Musterbildung in der Wachstumszone des Kurzkeim-Embryos von Tribolium: "segmentation clock" oder Gapgen-Mechanismus?
Final Report Abstract
In contrast to long germ insects like Drosophila, where all segments are formed during the blastoderm stage, in short germ insects the abdominal segments arise during subsequent growth stages. In basal arthropods, and probably also in the short germ beetle Tribolium, these segments are generated through a periodic mechanism, similarly to the segmentation clock of vertebrates. While a pair-rule gene based mechanism has been proposed for the beetle, also gap genes are known to be essential for posterior patterning. One main goal of this project was to employ mis-expression experiments to probe the function of gap genes in the growth zone, or segment addition zone, since loss-of-function experiments using RNAi usually result in the loss of germ band extension, thus obscuring the function of gap genes during germband growth (A). In addition, we investigated the function of the atypical gap gene mille-pattes, whose phenotype ressembles that of other gap genes, but which encodes several short peptides rather than a transcription factor (B). Project part (A): In this part of the project, we followed two approaches to disturb short germ segmentation by temporal, respectively spatial, misexpression of Tribolium gap gene homologs. Interestingly, we find that overexpression of the gap gene Tc-hunchback using a heat shock promoter, results in up to 17 (vs. normally 13) trunk segments. This phenotype throws light on the mechanisms of short germ segmentation in Tribolium in that we show pair-rule genes (as well as other gap genes) to be overexpressed in this situation. If segmentation in Tribolium indeed is generated through the action of a segmentation clock, our data indicate that pair-rule genes nevertheless also receive gap gene input. However, supernumerary segments seem to be formed through pattern repair of fragmented segment-polarity stripes, not via additional cycles of a segmentation clock, as one model for Tribolium segmentation would have predicted. Another approach, to mis-express segmentation genes along a longitudinal line using the single-minded promoter, has not been successful so far, since the constructs tested - while reproducing the pattern of the endogenous gene - appear not to drive sufficient levels of expression. This approach should eventually allow to test interaction with all emerging pair-rule stripes in the segment addition zone, and it should allow to test if such interactions are cell-autonomous. Thus, this approach will be continued but requires additional efforts. Project part (B): Here we could show that the polycistronic gene mille-pattes, which expresses several short peptides, functions by regulating the transcription factor shavenbaby also during Tribolium segmentation, an interaction previously found for most mille-pattes functions in Drosophila.
