UC Berkeley

Crustaceans are a group of organisms with tremendous diversity in the organization of limbs alongtheir body, also known as the bauplan, or body plan. This diversity is hypothesized to arise from evolutionary changes to the expression domains of the Hox genes, an ancient gene family conserved to most animals. Previous researchers have used the amphipod crustacean Parhyale hawaiensis as a genetic model organism to examine the expression and function of Hox genes within crustaceans. This work revealed that each of the Parhyale Hox genes is expressed in a unique domain of expression that prefigures the ultimate arrangement of different limb types within the Parhyale body plan. Moreover, RNA interference, CRISPR-Cas9 mutagenesis, and heat shock overexpression experiments revealed that each of the Hox genes in Parhyale is required – or sufficient – for the development of different types of limbs. Taken in the context of changing Hox expression patterns, these results suggest that crustacean body plan evolution may be driven by changes to Hox gene regulation between different crustacean groups. In this thesis, I present several avenues of investigation into the genetic and genomic basis of crustacean body plan evolution. First, I describe the generation of a developmental functional genomic database for the amphipod crustacean Parhyale hawaiensis. Using Omni-ATAC-Seq and short- and long-read RNA-Seq, I generate an improved genome annotation for Parhyale, discover hundreds of thousands of candidate cis-regulatory elements across the Parhyale genome, and infer additional features of the chromatin landscape of Parhyale development. Using this dataset, I demonstrate the potential to generate novel reporter genes. This dataset will serve as a platform for future researchers to investigate cis-regulation across the genome during Parhyale development, and will also enable the discovery of Hox cis-regulatory mechanisms. Second, I describe experiments that reveal the role of two families of general transcriptional regulators, the Polycomb Group and Trithorax Group proteins, in regulation of the Parhyale Hox genes. Loss of function of Polycomb Group genes in particular results in homeotic transformations and changes to Hox expression. By examining the timing of these changes and synthesizing these data with other work, 2 I demonstrate that Parhyale Hox regulation occurs via at least three distinct mechanisms: early PcG-independent boundary establishment, PcG-dependent boundary maintenance, and Hox cross-regulatory interactions. I propose that crustacean Hox genes have evolved decoupled regulation at the level of developmental timing, tissue type, and specific PcG gene function. These results provide some of the first insights into the genetic mechanisms of Parhyale Hox regulation. Moreover, these data provide new mechanistic explanations for how crustaceans may have evolved their tremendous body plan diversity. Finally, I describe efforts to establish animal husbandry, injection, and dissection techniques for the isopod crustacean Asellus aquaticus and the decapod crustacean Neocaridina davidi. I provide a developmental staging guide for each crustacean. I also describe the methods I have used to isolate early embryos for injection and the various types of troubleshooting I have performed in an attempt to establish these organisms as new genetic model systems. The work presented here will prove useful in future efforts to establish new crustacean genetic model systems.