Dorsoventral (DV) patterning in Drosophila melanogaster is one of the most well-known gene regulatory networks (GRN) in biology. To investigate if this GRN is conserved during insect evolution, functional analysis of TGF-beta and Toll pathways in the short-germ beetle Tribolium castaneum was performed. In the first part, the function of several BMP/Dpp extracellular modulators, including the products of Tolloid (Tld) and Twisted-gastrulation/Crossveinless (Tsg-Cv), was investigated in Tribolium via parental RNAi (pRNAi). While Tc-tld pRNAi knock-down decreases embryonic BMP activity, Tc-tsg(cv) knock-down completely abolishes it. These observations are strikingly different from those in Drosophila, where tsg is only required for a subset of Dpp activity. These results suggest that Tsg/Cv-like proteins are essential for BMP signalling in Tribolium. Since duplicated copies of tsg(cv)- and tld-related genes are present in the Drosophila melanogaster genome, duplication followed by sub-functionalization of these modulators might have changed the BMP/Dpp gradient during evolution of the dipteran Drosophila lineage. In the second part, a functional analysis of the Toll pathway was performed. This analysis addressed the question of why the Tribolium Dorsal nuclear gradient is not stable, but rapidly shrinks and disappears, in contrast to the stable Drosophila gradient. Negative feedback accounts for this dynamic behavior: Tc-Dorsal and one of its target genes (Tc-Twist) activate transcription of the I-KappaB homolog Tc-cactus, which in turn terminates Dorsal function. Despite its transient role, Tc-Dorsal is strictly required to initiate DV polarity, as in Drosophila. However, unlike Drosophila, embryos lacking Tc-Dorsal display a periodic pattern of DV cell fates along the AP axis, indicating that a self-organizing ectodermal patterning system operates independently of mesoderm or maternal DV polarity cues. The presence of self-organizing patterning systems in short-germ insects like Tribolium is in agreement with a regulative type of embryogenesis proposed by classical fragmentation studies on hemimetabolous insects. These results also elucidate how extraembryonic tissues are organized in short-germ embryos, and how patterning information is transmitted from the early embryo to the growth zone. Altogether, the functional analysis of the TGF-betA and Toll pathways in Tribolium dorsoventral patterning suggests that extensive changes in this GRN have occurred during insect evolution
