphogenesis.
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