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Scholarly Works (3 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2022)

This thesis concerns aspects of the development of the sea urchin Lytechinus pictus, a species that can be cultured from egg to breeding adult in six months. The first experimental portion of this thesis (Chapter 2) examines the developmental role of the enzyme phosphatidic acid phosphatase 2B (Pap2b). Fluorescent in situ hybridization demonstrated that Pap2b is expressed ubiquitously in the larval skeletogenic mesenchyme, and not in migrating primordial germ cells where expression was initially expected by analogy with studies on flies and zebrafish. The specific inhibitor Propranolol was used to reduce the function of Pap2b. At a low dose (0.5 µM) the body rods and ventral transverse rods of the larval skeleton failed to fuse normally; higher concentrations resulted in stunted embryos with fragmented, unpatterned skeletal elements. The second major experimental part of this thesis (Chapter 3) concerns the adoption of CRISPR-Cas9 gene editing in L. pictus. The majority of sea urchin CRISPR studies have been done in species that cannot be easily cultured, and have therefore relied on analysis of mosaic F0 embryos. I used CRISPR to create a homozygous knockout sea urchin line, an accomplishment so far attained by only one other laboratory in the world. I mutated the ATP binding cassette (ABC) transporter ABCB1, a small molecule transporter involved in cell signaling, environmental chemical uptake, and immune biology. L. pictus embryos were injected with two guide RNAs against Lp-ABCB1, and genotyping of post-metamorphic juveniles confirmed their activity. Subsequently, non-lethal somatic genotyping was used to identify mutant post-metamorphic individuals. F0 mosaic animals were outbred to wild-type animals to generate F1 heterozygotes, and these were subsequently inbred to produce homozygous F2 mutants. These results provide a foundation for further studies on the function of ABCB1, and more generally for the broader adoption of genetic engineering in sea urchin research. Finally, Chapter 4 of this thesis presents several topics, generally on insertion of transgenes using gene editing, which do not merit a whole chapter individually. These observations may serve as a starting point for future researchers interested in expanding the repertoire of gene editing available in L. pictus.

    Cover page: Imaging and CRISPR mediated genetic modification of pathways involved in sea urchin primordial germ cell migration and protection.
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2019)
    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.
      Cover page: Xenobiotic transporter activity in zebrafish embryo ionocytes
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2018)
      The calcium carbonate skeletons of corals provide the underlying structure of coral reefs; however, the cellular mechanisms responsible for coral calcification remain poorly understood. In osteoblasts from vertebrate animals, a Na+/Ca2+ exchanger (NCX) present in the plasma membrane transports Ca2+ to the site of bone formation. The aims of this study were to establish whether NCX exists in corals and its localization within coral cells, which are essential first steps to investigate its potential involvement in calcification. Data mining identified genes encoding for NCX proteins in multiple coral species, a subset of which were more closely related to NCXs from vertebrates (NCXA). We cloned NCXA from Acropora yongei (AyNCXA), which, unexpectedly, contained a peptide signal that targets proteins to vesicles from the secretory pathway. AyNCXA subcellular localization was confirmed by heterologous expression of fluorescently tagged AyNCXA protein in sea urchin embryos, which localized together with known markers of intracellular vesicles. Finally, immunolabeling of coral tissues with specific antibodies revealed AyNCXA was present throughout coral tissue. AyNCXA was especially abundant in calcifying cells, where it exhibited a subcellular localization pattern consistent with intracellular vesicles. Altogether, our results demonstrate AyNCXA is present in vesicles in coral calcifying cells, where potential functions include intracellular Ca2+ homeostasis and Ca2+ transport to the growing skeleton as part of an intracellular calcification mechanism.
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