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,
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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.