Functional Divergence of the Tribolium castaneum engrailed and invected Paralogs

SIMPLE SUMMARY: The engrailed (en) and invected (inv) paralogs play a fundamental role in arthropod segmentation. Previous research suggests that knockdown of either en or inv in sequentially segmenting insects leads to an unexpected and variable loss of segments but does not mimic the segment polarity defects seen in Drosophila en mutants; the consequences for segmentation when both paralogs are lost have not been reported outside of Drosophila. We analyzed the phenotypes of single and double knockdowns in the flour beetle Tribolium castaneum. Unlike Drosophila, inv knockdowns are inviable, consistent with a functional divergence of the paralogs between Tribolium and Drosophila. We find the Tribolium paralogs are redundant and act synergistically to pattern trunk appendages and segments. The most common Tribolium double knockdown results in small, limbless larvae that suffer a loss of a portion of each trunk segment that shares characteristics with segment polarity mutants in Drosophila. Some of the double knockdown embryos arrest development before germband retraction, consistent with an underexplored early function for en and inv in the regulation of cell proliferation or death in sequentially segmenting insects. ABSTRACT: Engrailed (en) and invected (inv) encode paralogous transcription factors found as a closely linked tandem duplication within holometabolous insects. Drosophila en mutants segment normally, then fail to maintain their segments. Loss of Drosophila inv is viable, while loss of both genes results in asegmental larvae. Surprisingly, the knockdown of Oncopeltus inv can result in the loss or fusion of the entire abdomen and en knockdowns in Tribolium show variable degrees of segmental loss. The consequence of losing or knocking down both paralogs on embryogenesis has not been studied beyond Drosophila. To further investigate the relative functions of each paralog and the mechanism behind the segmental loss, Tribolium double and single knockdowns of en and inv were analyzed. The most common cuticular phenotype of the double knockdowns was small, limbless, and open dorsally, with all but a single, segmentally iterated row of bristles. Less severe knockdowns had fused segments and reduced appendages. The Tribolium paralogs appear to act synergistically: the knockdown of either Tribolium gene alone was typically less severe, with all limbs present, whereas the most extreme single knockdowns mimic the most severe double knockdown phenotype. Morphological abnormalities unique to either single gene knockdown were not found. inv expression was not affected in the Tribolium en knockdowns, but hh expression was unexpectedly increased midway through development. Thus, while the segmental expression of en/inv is broadly conserved within insects, the functions of en and inv are evolving independently in different lineages.