Clock-based segmentation in the red flour beetle Tribolium castaneum

Doctor of PhilosophyGenetics Interdepartmental ProgramSusan J. BrownIn Drosophila, all segments form in the blastoderm where morphogen gradients spanning the entire anterior-posterior axis of the embryo provide positional information. However, in the beetle Tribolium castaneum and most other insects, a number of anterior segments form in the blastoderm, and the remaining segments form sequentially from a posterior growth zone during germband elongation. In this work, I show that segmentation at both blastoderm and germband stages of Tribolium is based on a segmentation clock. Specifically, I show that the Tribolium primary pair-rule gene, Tc-even-skipped (Tc-eve), is expressed in waves propagating from the posterior pole and progressively slowing until they freeze into stripes; such dynamics are a hallmark of clock-based segmentation. Phase shifts between Tc-eve transcripts and protein confirm that these waves are due to expression dynamics. Such waves, like their counterparts in vertebrates, are assumed to arise due to the modulation of a molecular clock by a posterior-to-anterior frequency gradient. I provide evidence that the posterior gradient of Tc-caudal (Tc-cad) expression regulates the oscillation frequency of pair-rule gene expression in Tribolium. I show this by correlating the gradient of Tc-cad expression to the spatiotemporal dynamics of Tc-even-skipped expression in WT as well as in different knockdowns of Tc-cad regulators. Specifically, the spatial extent, frequency, and width of Tc-eve waves correlate with the spatial extent, expression level, and slope of Tc-cad gradient, respectively, as predicted by computer modeling. These results pose intriguing evolutionary questions, since Drosophila and Tribolium segment their blastoderms using the same genes but different mechanisms, and highlight the role of frequency gradients in pattern formation