Large scale RNAi screen in Tribolium reveals novel genes involved in dorsoventral pattern formation

The detailed knowledge about dorsoventral (DV) axis formation in Drosophila melanogaster provides an excellent platform for comparative studies of embryonic DV patterning within insects. In Drosophila, the ventral activation of Toll-receptor initiates the nuclear uptake of the transcription factor NF-kB/Dorsal resulting in a stable nuclear Dorsal gradient, which regulates zygotic target genes like the mesodermal gene twist and the neuroectodermal gene short gastrulation (sog) in a concentration dependent manner. In the red flour beetle Tribolium castaneum, NF-kB/Dorsal also forms a nuclear concentration gradient. This gradient, however, is highly dynamic due, to positive and negative feedback loops. Both Tc-Toll and the inhibitor Tc-cactus are activated zygotically by Toll signaling. In the past, the investigation of DV patterning genes in T. castaneum was based on a candidate gene approach. Through this comparative approach, only genes which were known to have a DV function in D. melanogaster were investigated in T. castaneum, which leads to biased conserved gene function analysis. Hence, novel DV patterning genes in T. castaneum that are not present in D. melanogaster, could not be identified through a candidate gene approach. Therefore, I have participated in a large scale RNAi screen (the iBeetle screen Phase-II). The unbiased iBeetle RNAi screen provided an excellent platform to overcome the limitations of the candidate gene approach and revealed novel DV patterning genes in T. castaneum. In the course of a large scale RNAi screen in T. castaneum, we have identified three novel DV patterning genes (two serine proteases and one serpin inhibitor) and the Toll ligand Spaetzle. The newly identified genes act upstream of Toll signaling and regulate the activity of NF-kB/Dorsal gradient formation and the activation of zygotic target genes. Knockdown of both iB-09824 (anionic trypsin-2-like serine protease) and iB-06774 (spaetzle) produced Toll-like dorsalized phenotype, whereas knockdown of iB-06699 (leukocyte elastase inhibitor-like) revealed ventralized phenotype. In addition, another positive feedback element, a new serine protease (iB-07888) was identified in the iBeetle screen. iB-07888 is likely to act upstream of Toll within the protease cascade activating the Toll ligand Spaetzle. The expression of this gene is activated ventrally during early blastoderm stage in a broad domain along the entire egg length. After iB-07888 knockdown, Tc-twist, Tc-sim and Tc-cactus lack early expression and are activated only during early gastrulation. This corresponds to a delayed formation of the nuclear Dorsal gradient. In contrast to Tc-twist Tc-sim and Tc-cactus, the expression of Tc-sog is completely lost in most knockdown embryos. This suggests that the new protease affects the timing of Dorsal gradient formation. Taken together, our results indicate that the Dorsal target genes in T. castaneum are activated in a temporal sequence to define their spatial expression domains at different time points. We propose that in contrast to D. melanogaster, where several Dorsal target genes are activated simultaneously in a concentration dependent manner, the Tribolium NF-kB Dorsal gradient acts by a temporal shift to regulate its zygotic target genes in a time-dependent manner