Developmental time-course study of Drosophila melanogaster, D. sechellia, D. simulans, and D. sim x D.s sec hybrids

In order to test the hypothesis that adult hybrid misexpression results from the cascading effect of earlier-expressed developmentally important improperly regulated genes, as well as address whether Von Baer’s 3rd law (suggesting that earlier stages of development should be more similar between species than later stages) holds at the level of gene expression, we conducted whole-transcriptome Drosophila melanogaster cDNA microarray-based expression profiling of males of D. melanogaster, D. sechellia, and D. simulans, at four synchronized developmental time-points (3rd instar larval [larval], early pupal, late pupal, and newly-emerged adult [adult]). D. simulans and D. sechellia shared a most recent common ancestor (MRCA) ~0.5 to 1.0 million years ago (MYA) and form a clade that shared an MRCA with D. melanogaster approximately 5.4 MYA. In addition, we also performed the same analysis on the male interspecific F1 hybrids of the D. simulans (♀) × D. sechellia (♂) cross. Males from each species and hybrids were synchronized within a 2h window (1.5h for adults) at the beginning of the stage during which they were collected (larval, early pupal, late pupal and adult) and 25 staged individuals were collected and their RNA extracted and amplified twice according to the protocols associated with each array. For the three pure species, 3 technical replicates were performed for each stage; however, in the case of the D. simulans x D. sechellia F1 hybrids, three separate extractions of 25 individuals were performed at each stage, such that they acted as biological as well as technical replicates. Arrays were scanned and analyzed as indicated in the descriptions of each array with the intent of profiling how gene expression changes over development within species as well as comparing how expression levels evolve during development between species. The hybrids were used in order to analyze how mechanisms of gene regulation diverge between species as manifested as misexpression of genes in the hybrids relative to their parents.