Shared and distinct mechanisms of atonal regulation in Drosophila ocelli and compound eyes

Abstract

The fruit fly Drosophila melanogaster has two types of external visual organs, a pair of compound eyes and a group of three ocelli. At the time of neurogenesis, the proneural transcription factor Atonal mediates the transition from progenitor cells to differentiating photoreceptor neurons in both organs. In the developing compound eye, atonal (ato) expression is directly induced by transcriptional regulators that confer retinal identity, the Retinal Determination (RD) factors. Little is known, however, about control of ato transcription in the ocelli. Here we show that a 2kb genomic DNA fragment contains distinct and common regulatory elements necessary for ato induction in compound eyes and ocelli. The three binding sites that mediate direct regulation by the RD factors Sine oculis and Eyeless in the compound eye are also required in the ocelli. However, in the latter, these sites mediate control by Sine oculis and the other Pax6 factor of Drosophila, Twin of eyeless, which can bind the Pax6 sites in vitro. Moreover, the three sites are differentially utilized in the ocelli: all three are similarly essential for atonal induction in the posterior ocelli, but show considerable redundancy in the anterior ocellus. Strikingly, this difference parallels the distinct control of ato transcription in the posterior and anterior progenitors of the developing compound eyes. From a comparative perspective, our findings suggest that the ocelli of arthropods may have originated through spatial partitioning from the dorsal edge of an ancestral compound eye.

Keywords: Eye evolution; RDN; Retina development; Transcriptional regulation; ath5; atoh7.

Figure 1. Ato in the development of eyes and ocelli

Where needed, orientation is shown by coordinates (A=anterior, P=posterior, D=dorsal, V=ventral). (A) Micrograph of dorsal fly head showing the ocellar complex. (B) Location of developing ocellar primordia (OCP) in the late L3 eye disc. Schematic of the larval eye-antennal imaginal disc with eye and ocellar fields shaded in gray. (C) Evolutionary relationship of eyes and ocelli as derived from a precursor organ devoted to light sensation in a common ancestor of arthropods (Mya= million years ago). (D) Diagram of ato expression pattern during ocellar and retinal development. In both, founder neuronal precursors are selected from the larger pool of Ato-positive proneural cells. Ato expression in the proneural domain is Ato-independent, but primary precursors maintain Ato-expression through feedback regulation. Neurogenesis in surrounding cells is induced through an ato-independent process in the eye. As suggested by the lineage tracing experiment (Fig. S1), in the ocelli, ato-dependent precursors may recruit additional photoreceptor neurons in Ato-independent fashion, similarly to the eye. However, an Ato-dependent recruitment step of the type described for the femoral chordotonal organ cannot be entirely excluded (zur Lage et al., 2004). An accurate understanding of ocellar development awaits a detailed analysis of the neurogenetic process in this organ. (E) Summary diagram of the ato gene and its regulatory regions based on published reports. The single exon of the gene is shown as a thick black rectangle marked ‘ato’ in white, black arrow above shows direction of transcription. The 1.1 kb genomic segment, shown as a thick line, includes the promoter region and extends into the exon to include the short ato 5’UTR. This segment does not drive expression in eyes or ocelli but was used as ‘endogenous promoter’ fragment in all constructs analyzed. Fragments implicated in the regulation of ato expression in eyes and ocelli are shown, numbers to the right refer to relevant references (1=Sun et al. 1998; 2=Zhang et al., 2006; 3=Tanaka-Matakatsu and Du., 2008; 4=Zhou et al., 2014). (F-F’) Confocal image of a late L3 eye disc to highlight relative position of the large eye field and the ocellar primordial (F). Enlarged view of ocellar region is shown in F’. Disc is stained for Eya (blue), which marks both the eye field and the ocellar primordia, and for Sens (red), a direct target of Ato in the eye that is also expressed in the ocellar region very late in L3. Here and in the following figures, the ocellar primordia are marked by dashed lines, orange for the OCP^P and yellow for the OCP^A.

Figure 2. The 3’ENH^I and 3’ENH^II retinal enhancers are required but not sufficient for expression in the ocelli

All constructs compared carry the β-Galactodidase (cytoplasmic) reporter. (A-A’) Diagram of constructs analyzed with summary results for relative expression level. Standard deviation is shown. Student’s t-test p-values in relation to M”-lacZ were p≤0.01 for M’-D183, M”-D300 and EI+EII, and <0.05 for 1.2. (B–F’) Confocal images of representative samples with immunostaining for Eya (blue) to identify the ocellar primordia, Sens (red) to confirm late L3 stage, and β-Galactosidase (green) to assess reporter constructs expression. In all panels, orientation is as in Fig.1F-F’ and posterior and anterior ocellar primordia are marked by orange and yellow dashed lines, respectively.

Figure 3. Pax6 and So sites are required and differentially utilized in posterior versus anterior ocelli

All constructs compared carry the eGFP (nuclear) reporter. (A-A’) Symbolic representation of M”-reporter constructs (wt and altered) with summary results of relative expression levels. Filled shapes indicate normal binding sites, unfilled shapes indicate deleted binding sites. Letters on the left correspond to following panels B-I’. Standard deviation is shown. Student’s t-test p-values in relation to M”-GFP were p≤0.01 for all modified reporters except for M”-ΔPax6^EII and M”-ΔSo^EII in the OC^A, which showed no change; p-value for M”-ΔPax6^EIPax6^EII or M”-ΔPax6^EISo^EII compared to M”-ΔPax6^EI is p≤0.01. (B–I’) Confocal images of representative samples with immunostaining for Eya (blue) and Sens (red), as explained in legend of Fig. 2, and GFP (green) to assess reporter constructs expression. In all panels, orientation is as in Fig.1F-F’ and posterior and anterior ocellar primordia are marked by orange and yellow dashed lines, respectively. (J) EMSA for Toy protein binding at the Pax6^EI site. From left to right: a shift of the 120 bp fragment spanning part of 3’ENH^I is observed when Toy protein is present (compare lane 1 to lane 2). The shift is suppressed by cold DNA competitor with the Pax6^EI binding site (wt), but not by DNA competitor with a mutated Pax6^EI sequence (mut). Arrow marks position of Toy-bound DNA probe.

Figure 4. Summary of ato regulation and proposed evolution of eye and ocelli from a compound-eye-like ancestral organ

(A) Diagram of regulatory region for the onset of ato expression in eyes and ocelli. Position relative to transcription unit is shown above and contribution of DNA segments and binding sites to reporter gene expression is summarized below. Orientation of arrows reflects predicted effect on gene expression based on report constructs analyses, up- or down-regulation. The three binding sites function cooperatively in posterior retinal progenitors and OCP^P, but largely additively in anterior retinal progenitors and OCP^A. (B) Evolution of the compound eye and the ocelli from an already complex ancestral visual organ, a compound eye with genetically distinguishable posterior and anterior ommatidia. (C) Proposed evolution of ocelli by dorsal partitioning from of an ancestral compound eye, with posterior (green) and anterior (magenta) ommatidia.