title: Manipulation of developing juvenile structures in purple sea urchins (Strongylocentrotus purpuratus) by morpholino injection into late stage larvae.
abstract: Sea urchins have been used as experimental organisms for developmental biology for over a century. Yet, as is the case for many other marine invertebrates, understanding the development of the juveniles and adults has lagged far behind that of their embryos and larvae. The reasons for this are, in large part, due to the difficulty of experimentally manipulating juvenile development. Here we develop and validate a technique for injecting compounds into juvenile rudiments of the purple sea urchin, Strongylocentrotus purpuratus. We first document the distribution of rhodaminated dextran injected into different compartments of the juvenile rudiment of sea urchin larvae. Then, to test the potential of this technique to manipulate development, we injected Vivo-Morpholinos (vMOs) designed to knock down p58b and p16, two proteins involved in the elongation of S. purpuratus larval skeleton. Rudiments injected with these vMOs showed a delay in the growth of some juvenile skeletal elements relative to controls. These data provide the first evidence that vMOs, which are designed to cross cell membranes, can be used to transiently manipulate gene function in later developmental stages in sea urchins. We therefore propose that injection of vMOs into juvenile rudiments, as shown here, is a viable approach to testing hypotheses about gene function during development, including metamorphosis.
authors.affiliation: Integrative Biology, University of Guelph, Guelph, Ontario, Canada.
authors.forename: Andreas
authors.initials: A
authors.lastname: Heyland
authors.affiliation: Hopkins Marine Station, Stanford University, Pacific Grove, CA, United States of America.
authors.forename: Jason
authors.initials: J
authors.lastname: Hodin
authors.affiliation: Department of Biology, St. Francis Xavier University, Antigonish, NS, Canada.
authors.forename: Cory
authors.initials: C
authors.lastname: Bishop
mesh_terms: D000818:Animals; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics; D003911:Dextrans / Q000494:pharmacology; D005260:Female; D005456:Fluorescent Dyes / Q000494:pharmacology; D055785:Gene Knockdown Techniques / Q000379:methods*; D007814:Larva / Q000254:growth & development; D008297:Male; D008954:Models, Biological; D060172:Morpholinos / Q000494:pharmacology*; D012235:Rhodamines / Q000494:pharmacology; D047270:Strongylocentrotus purpuratus / Q000187:drug effects / Q000235:genetics / Q000254:growth & development*
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16; D003911:Dextrans; D005456:Fluorescent Dyes; D060172:Morpholinos; D012235:Rhodamines; C051311:rhodamine dextran