Molecular, Morphological, and Functional Characterization of Pigmented Cells in the Sea Urchin Embryo

Abstract

This dissertation combines live imaging, embryological manipulations and computational analysis to understand the development of pigmented immunocytes in the sea urchin. Three areas received special attention: the mechanism and timing of the epithelial to mesenchymal transition, the function of pigment cells in larvae, and identification of new candidate molecules that may participate in those activities.Chapter 2 is a methods chapter combining embryo cut and paste approaches with live imaging. Improvements over earlier methods including development of techniques to better image sea urchin embryos in vivo are described. One of the primary strengths of the sea urchin is its optical clarity and simplistic developmental morphogenetic movements. The embryo develops cilia prior to undergoing morphogenetic movements. Consequently, long-term live imaging is a challenge. This can be overcome through multiple treatments and image processing techniques, resulting in the ability to acquire time-lapse movies for up to 24hrs.Chapter 3 describes an investigation of pigment cells epithelial-to-mesenchymal transition (EMT) a dynamic cellular process during which cells must change polarity, de-adhere from neighboring cells, breach through the basement membrane and become motile. Building off previous work in the lab, a comparison of the gene regulatory network that directs this process was identified and compared with other cell types undergoing an EMT to learn whether the same, or a different network is deployed in different cell types. I show that most of the TFs deployed in the skeletogenic EMT are not expressed in pigment cells before or during EMT, suggesting that there are different mechanisms controlling EMT in the pigment cells. There is some overlap, however, in that morpholino knock down of the TF twist disrupts EMT in pigment cells as it does in skeletogenic cells.Chapter 4 is a study of pigment cell biology. A number of questions were addressed. We learned where pigment cells incorporate into the ectoderm, how are they are patterned in the ectoderm and demonstrate several components of their function. Pigment cell incorporation initially occurs into the posterior dorsal ectoderm and the cells then migrate in the dorsal ectoderm to spread into anterior regions of that tissue. Observations were made describing how the cells move with the ectoderm. Functionally, pigment cells are shown to reversibly respond to light exposure. They also are shown to respond to wounding. Further, the immunosurveillance activities are recorded revealing populations of immobile and migratory pigment cells, suggesting that the population may diversify functionally.Computational and molecular approaches were used in Chapter 5 to identify new candidate molecules, specific to pigment cells, to begin to understand molecular processes underlying pigment cell behaviors. This approach provides the largest expansion to date of previously un-described genes expressed in pigment cells.Pigment cells serve in a protective role during larval development. This project has advanced an understanding of how those cells work, and it opens new possibilities for further in depth approaches toward molecular explanations of function. The final synthesis addresses unanswered questions in the development, differentiation and function of pigment cells and provides insight into how these activities can now be explored.