Abstract
The beetle Tribolium castaneum has increasingly become a powerful model for comparative research on insect development. One recent resource is a collection of piggyBac transposon-based enhancer trap lines. Here, we provide a detailed analysis of three selected lines and demonstrate their value for investigations in the second half of embryogenesis, which has thus far lagged behind research on early stages. Two lines, G12424 and KT650, show enhanced green fluorescent protein (EGFP) expression throughout the extraembryonic serosal tissue and in a few discrete embryonic domains. Intriguingly, both lines show for the first time a degree of regionalization within the mature serosa. However, their expression profiles illuminate distinct aspects of serosal biology: G12424 tracks the tissue’s rapid maturation while KT650 expression likely reflects ongoing physiological processes. The third line, G04609, is stably expressed in mesodermal domains, including segmental muscles and the heart. Genomic mapping followed by in situ hybridization for genes near to the G04609 insertion site suggests that the transposon has trapped enhancer information for the Tribolium orthologue of midline (Tc-mid). Altogether, our analyses provide the first live imaging, long-term characterizations of enhancer traps from this collection. We show that EGFP expression is readily detected, including in heterozygote crosses that permit the simultaneous visualization of multiple tissue types. The tissue specificity provides live, endogenous marker gene expression at key developmental stages that are inaccessible for whole mount staining. Furthermore, the nonlocalized EGFP in these lines illuminates both the nucleus and cytoplasm, providing cellular resolution for morphogenesis research on processes such as dorsal closure and heart formation. In future work, identification of regulatory regions driving these enhancer traps will deepen our understanding of late developmental control, including in the extraembryonic domain, which is a hallmark of insect development but which is not yet well understood.
Highlights
After the fruit fly Drosophila melanogaster, the red flour beetle, Tribolium castaneum, is one of the most established models for studying insect development
We discuss four aspects of the enhanced green fluorescent protein (EGFP) expression in the enhancer trap lines: (i) characteristics of the transgenic promoter, (ii) new insights into the biology of the serosa, (iii) how expression in the mesodermal line G04609 compares to the expression of genes near the transposon insertion site, and (iv) some general features that make these lines valuable visualization tools
Our analyses involved the definition of morphological landmarks as well as their timing and variability under live imaging conditions (Fig. 1H, Table 1)
Summary
After the fruit fly Drosophila melanogaster, the red flour beetle, Tribolium castaneum, is one of the most established models for studying insect development. Many advances have been made in building the toolkit for Tribolium research, including genomic resources, forward and reverse genetics techniques, and transgenesis for both visualization and mis-expression applications [1,2,3,4,5,6] These tools make Tribolium a powerful resource for comparative developmental genetics, as the biology of this species is less derived and more broadly representative of insect development than Drosophila. While Drosophila eggs have an amnioserosa, a monolayered epithelium that covers the yolk dorsally, most insects, including Tribolium, have distinct amniotic and serosal tissues that cover both the embryo and yolk [13,14] Their late morphogenetic movements to withdraw from the embryo are both essential for development in Tribolium and more representative of how these tissues play key roles in positioning the embryo itself, in the more basally branching hemimetabolous insects [13,15,16]
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