RNA in situ hybridization (ISH) is used to visualize spatio-temporal gene expression patterns with broad applications in biology and biomedicine. Here we provide a protocol for mRNA ISH in developing pupal wings and abdomens for model and non-model Drosophila species. We describe best practices in pupal staging, tissue preparation, probe design and synthesis, imaging of gene expression patterns, and image-editing techniques. This protocol has been successfully used to investigate the roles of genes underlying the evolution of novel color patterns in non-model Drosophila species.

The complex color patterns on the wings and body of Drosophila guttifera (D. guttifera) are emerging as model systems for studying evolutionary and developmental processes. Studies regarding these processes depend on overexpression and downregulation of developmental genes, which ultimately rely upon an effective transgenic system. Methods describing transgenesis in Drosophila melanogaster (D. melanogaster) have been reported in several studies, but they cannot be applied to D. guttifera due to the low egg production rate and the delicacy of the eggs. In this protocol, we describe extensively a comprehensive method used for generating transgenic D. guttifera. Using the protocol described here, we are able to establish transgenic lines, identifiable by the expression of enhanced green fluorescent protein (EGFP) in the eye disks of D. guttifera larvae. The entire procedure, from injection to screening for transgenic larvae, can be completed in approximately 30 days and should be relatively easy to adapt to other non-model Drosophila species, for which no white-eyed mutants exist.

Changes in cis -regulatory modules (CRMs) that control developmental gene expression patterns have been implicated in the evolution of animal morphology ^1-6 . However, the genetic mechanisms underlying complex morphological traits remain largely unknown. Here we investigated the molecular mechanisms that induce the pigmentation gene yellow ( y ) in a complex spot and shade pattern on the abdomen of the quinaria group species Drosophila guttifera . We show that the y expression pattern is controlled by only one CRM, which contains a stripe-inducing CRM at its core. We identified several developmental genes that may collectively interact with the CRM to orchestrate the patterning in the pupal abdomen of D. guttifera . We further show that the core CRM is conserved among D. guttifera and the closely related quinaria group species Drosophila deflecta , which displays a similarly spotted abdominal pigment pattern. Our data suggest that besides direct activation of patterns in distinct spots, abdominal spot patterns in Drosophila species may have evolved through partial repression of an ancestral stripe pattern, leaving isolated spots behind. Abdominal pigment patterns of extant quinaria group species support the partial repression hypothesis and further emphasize the modularity of the D. guttifera pattern.

To understand how novel animal patterning emerged, one needs to ask how the development of color patterns has changed among diverging species. Here we examine three species of fruit flies – Drosophila guttifera ( D. guttifera ), Drosophila palustris ( D. palustris ), and Drosophila subpalustris ( D. subpalustris ) – displaying a varying number of abdominal spot rows that were either gained or lost throughout evolutionary time. Through in situ hybridization, we examine the mRNA expression patterns for the pigmentation genes Dopa decarboxylase ( Ddc ), tan ( t ), and yellow ( y ) during pupal development. Our results show that Ddc , t , and y are co-expressed in identical patterns, each foreshadowing the adult abdominal spots in D. guttifera , D. palustris , and D. subpalustris .