Sea urchins are premier model organisms for the study of early development. However, the lengthy generation times of commonly used species have precluded application of stable genetic approaches. Here, we use the painted sea urchin Lytechinus pictus to address this limitation and to generate a homozygous mutant sea urchin line. L. pictus has one of the shortest generation times of any currently used sea urchin. We leveraged this advantage to generate a knockout mutant of the sea urchin homolog of the drug transporter ABCB1, a major player in xenobiotic disposition for all animals. Using CRISPR/Cas9, we generated large fragment deletions of ABCB1 and used these readily detected deletions to rapidly genotype and breed mutant animals to homozygosity in the F2 generation. The knockout larvae are produced according to expected Mendelian distribution, exhibit reduced xenobiotic efflux activity and can be grown to maturity. This study represents a major step towards more sophisticated genetic manipulation of the sea urchin and the establishment of reproducible sea urchin animal resources.

Background: Small micromeres are produced at the fifth cleavage of sea urchin development. They express markers of primordial germ cells (PGCs), and are required for the production of gametes. In most animals, PGCs migrate from sites of formation to the somatic gonad. Here, we investigated whether they also exhibit similar migratory behaviors using live-cell imaging of small micromere plasma membranes. Results: Early in gastrulation, small micromeres transition from non-motile epithelial cells, to motile quasi-mesenchymal cells. Late in gastrulation, at 43 hr post fertilization (HPF), they are embedded in the tip of the archenteron, but remain motile. From 43-49 HPF, they project numerous cortical blebs into the blastocoel, and filopodia that contact ectoderm. By 54 HPF, they begin moving in the plane of the blastoderm, often in a directed fashion, towards the coelomic pouches. Isolated small micromeres also produced blebs and filopodia. Conclusions: Previous work suggested that passive translocation governs some of the movement of small micromeres during gastrulation. Here we show that small micromeres are motile cells that can traverse the archenteron, change position along the left-right axis, and migrate to coelomic pouches. These motility mechanisms are likely to play an important role in their left-right segregation. Developmental Dynamics 243:917-927, 2014. V C 2014 Wiley Periodicals, Inc.

Ionocytes are specialized cells in the epidermis of embryonic zebrafish (Danio rerio) that play important roles in ion homeostasis and have functional similarities to mammalian renal cells. Here, we examined whether these cells might also share another functional similarity with renal cells, which is the presence of efflux transporter activities useful for elimination of toxic small molecules. Xenobiotic transporters (XTs), including the ATP-Binding Cassette (ABC) family, are a major defense mechanism against diffusible toxic molecules in aquatic embryos, including zebrafish, but their activity in the ionocytes has not previously been studied. Using fluorescent small molecule substrates of XT, we observed that specific populations of ionocytes uptake and efflux fluorescent small molecules in a manner consistent with active transport. We specifically identified a P-gp/ABCB1 inhibitor-sensitive efflux activity in the H +-ATPase-rich (HR) ionocytes, and show that these cells exhibit enriched expression of the ABCB gene, abcb5. The results extend our understanding of the functional significance of zebrafish ionocytes and indicate that these cells could play an important role in protection of the fish embryo from harmful small molecules.