FATE MAP OF THE BLASTODERM TO DETERMINE SEGMENTAL FATE IN TRIBOLIUM CASTANEUM

Segmentation in arthropods has been modeled on the well-defined segmentation patterns found in Drosophila. In Drosophila, segments form simultaneously in the blastoderm where morphogenic gradients spanning the AP axis provide patterning inputs. However, in most arthropods, segments form sequentially from a posterior growth zone. Sequential segmentation in arthropods has recently been demonstrated to use a vertebrate- like segmentation clock (Sarrazin et al. 2012). The vertebrate segmentation clock is a molecular oscillator that regulates periodic somite formation (Gibb 2010). In the red flour beetle, Tribolium castaneum, the segmentation clock is coordinated by traveling waves of expression generated by a pair-rule gene oscillator. For this study, we aimed to identify and describe regulatory controls of the Tribolium clock and construct a fate map of the blastoderm. We particularly focus on whether the output of the clock at the blastoderm stage - prior to the striking rearrangements of germband formation - actually determines cell fate. From preliminary studies we know that the caudal and even-skipped genes are two key genes of the Tribolium segmental clock. To understand how the clock is regulated, we identified and isolated 2 kb upstream of the caudal promoter region. We have cloned and sequenced this fragment to use in building reporter constructs that will be used to identify the cis- regulatory region driving wild type caudal expression. A deletion series of those regions showing enhancer activity will be completed to resolve the cis-regulatory regions to smaller domains (~500 bp-1.5 kb). In efforts to generate a fate map of the blastoderm, we created the T3-Nls-Eos DNA template for later mRNA synthesis. We will perform embryo injections with mRNA encoding for EosFP to determine the degree to which segmental fate is determined at the blastoderm stage of development