Formation of the Clypeolabral Region During Embryonic Head Development of the Red Flour Beetle Tribolium castaneum

Abstract

Insects are segmented organisms but the number of segments contributing to the head is disputed. The most anterior head region is patterned differently from the rest of the body and, therefore, is considered to be non-segmental. During embryogenesis, the non-segmental region can be subdivided into an outer neurogenic region and an inner triangular part which is mostly free of neurogenic precursor cells. Due to the similarities with a structure known from embryonic development of the vinegar fly Drosophila melanogaster (MEIGEN, 1830), this median region will be referred to as the clypeolabral region (CLR). It gives rise to the larval clypeolabrum and comprises the anlagen of the anterior gut and the stomatogastric nervous system. The red flour beetle Tribolium castaneum (HERBST, 1797) has been established as an important model organism for insect head development due to its insect-typical head and its amenability to functional studies. The formation of the CLR in Tribolium involves a previously unnoticed connection between mesodermal cells and the extraembryonic amnion as well as the formation of a fold in the anterior head. The anterior fold separates the ectoderm of both head sides during early embryogenesis and apparently accounts for the generation of the lateral head lobes found in insect embryos. It is probably also responsible for the relocation of anterior cells to a more posterior sub-terminal position. Moreover, it may be involved in the formation of the stomodaeum. Seven genes were found to show a rather exclusive expression pattern in the CLR during embryogenesis. Surrounding head regions are marked by a distinct set of genes, as well. RNA interference-mediated knockdown of several genes expressed in the anterior head and the subsequent analysis of developing larvae and embryonic gene expression patterns led to the establishment of a model for a gene regulatory network of the CLR. While the late expression aspects of the analyzed genes show a high degree of evolutionary conservation throughout arthropods, the genetic interactions during CLR development appear to be only partly conserved between Drosophila and Tribolium. Three genes, Tc-six3, Tc-crocodile (Tc-croc) and Tc-cap'n'collar (Tc-cnc) were identified as most important upstream regulators of CLR development. Tc-six3 is necessary for all anterior ectodermal derivatives, Tc-croc for development of the foregut and Tc-cnc for clypeolabrum formation. I established transgenic animals for ubiquitous expression of Tc-croc and Tc-six3 by using the φC31-based site specific integration system to further test the function of these genes during embryonic head development.