Dw. Piston et al., Characterization of involution during sea urchin gastrulation using two-photon excited photorelease and confocal microscopy, MICROS MICR, 4(4), 1998, pp. 404-414
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.