Abstract
Gene Regulatory Networks (GRN) regulating embryogenesis are complex and multifaced; how they evolve to produce new morphologies is mainly unknown. GRNs must be robust, producing the same output in each embryo regardless of environment but also flexible enough to support evolutionary change. Studies in model insects for segmentation are beginning to reveal slight regulatory adjustments to GRNs that share the same genes can generate the significant interspecies variation observed today.
Insects are segmented organisms, a feature that underpins their diversity and morphology. The process of dividing the embryo into segments, segmentation, has been well studied at the genetic level in Drosophila melanogaster. One key aspect of segmentation is the switch from double-segment to single-segment periodicity. This switch, in Drosophila, involves two genes, caudal and odd-paired, acting to switch segmentation from one GRN that produces double segments to another that produces single segments. Drosophila segment in what is known as a long germ manner, with segmentation occurring simultaneously across the embryo. Many other insects, including Nasonia vitripennis (Jewel Wasp), segment in a progressive way, with segmentation occurring in an anterior-to-posterior direction. This well- studied GRN control mechanism, and interspecies variation among insects, allows us to study the evolution of a GRN which is crucial for translating data from model organisms to human health.
My work aimed to knock out the odd-paired and caudal genes in Nasonia embryos using a maternally injected mutagenesis tool. This tool, named Remote-Crispr, is created from a peptide (P2C3) that transduces Cas9 into the ovaries where, with a guide RNA, it can cut its targets. By causing double-strand breaks, and mutations in target genes, I hoped to assess the 2 function of these genes in comparison to Drosophila and build a platform for testing the function of other genes in the GRN network. In vitro analysis found the P2C3-Cas9 system able to cut the target DNA, however, no mutants were found in cuticle preparation screens. Immunohistochemistry found the P2C3 system to be present in the ovaries and in situ Hybridisation Chain Reaction v.3 HCR found the target genes were expressed in the supporting ovary cells. For the caudal gene, one-hour-old Cas9 embryonic injections were performed, but no mutants were found. Overall this thesis found that P2C3 is not a successful mutagenesis mechanism for studying the Nasonia segmentation GRN.