Temporal restriction of RNAi reveals breakdown of the segmentation clock is reversible after knock down of primary pair rule genes but not Wnt-signaling in the red flour beetle

Animals from all major clades have evolved a segmented trunk, reflected for instance in the repetitive organization of the human spine or the insect segments. These units emerge during embryonic segmentation from a posterior segment addition zone, where repetitive gene activity is regulated in a spatiotemporal dynamic described by the clock and wavefront/speed gradient model. This model has been tested in the red flour beetle Tribolium castaneum and other insects by studying the effect of the RNAi knockdown of segmentation genes. For upstream components such as primary pair rule genes, caudal or Wnt pathway components, this treatment often led to the breakdown of segmentation. However, it has remained untested, how the system would react to a temporally limited interruption of gene function. In order to ask such questions, we established a novel experimental system in T. castaneum , which allows blocking an ongoing RNAi effect with temporal control by expressing a viral inhibitor of RNAi. We show that the T. castaneum segmentation machinery re-established after we blocked an ongoing RNAi response targeting the primary pair rule genes Tc-eve, Tc-odd and Tc-runt . However, we observed no rescue after blocking RNAi responses targeting Wnt pathway components. We conclude that the insect segmentation system contains both, robust feedback-loops that can re-establish and labile feedback loops that can breakdown irreversibly. This combination may reconcile two partially conflicting needs of the embryonic regulation of segmentation: A tightly controlled initiation and maintenance of the SAZ by labile feedback-loops ensures that only one segment addition zone is formed. Conversely, robust feedback-loops confer developmental robustness required for proper segmentation, which may be challenged by internal or external disturbances. Our results ponder the insect segmentation machinery from a different angle and introduce a new experimental tool for temporal control on RNAi. Significance statement The generation of repetitive body parts during embryonic segmentation has been of key interest to developmental biologists, who usually used permanent knock-down of gene function for their studies. Using a new tool to temporally stop a gene knock-down effect, we find both robust and labile feedback-loops within the segmentation machinery. Thereby, the embryo may ensure that only one trunk is formed but that trunk formation is robust against external disturbance. ### Competing Interest Statement The authors have declared no competing interest.