Imaging and CRISPR mediated genetic modification of pathways involved in sea urchin primordial germ cell migration and protection.
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Imaging and CRISPR mediated genetic modification of pathways involved in sea urchin primordial germ cell migration and protection.


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.

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