Genetic Changes to a Transcriptional Silencer Element Confers Phenotypic Diversity within and between Drosophila Species

Abstract

The modification of transcriptional regulation has become increasingly appreciated as a major contributor to morphological evolution. However, the role of negative-acting control elements (e.g. silencers) in generating morphological diversity has been generally overlooked relative to positive-acting “enhancer” elements. The highly variable body coloration patterns among Drosophilid insects represents a powerful model system in which the molecular alterations that underlie phenotypic diversity can be defined. In a survey of pigment phenotypes among geographically disparate Japanese populations of Drosophila auraria, we discovered a remarkable degree of variation in male-specific abdominal coloration. In testing the expression patterns of the major pigment-producing enzymes, we found that phenotypes uniquely correlated with differences in the expression of ebony, a gene required for yellow-colored cuticle. Assays of ebony’s transcriptional control region indicated that a lightly pigmented strain harbored cis-regulatory mutations that caused correlated changes in its expression. Through a series of chimeric reporter constructs between light and dark strain alleles, we localized function-altering mutations to a conserved silencer that mediates a male-specific pattern of ebony repression. This suggests that the light allele was derived through the loss of this silencer’s activity. Furthermore, examination of the ebony gene of D. serrata, a close relative of D. auraria which secondarily lost male-specific pigmentation revealed the parallel loss of this silencer element. These results demonstrate how loss-of-function mutations in a silencer element resulted in increased gene expression. We propose that the mutational inactivation of silencer elements may represent a favored path to evolve gene expression, impacting morphological traits.

The Genetic Basis of Pigmentation Differences Within and Between Drosophila Species

“…Genetic differences in ebony cis -regulatory sequences also appear to contribute to variable abdominal pigmentation in other populations of D. melanogaster and other species (Bastide et al 2013; Johnson et al 2015; Dembeck et al 2015b; Endler et al 2016). For example, an association study using the Drosophila Genetic Reference Panel (DGRP) of D. melanogaster strains isolated from a population in Raleigh, North Carolina (Mackay et al 2012) found a significant correlation between a noncoding variant located within a known cis- regulatory element of ebony and pigmentation variation within this population (Dembeck et al 2015b).…”

“…Weak associations with noncoding SNPs in ebony cis -regulatory elements were also observed for European populations of D. melanogaster (Bastide et al 2013; Dembeck et al 2015a; Endler et al 2016), with the most highly ranked SNP associated with ebony in Bastide et al (2013) located in a sequence that inhibits ebony expression in male abdominal segments during development. Outside of D. melanogaster , genetic variation linked to ebony has been shown to be associated with polymorphic abdominal pigmentation within Drosophila americana (Wittkopp et al 2009) and Drosophila auraria (Johnson et al 2015). In this latter species, specific alleles of ebony cis -regulatory sequences were identified in light and dark individuals, and transgenic analyses of reporter genes were used to demonstrate the effects of these variable sites on ebony expression (Johnson et al 2015).…”

“…Outside of D. melanogaster , genetic variation linked to ebony has been shown to be associated with polymorphic abdominal pigmentation within Drosophila americana (Wittkopp et al 2009) and Drosophila auraria (Johnson et al 2015). In this latter species, specific alleles of ebony cis -regulatory sequences were identified in light and dark individuals, and transgenic analyses of reporter genes were used to demonstrate the effects of these variable sites on ebony expression (Johnson et al 2015). …”

“…Variation at ebony also appears to be important for abdominal pigmentation differences between the montium subgroup species D. auraria and D. serrata in the melanogaster group (Johnson et al 2015), which last shared a common ancestor approximately as long ago as D. melanogaster and D. simulans (Nikolaidis and Scouras 1996), or ~1.5 million years ago (Cutter 2008) (Figure 3). In D. auraria , males have a stripe of pigment in each abdominal segment similar to D. melanogaster , but the more complete pigmentation of male abdominal segments is seen only on A6 rather than in A5 and A6 (Johnson et al 2015).…”

Hox genes, evo-devo, and the case of the ftz gene

“…For example, expression of ftz in Artemia was homeotic-like but so weak as to be almost undetectable (Heffer et al 2010). Alternatively, acquisition of silencers may have led to loss of expression, as seen recently for color patterning genes (Johnson et al 2015). We previously proposed that it was loss- or low-level expression of ftz in embryos that was permissive for gain-of-function changes in its protein function.…”