Deciphering Segmentation: Identifying Eve Enhancers in Tribolium Castaneum

Abstract

Arthropods are a phylum of invertebrates that are distinguished by their ability to generate segments in their body plan, utilizing at least two modes of development to do so. Drosophila melanogaster is a long-germband insect that can give rise to its segments in a simultaneous manner. The segmental development of Tribolium castaneum is characteristic of a short-germ band insect that develops its segments sequentially through the regulation of a molecular oscillator. These two arthropods have in common a segmentation gene termed even-skipped (eve) that oversees how the segments are created. Using this gene, we can examine how it is that these two different mechanisms of segmentation came to exist. In Drosophila, stripe-specific and modular enhancers regulate and drive expression of eve; the enhancers of Tribolium eve are unknown. To study the enhancers that regulate eve in Tribolium, we used a bioinformatics tool to aid us in predicting putative enhancers. Tribolium and Drosophila transgenics were then created that have one of five constructs integrated into their genome and enhancer-mCherry fusions were utilized to visualize the expression patterns. Immunohistochemistry revealed that the Tc-eve intron 2 enhancer drives expression in the posterior growth zone, hindgut, and nervous system in Tribolium, whereas in Drosophila, the Tribolium enhancer drove expression in the dorsal vessel (heart) and consistently the hindgut. To date, the transgenics and wild-type endogenous eve share expression only in the hindgut. Our current data suggests that the candidate Tc-eve intron 2 enhancer regulates eve during posterior growth, a function unique to sequentially segmenting insects and later-hindgut expression of eve - a function shared more broadly throughout metazoans.