Enhancer diversity and the control of a simple pattern of Drosophila CNS midline cell expression

Abstract

Transcriptional enhancers integrate information derived from transcription factor binding to control gene expression. One key question concerns the extent of trans- and cis-regulatory variation in how co-expressed genes are controlled. The Drosophila CNS midline cells constitute a group of neurons and glia in which expression changes can be readily characterized during specification and differentiation. Using a transgenic approach, we compare the cis-regulation of multiple genes expressed in the Drosophila CNS midline primordium cells, and show that while the expression patterns may appear alike, the target genes are not equivalent in how these common expression patterns are achieved. Some genes utilize a single enhancer that promotes expression in all midline cells, while others utilize multiple enhancers with distinct spatial, temporal, and quantitative contributions. Two regulators, Single-minded and Notch, play key roles in controlling early midline gene expression. While Single-minded is expected to control expression of most, if not all, midline primordium-expressed genes, the role of Notch in directly controlling midline transcription is unknown. Midline primordium expression of the rhomboid gene is dependent on cell signaling by the Notch signaling pathway. Mutational analysis of a rhomboid enhancer reveals at least 5 distinct types of functional cis-control elements, including a binding site for the Notch effector, Suppressor of Hairless. The results suggest a model in which Notch/Suppressor of Hairless levels are insufficient to activate rhomboid expression by itself, but does so in conjunction with additional factors, some of which, including Single-minded, provide midline specificity to Notch activation. Similarly, a midline glial enhancer from the argos gene, which is dependent on EGF/Spitz signaling, is directly regulated by contributions from both Pointed, the EGF transcriptional effector, and Single-minded. In contrast, midline primordium expression of other genes shows a strong dependence on Single-minded and varying combinations of additional transcription factors. Thus, Single-minded directly regulates midline primordium-expressed genes, but in some cases plays a primary role in directing target gene midline expression, and in others provides midline specificity to cell signaling inputs.

Redeployment of a conserved gene regulatory network during Aedes aegypti development

“…We refer to these three stages of expression as “mesectoderm,” “midline primordium,” and “late midline,” respectively. Although the mechanisms responsible for the late midline stage of sim expression have not been definitively established, it has been proposed to be dependent on Notch signaling and possibly autoregulation (Pearson and Crews, 2014). …”

“…Toward the late primordium stage and into the late midline stage, Sim’s role is more variable: while CRMs for some genes are strongly affected by loss of Sim binding, others show relatively minor effects (Pearson and Crews, 2014). This suggests that while Sim remains a necessary component of midline gene expression, at the later stages additional transcription factors (e.g., Su(H)) may also be playing important roles.…”

“…In order to test whether a change in the Sim expression pattern alone could account for the lateral relocation observed in the A. aegypti CNS, we expressed Sim in stripes in lateral CNS regions using prd-Gal4/UAS-sim (Pearson and Crews, 2014). Previous studies have shown that a number of Sim target genes, including rho, sog, and slit, as well as reporter gene expression driven by the rho-365 CRM, are induced in response to ectopic Sim CNS expression at the midline primordium stage (Nambu et al, 1991; Pearson and Crews, 2014; Zinzen et al, 2006).…”

“…sim expression persists in all midline cells until germband retraction, after which it is maintained in most although not all of the lineage (see also Results). Sim binding sites have been identified in almost all known midline CRMs, and Sim binding contributes to midline gene expression in all cases tested to date (Pearson and Crews, 2014). Although the overall structure of the GRN regulating midline development remains to be elucidated, sim appears to be a “master regulatory gene” that sits at the head of this GRN and is directly or indirectly responsible for many of the subsequent developmental events.…”

Chromatin profiling of Drosophila CNS subpopulations identifies active transcriptional enhancers

“…The Gal4 lines simC2.3, simD2.1 and simE2.3 were described previously (Freer et al, 2011). FlyLight Gal4 enhancer lines were obtained from the Bloomington Drosophila Stock Center, and embryos were subjected to fluorescent ISH with a Gal4 probe or crossed to UAS-mCD8::GFP and immunostained with anti-GFP as previously described (Pearson and Crews, 2014;Wheeler et al, 2006).…”