Head versus tail: germ cell-less initiates axis formation via homeobrain and zen1 in a beetle

The iBeetle screen project: Christiane Nusslein-Volhard and Eric Wieschaus performed the first saturated forward genetic screen using random mutagenesis to identify genes important for embryonic development in Drosophila melanogaster. However, the Drosophila larval head turns outside in during embryogenesis as a result this involuted head lacks phenotypic markers for identification of defects. Moreover, perturbation of Drosophila head development often interferes with head involution making it difficult to identify the primary phenotype. Therefore, we decided to study head development in an alternative model organism with an insect typical head, Tribolium castaneum. In order to identify all genes required for head development, the genome-wide iBeetle RNAi screen has been performed. I participated in the second phase of this screen to not only identify head related phenotypes but also muscle, ovary, stink gland and other phenotypes. After completion of screening phase with 865 screened genes, I was able to identify eight novel head patterning genes from 3,500 genes during the rescreening phase. Moreover, I performed the preliminary study of all eight genes which include phylogenetic analysis, RNAi phenotype and expression pattern analysis. The Gcl project: Axis formation is an essential, early processes during bilaterian development. Classical manipulation like cytoplasm leakage, cytoplasm ligation, UV irradiation and RNase treatment showed that global organizing centres operate from both ends of insect eggs and establish the anterior-posterior (A-P) axis. Christiane Nusslein-Volhard and co-workers identified the first anterior global pattern organizer (-bicoid) from Drosophila melanogaster in 1987. Bicoid is a morphogen that autonomously patterns the Summary 2 anterior structure in a concentration dependent manner to pattern the A-P axis. However, bicoid is limited to higher flies and neither anterior determinants nor a molecular mechanism that establishes A-P polarity in less short germ insects like beetles have been discovered. In an ongoing genome-wide RNAi screen in the short germ beetle Tribolium castaneum, two genes namely Tc-germ cell-less (Tc-gcl) and Tc-homeobrain (Tc-hbn) were identified whose knockdown resulted in larvae with a double abdomen phenotype similar to Dm-bicoid and Dm-hunchback double mutants in Drosophila. Dm-gcl is known to be involved in germ cell development at the posterior pole, but not for anterior patterning in Drosophila. Surprisingly, I found that Tc-gcl is involved in A-P axis formation at the anterior pole in Tribolium. The Drosophila ortholog of the second gene (-Hbn) did not have phenotypic or functional information available. I was able to show that Tc-hbn plays an important role in axis formation in Tribolium. This is the first report where zygotic genes are required for axis formation in insects as they do in vertebrates. Specifically, duplication of functional SAZ (segment addition zone) either during blastoderm stage or postblastoderm stage provide the evidence of zygotic control of axis formation. The first double anterior phenotype for short germ insects was observed after double knockdown of Tc-caudal and Tc-pangolin. Moreover, Wnt signaling needs to be repressed anteriorly during early embryogenesis for proper anterior development in Tribolium similar to vertebrates (but not in Drosophila). We introduce a novel technique that will help overcoming a major problem of RNAi studies in the emerging model organisms. Often, RNAi leads to lethality or sterility of the mother before offspring can be analyzed. In this study, I was able to show that VSR (viral suppressor of RNAi) transgenic line (developed by Julia Ulrich), which ubiquitously expresses an RNAi inhibitor, can rescue the sterility of the mother without compromising the zygotic phenotypes. Based on my finding, I established the most comprehensive model for insect axis formation outside of Drosophila.