Characterization of involution during sea urchin gastrulation using two-photon excited photorelease and confocal microscopy

Sea urchin embryos have served as a model system for the investigation of m any concepts in developmental biology. Their gastrulation consists of two s tages; primary invagination, where part of the epithelium invaginates into the blastocoel, and secondary invagination, where the archenteron elongates to completely traverse the blastocoel. Primary invagination involves proli feration of cells within the vegetal plate during primary invagination, but until recently, it was assumed that the larval gastrointestinal (GI) tract developed without further involution of epithelial cells. To investigate r igorously the contribution of epithelial cell involution during archenteron and GI tract development in the sea urchin, Lytechinus variegatus, We deve loped a new method for cell tracking based on two-photon excited photorelea se of caged fluorophores. Single-cell embryos were injected with caged dye and two-photon excitation uncaging was employed to mark small groups of cel ls throughout gastrulation. Two-photon excitation allowed for noninvasive, three-dimensionally resolved uncaging inside living cells with minimal biol ogical damage. Cellular involution into the archenteron was observed throug hout primary and secondary invagination, and the larval intestine was forme d by further involution of cells following secondary invagination, which is inconsistent with the traditional model of sea urchin gastrulation. Furthe r, as two-photon excitation microscopy becomes accessible to many researche rs, the novel techniques described here will be broadly applicable to devel opment of other invertebrate and vertebrate embryos.