The Regulation and Role of the Hox Gene Ultrabithorax in Hindwing Identity in Lepidoptera
Open AccessThe origin and evolution of Hox genes was a major event in the diversification of animal body plans. These deeply conserved developmental genes bring about differentiation of serial homologs in bilateral animals. Mutations in Hox genes result in homeosis - loss of one body identity and gain of another. My research focuses on the Hox gene Ultrabithorax (Ubx) and its role in insect wing diversification. Wings show structural differences among insect orders - balancing halteres in flies, protective hardened shells in beetles and intricate patterns in butterflies. In insects, Ubx is expressed in the third thoracic segment and is responsible for the ontogenesis of the hindwing. The role of Ubx in hindwing differentiation has been studied in flies, beetles and planthoppers; however, description of its role in butterflies and moths is limited. I used CRISPR targeted mutagenesis to generate Ubx loss-of-function somatic mutations in two nymphalid butterflies (Junonia coenia, Vanessa cardui) and a pyralid moth (Plodia interpunctella). The resulting mosaic clones yielded hindwing- to-forewing transformations in scale shape and size, and color patterns. Additionally, in the moth P. interpunctella, loss of Ubx generated wing venation defects and ectopic forewing secondary sexual traits on the hindwing, showing that Ubx is necessary for specifying many aspects of hindwing-vi specific identities. My study establishes Ubx as the micromanager of hindwing identity, a result that parallels observations in other insects. To disentangle the various Ubx functions, I used conservation analysis and published ATAC-seq data to identify open chromatin regions within the Ubx Topologically Associating Domain (TAD) conserved within Lepidoptera. To functionally test these putative regulatory regions, I generated mutations of putative CREs using CRISPR-Cas9. I identified various regulatory regions within the TAD that have an activating and/or a repressive effect on Ubx expression. I also identified a region at the TAD boundary between Ubx and its upstream neighbor Antennapedia (Antp), disruption of which caused transformation of forewings into hindwings at high-frequency. These data suggest that mutational perturbation of the TAD boundary results in a misexpression of Ubx in the normally Ubx-free mesothorax, possibly due to a de-insulation of the TAD regulatory environment. Since genomic knock-outs can be lethal due to gene pleiotropy, I also implemented and optimized an RNA interference method in J. coenia to study wing specific functions of Ubx. This study revealed that Ubx input is required at early pupal stages to impart hindwing identity during pigment deposition. My study provides insight into the different aspects of lepidopteran hindwing that Ubx controls and contributes knowledge to compare lepidopteran hindwing development with other insects.
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Tendolkar_gwu_0075A_16488.pdf | 2023-11-14 | Open Access |
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