An arthropod cis-regulatory element functioning in sensory organ precursor development dates back to the Cambrian

Abstract

Background: An increasing number of publications demonstrate conservation of function of cis-regulatory elements without sequence similarity. In invertebrates such functional conservation has only been shown for closely related species. Here we demonstrate the existence of an ancient arthropod regulatory element that functions during the selection of neural precursors. The activity of genes of the achaete-scute (ac-sc) family endows cells with neural potential. An essential, conserved characteristic of proneural genes is their ability to restrict their own activity to single or a small number of progenitor cells from their initially broad domains of expression. This is achieved through a process called lateral inhibition. A regulatory element, the sensory organ precursor enhancer (SOPE), is required for this process. First identified in Drosophila, the SOPE contains discrete binding sites for four regulatory factors. The SOPE of the Drosophila asense gene is situated in the 5' UTR.

Results: Through a manual comparison of consensus binding site sequences we have been able to identify a SOPE in UTR sequences of asense-like genes in species belonging to all four arthropod groups (Crustacea, Myriapoda, Chelicerata and Insecta). The SOPEs of the spider Cupiennius salei and the insect Tribolium castaneum are shown to be functional in transgenic Drosophila. This would place the origin of this regulatory sequence as far back as the last common ancestor of the Arthropoda, that is, in the Cambrian, 550 million years ago.

Conclusions: The SOPE is not detectable by inter-specific sequence comparison, raising the possibility that other ancient regulatory modules in invertebrates might have escaped detection.

Figure 1

Conserved domains of the arthropod ASH and Ase proteins. Alignment of the bHLH domain, Ase motif and carboxy-terminal motif of ASH and Ase. Am, Apis mellifera; Cs, Cupiennius salei; Dm, Drosophila melanogasster; Dp, Daphnia pulex; Gm, Glomeris marginata; Sm, Strigamia maritima; Tc, Tribolium castaneum.

Figure 2

Phylogenetic analysis of the arthropod ASH and Ase-like proteins. ASH and Ase of insects group together, as do those of crustaceans, while the ASH proteins of myriapods and of chelicerates are arranged in a single group. Both the insect and the crustacean proteins are clearly subdivided into ASH and Ase groups. The spider proteins CsASH1 and CsASH2 are arranged in a group with the single myriapod orthologues. Cs, Cupiennius salei; Dm, Drosophila melanogaster; Dp, Daphnia pulex; Gm, Glomeris marginata; Sm, Strigamia maritima; Tc, Tribolium castaneum.

Figure 3

C. salei CsASH2 and T. castaneum ase can both rescue the bristle phenotype in D. melanogaster ase¹ mutants. (A) Bar chart showing the extent to which Dm-ase, Tc-ase and CsASH2 can rescue the phenotype when ectopically expressed (hsp70Gal4 > UAS Dm-sc/Dm-ase/Tc-ase/CsASH2). In contrast, Dm-sc enhances the ase¹ phenotype (hsp70Gal4 > UAS Dm-sc). The number of bristles affected is given on the x-axis. Error bars indicate the standard error of the mean (see Additional file 1 for details). (B-G) Abnormally differentiated bristles (asterisks) on the anterior wing margin are shown for the genotypes indicated (genotypes as in (A)). The number of abnormal bristles is enhanced in (D) and reduced in (F, E, G). (H-K) Detail of the abnormalities at higher magnification.

Figure 4

Arrangement of transcription factor binding sites in the UTR of the arthropod ase-like genes. Note that the SOPE covers a larger area in D. pulex (882 bp) and in S. maritima (1,052 bp) compared to D. melanogaster (297 bp), T. castaneum (247 bp) and C. salei (246 bp). See text for details. Yellow, E box; green, β box; pink, α box; blue, N box. Dm, D. melanogaster; Tc, T. castaneum; Dp, D. pulex; Cs, C. salei; Sm, S. maritima.

Figure 5

Comparison of the number of ectopic bristles (macrochaetes) displayed by transgenic flies after ectopic expression. UAS constructs containing D. melanogaster ase, T. castenum ase and C. salei CsASH2 (ORF alone or the entire transcribed region (ORF+SOPE) of ase or CsASH2) were each crossed to four different Gal4 drivers allowing expression in different parts of the thorax (see Materials and methods). (A) The number of ectopic bristles was counted in the respective Gal4 expression domains of each of the driver lines. Columns give the number of ectopic bristles and data from all four crosses have been pooled for each UAS construct (UAS-ORF+SOPE or UAS-ORF). The number of ectopic bristles is significantly reduced in flies carrying the UAS-ORF+SOPE constructs (see Additional file 4 for details). The error bars give the standard error of the mean. (B-G) Thoraces illustrating the phenotypes obtained: (B, C) ptc-Gal4 > UAS Dm-ase; (D, E) sca-Gal4 > UAS Tc-ase; (F, G) MD806-Gal4 > UAS CsASH2.