Ariane Ramaekers
email: ariane.ramaekers@curie.frbassem.hassan@icm-institute.org

Annelies Claeys

Martin Kapun

Emmanuele Mouchel-Vielh

Delphine Potier

Simon Weinberger

Nicola Grillenzoni

Delphine Dardalhon-Cumenal

Jiekun Yan

Reinhard Wolf

Emmanuèle Mouchel-Vielh

Delphine Dardalhon-Cuménal

Jiekun Jan

Thomas Flatt

Erich Buchner

Bassem A Hassan

Altering the Temporal Regulation of One Transcription Factor Drives Evolutionary Trade-Offs between Head Sensory Organs

Keywords: Sensory trade-offs, Eye size, sensory development, temporal regulation, non-coding SNP, Eyeless/Pax6, Cut, Transcription factor, Drosophila, Evolution

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INTRODUCTION

The senses animals rely on have been shaped during evolution to better navigate and exploit the environment. As a result, even closely related species living in different ecological niches show variation in the sizes and shapes of their sensory structures. Adaptive variation in visual sensory organs is a fascinating case in point and ranges from almost complete loss of the eyes in darkness-adapted animals [START_REF] Partha | Subterranean mammals show convergent regression in ocular genes and enhancers, along with adaptation to tunneling[END_REF][START_REF] Retaux | Evolution of eye development in the darkness of caves: adaptation, drift, or both?[END_REF] to the expansion of visual organs and processing areas in some other groups such as tree-dwelling mammals [START_REF] Campi | Comparison of area 17 cellular composition in laboratory and wild-caught rats including diurnal and nocturnal species[END_REF][START_REF] Campi | Comparative studies of diurnal and nocturnal rodents: differences in lifestyle result in alterations in cortical field size and number[END_REF] and predator insects [START_REF] Elzinga | Fundamentals of entomology[END_REF].

A striking, yet poorly understood feature of natural variation in eye size is that it often occurs as a trade-off between the visual organs and other head sensory structures such as olfactory organs.

This was described in a large variety of animal groups including mammals [START_REF] Nummela | Exploring the mammalian sensory space: co-operations and trade-offs among senses[END_REF], and fishes [START_REF] Retaux | Evolution of eye development in the darkness of caves: adaptation, drift, or both?[END_REF]. In arthropods as well, trade-offs between the size of the eyes and of the antennae, where most olfactory organs are located, are pervasive.

Examples include beetle species with different life-styles (nocturnal vs diurnal; visual hunters or not; [START_REF] Bauer | Morphology of the compound eyes as an indicator of life-style in carabid beetles[END_REF]; fireflies emitting or not emitting light signals [START_REF] Stanger-Hall | The evolution of sexual signal modes and associated sensor morphology in fireflies (Lampyridae, Coleoptera)[END_REF]; surface and cave crustaceans [START_REF] Protas | Evolution and development in cave animals: from fish to crustaceans[END_REF] and millipedes [START_REF] Liu | Convergent Evolution of Unique Morphological Adaptations to a Subterranean Environment in Cave Millipedes (Diplopoda)[END_REF]. This is also the case between and within species of fruit flies, in which eye size often anticorrelates with the size of the face and/or of the antennae [START_REF] Arif | Genetic and developmental analysis of differences in eye and face morphology between Drosophila simulans and Drosophila mauritiana[END_REF][START_REF] Gaspar | Characterisation of the genetic architecture underlying eye size variation within Drosophila melanogaster and Drosophila simulans[END_REF][START_REF] Keesey | Inverse resource allocation between vision and olfaction across the genus Drosophila[END_REF][START_REF] Norry | Quantitative Trait Loci and Antagonistic Associations for Two Developmentally Related Traits in the Drosophila Head[END_REF][START_REF] Posnien | Evolution of eye morphology and rhodopsin expression in the Drosophila melanogaster species subgroup[END_REF]. However, the developmental mechanisms that govern such trade-offs are essentially unknown.

A commonly observed property of sensory organ formation is the shared developmental origin of most head sensory structuressuch as eyes and nosesthat derive from the subdivision of a single multipotent primordium. In vertebrates, the olfactory and lens placodes derive from the subdivision of the anterior aspect of a multipotent preplacodal ectoderm [START_REF] Grocott | The peripheral sensory nervous system in the vertebrate head: a gene regulatory perspective[END_REF][START_REF] Singh | The molecular basis of craniofacial placode development[END_REF]. Similarly, during Drosophila development, the ectodermal eye-antennal imaginal disc (EAD) gives rise to all external sensory, including the visual (compound eyes and ocelli) and olfactory (antennae and maxillary palps) sense organs, and nonsensory head cuticle. In vertebrates and in flies, antagonistic relationships between gene regulatory networks (GRNs) and signaling pathways that promote different sensory identities regulate the subdivision of the multipotent primordium [START_REF] Grocott | The peripheral sensory nervous system in the vertebrate head: a gene regulatory perspective[END_REF][START_REF] Singh | The molecular basis of craniofacial placode development[END_REF][START_REF] Wang | Segregation of eye and antenna fates maintained by mutual antagonism in Drosophila[END_REF][START_REF] Weasner | Competition among gene regulatory networks imposes order within the eye-antennal disc of Drosophila[END_REF]. First active in the entire tissue, their expression segregates as the visual and non-visual territories become distinct [START_REF] Bhattacharyya | Segregation of lens and olfactory precursors from a common territory: cell sorting and reciprocity of Dlx5 and Pax6 expression[END_REF] , [START_REF] Kenyon | Coordinating proliferation and tissue specification to promote regional identity in the Drosophila head[END_REF]. In addition to promoting eye fate, the transcription factor (TF) Pax6 and its two Drosophila orthologues Eyeless (Ey) and Twin-of-Eyeless (Toy), play a key role in the growth and the subdivision of the multipotent primordium [START_REF] Zhu | Drosophila Pax6 promotes development of the entire eye-antennal disc, thereby ensuring proper adult head formation[END_REF]. In Drosophila, at early developmental stages, Ey and Toy are co-expressed in the entire EAD with antennal TFs, such as Homothorax (Hth). The expression of these eye and antenna-promoting TFs progressively segregates along the EAD"s anterior-posterior axis, delineating the posterior eye and anterior antennal compartments. Eye and antennal TFs mutually repress each other: the antennal TFs Hth and Cut (Ct) directly repress ey expression while Sine oculis, another eye promoting TF, represses ct [START_REF] Anderson | Dual transcriptional activities of SIX proteins define their roles in normal and ectopic eye development[END_REF][START_REF] Wang | Segregation of eye and antenna fates maintained by mutual antagonism in Drosophila[END_REF][START_REF] Weasner | Competition among gene regulatory networks imposes order within the eye-antennal disc of Drosophila[END_REF]. Consequently, loss or gain of function of these selector TFs leads to the transformation of most of the head tissue into visual or olfactory organs at the expense of the other sensory structure (e.g. [START_REF] Anderson | Dual transcriptional activities of SIX proteins define their roles in normal and ectopic eye development[END_REF][START_REF] Czerny | twin of eyeless, a second Pax-6 gene of Drosophila, acts upstream of eyeless in the control of eye development[END_REF][START_REF] Halder | Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila[END_REF]).

Therefore, the subdivision of a single multipotent primordium into distinct territories through mutual repression by antagonistic TFs is a shared step of the development of head sensory organs across animals. It is thus tempting to speculate that evolutionary mechanisms have exploited this process leading to natural sensory size trade-offs between visual and olfactory organs. A hint in that direction comes from studies on Astyanax fishes, which live as cave or surface-dwelling morphs [START_REF] Retaux | Evolution of eye development in the darkness of caves: adaptation, drift, or both?[END_REF]. Cave morphs have small lenses and large olfactory placodes, while surface-dwellers show the reciprocal ratio. Chemical manipulation of signaling pathways that regulate the subdivision of the lens versus olfactory territories mimics the differences observed between natural morphs [START_REF] Hinaux | Sensory evolution in blind cavefish is driven by early embryonic events during gastrulation and neurulation[END_REF].

Whether this is a mechanism of natural variation in sensory trade-offs is unknown.

Demonstrating a direct link requires the identification of naturally occurring causal genetic variants and the elucidation of their effect on the GRNs that regulate visual and olfactory sensory organ development. The paucity of model systems amenable to combining comparative, genetic, molecular and developmental analyses has thus far hindered such an endeavor.

We reasoned that natural variation in eye size between and within Drosophila species may offer precisely such a model. We therefore used comparative analyses combined with developmental, molecular and genome editing approaches to tackle this question. We show that differential subdivision of the EAD, resulting in different proportions of eye and antennal compartments, underlies eye size variation between and within Drosophila species. In both cases, this is associated with changes in the temporal regulation of the expression of ey during EAD subdivision. We also demonstrate that in D. melanogaster (D. mel.), this is caused by a non-coding single polymorphic nucleotide (SNP) present in most natural populations of D. mel. This SNP is located in a binding site for the antennal factor Ct within the eye enhancer of ey.

Using CRISPR/Cas 9 genome editing, we show that this SNP is causal to temporal changes in ey expression and to facet number variation. Thus, changes in the subdivision of a multipotent primordium, caused by subtle alterations of the mutual repression between distinct fates, underlies natural variation in sensory trade-offs.

RESULTS

Reciprocal changes in the sizes of visual and non-visual head structures

The insect compound eye is composed of a crystalline array of small units, named facets or ommatidia. In insects, and specifically in Drosophilids, eye size depends both on the number and diameter of the ommatidia and is often negatively correlated with face and/or antenna size [START_REF] Arif | Genetic and developmental analysis of differences in eye and face morphology between Drosophila simulans and Drosophila mauritiana[END_REF][START_REF] Norry | Quantitative Trait Loci and Antagonistic Associations for Two Developmentally Related Traits in the Drosophila Head[END_REF][START_REF] Posnien | Evolution of eye morphology and rhodopsin expression in the Drosophila melanogaster species subgroup[END_REF] [START_REF] Gaspar | Characterisation of the genetic architecture underlying eye size variation within Drosophila melanogaster and Drosophila simulans[END_REF][START_REF] Keesey | Inverse resource allocation between vision and olfaction across the genus Drosophila[END_REF]. In this study, we selected four Drosophila species, which presented a larger eye to face width ratio as compared to D. mel. (Figure 1A-1A"; Figure S1). All subsequent morphological measurements were performed on females raised in density-controlled conditions. We focused on Drosophila pseudoobscura (D. pse.), which had the largest difference in terms of ommatidia number, an increase of 35% as compared to D. mel. (Figures 1B and 1B") while sharing similar facet diameters (Figures 1C and 1C"). Interestingly, the third antennal segment, which hosts the olfactory sensillae, was thinner in D. pse. as compared to D. mel. (Figures 1D and1E). As a control, we measured tibia length as a proxy to body size [START_REF] Arif | Genetic and developmental analysis of differences in eye and face morphology between Drosophila simulans and Drosophila mauritiana[END_REF][START_REF] Posnien | Evolution of eye morphology and rhodopsin expression in the Drosophila melanogaster species subgroup[END_REF] (Figure S2). In line with previous studies e. g. [START_REF] Arif | Genetic and developmental analysis of differences in eye and face morphology between Drosophila simulans and Drosophila mauritiana[END_REF][START_REF] Gaspar | Characterisation of the genetic architecture underlying eye size variation within Drosophila melanogaster and Drosophila simulans[END_REF][START_REF] Keesey | Inverse resource allocation between vision and olfaction across the genus Drosophila[END_REF][START_REF] Posnien | Evolution of eye morphology and rhodopsin expression in the Drosophila melanogaster species subgroup[END_REF], our results suggest that variation in eye, face or antennal size cannot be explained solely by variation in body size.

Increased facet number has been associated with higher visual acuity in predator flies [START_REF] Elzinga | Fundamentals of entomology[END_REF][START_REF] Gonzalez-Bellido | Compound eyes and retinal information processing in miniature dipteran species match their specific ecological demands[END_REF]. We thus tested whether a modest variation such as the one observed between D. pse. and D. mel. was potentially relevant to visual function. We measured the minimal angular distance between two successive vertical black stripes resolved by the flies as a read-out of their visual acuity (Figure 1F) [START_REF] Buchner | Elementary Movement Detectors in an Insect Visual-System[END_REF][START_REF] Gotz | Optomoter studies of the visual system of several eye mutants of the fruit fly Drosophila[END_REF]. D. pse.

were able to distinguish between more closely juxtaposed stripes (minimal angle = 7.0°) as compared to D. mel. (minimal angle = 8.51°) (Figure 1F"). Thus, D. pse. have a better visual acuity correlating with an increased number of facets.

A trade-off between eye and non-eye progenitor fields

What is the developmental origin of facet number variation between D. mel. and D. pse. and why does it inversely correlate with the size of non-visual structures? All external structures of the head of the adult fly including the sensory organs develop from the EAD (Figure 2A). The eye field occupies most of the posterior EAD compartment and is marked by the expression of Eyes Absent (Eya; Figure 2B) [START_REF] Roignant | Pattern formation in the Drosophila eye disc[END_REF]. We measured the surface of the eye field in late, fully-grown, EADs (stage P0) using Eya and found that the eye field was 31% larger in D. pse. than in D. mel. (Figure 2B"). This is very close to the 35% difference in the number of adult eye facets. We therefore queried the developmental origin of the difference in eye field size between the two species and considered several possibilities. A first possibility is that the initial pool of embryonic cells forming the EAD differs between the two species. In the late embryo (stage 17), the EADs is composed of a few dozen closely juxtaposed cells located anterior to the brain. Using Ey as a marker, we quantified and compared the number of embryonic EAD cells between the two species (Figures 2D and2D") but found no significant difference in the number of EAD progenitors, ruling out this first possibility. Variation in eye field size could also originate from different rates of proliferation. However, the similar density of mitotic cells in the proliferating eye field in EADs of the two species did not support this hypothesis (Figures 2F and 2F"). In addition, the density of ommatidia progenitor cells in the eye field, characterized by the expression of the proneural factor Atonal, was similar between the two species (Figures S3A-S3A""). Finally, variation in eye field size could also derive from a change in the subdivision of the EAD between eye and non-eye fields. To test this possibility, we compared the proportion of the EAD occupied by the eye field in early L3 imaginal discs, after the subdivision between the fields is completed (Figure 2E). The total EAD size was similar between the two species, confirming that it underwent similar growth during prior larval development (Figure 2E"). In contrast, already at this early stage, the eye field was proportionally larger in D. pse. than in D. mel. (Figure 2E""). Thus, the two species differ by the proportion of the multipotent EAD dedicated to the eye versus non-eye tissues, resulting in different proportions of the head structures in the adult. Therefore, the species variation in eye size involves a developmental trade-off between eye and non-eye primordia.

Temporal regulation of EAD subdivision governs the trade-off between eye and non-eye fields

What are the regulatory mechanisms governing this developmental trade-off? EAD subdivision requires the temporally progressive restriction of selector TFs expression to the anterior "antennal" or posterior "eye" compartments, a process completed by mid to late second instar larval stage (L2) [START_REF] Kenyon | Coordinating proliferation and tissue specification to promote regional identity in the Drosophila head[END_REF]. At this developmental time point, the mutually exclusive expression domains of antenna and eye selectors define the relative sizes of the compartments. In D. mel., a 3.2 kb cis-regulatory intron governs ey expression during eye development (Figure 3A). We cloned the orthologous intron from D. pse. based on the conservation of the flanking exons. The D. pse. intronic sequence is slightly shorter (3.0 kb) with 22% of the intron from D. mel. aligning to the corresponding sequences in D. pse. (Figure 3B). Nonetheless, when inserted at the same position in D. mel. genome, both D. mel. and D. pse. introns were able to drive GFP expression in the EAD throughout eye development, revealing global functional conservation (not shown).

We tested whether, despite their overall functional conservation, subtle changes in ey regulation exist between D. mel. and D. pse. introns (Figures 3C-3D"). In early EADs (late embryos and in L1), both D. mel. and D. pse. enhancers drove GFP expression across the entire disc (Figure S4). At the L2 stage, we noted that GFP expression driven by the melanogaster enhancer (D.m.ey3.5) (Figures 3C and3C") extended further anteriorly into the antennal compartment as compared to the pseudoobscura enhancer (D.p. ey3.3) (Figures 3D and3D"). This means that the posterior retraction of expression driven by the two ey enhancers occurs at different velocities. To quantify this effect, we generated two lines of transgenic D. mel. flies.

The first line carries two transgenes driving the expression of the red fluorescent protein mCherry and of the green fluorescent protein (GFP), respectively, both under the control of D. mel. ey enhancer. In this control line, any difference in the expression of mCherry and GFP driven by the same enhancer must only be caused by different dynamics of the two fluorescent proteins. In the second line, mCherry was driven by the D. mel. ey enhancer, while GFP was driven by D. pse. ey enhancer. In this case, the differences in expression between the GFP and mCherry is caused both by different dynamics of the fluorescent proteins as well as differences in their transcriptional regulation. Thus, to detect differences in the activity of D. mel. and D.

pse. ey enhancers, we performed pairwise comparisons of the difference between GFP and mCherry expression in line 1 versus line 2. In early L3 discs, when the antennal and eye compartments have already segregated, the co-expression of mCherry and GFP driven by either D. mel. or D. pse. regulatory sequences were indistinguishable (Figures 3F and 3F"). In contrast, at L2, during the process of ey retraction, the posterior retraction of the GFP was more posteriorly advanced when driven by the D. pse. than by the D. mel. enhancer (Figures 3E and 3E"). Therefore, the partitioning of the EAD into eye and non-eye fields occurs at an earlier time point in D. pse. compared to D. mel. Since Ey positive cells proliferate more than Ey negative cells [START_REF] Zhu | Drosophila Pax6 promotes development of the entire eye-antennal disc, thereby ensuring proper adult head formation[END_REF], earlier establishment of the two sensory fields would drive greater differential growth.

A conserved mechanism of sensory trade-offs

To understand the genetic basis of sensory trade-off in Drosophila, we exploited the fact such trade-offs have also been observed within single fly species [START_REF] Arif | Genetic and developmental analysis of differences in eye and face morphology between Drosophila simulans and Drosophila mauritiana[END_REF][START_REF] Cowley | Development and Quantitative Genetics of Correlation Structure Among Body Parts of Drosophila melanogaster[END_REF][START_REF] Gaspar | Characterisation of the genetic architecture underlying eye size variation within Drosophila melanogaster and Drosophila simulans[END_REF][START_REF] Norry | Quantitative Trait Loci and Antagonistic Associations for Two Developmentally Related Traits in the Drosophila Head[END_REF][START_REF] Posnien | Evolution of eye morphology and rhodopsin expression in the Drosophila melanogaster species subgroup[END_REF]. We found that two wild-type D. mel. laboratory strains, Canton-S and Hikone-AS, show different eye-to-face ratios. This is associated with changes in ommatidia number (12, 5 % more facets in Canton-S) and diameter (Figures 4A and4B, Figures S1A -S1D, Table 1) as well as variation of antennal width (Figure 4C). F1 progeny of Canton-S and Hikone-AS parents presented intermediate ommatidia numbers relative to their parents, demonstrating the heritable nature of this trait (Figure 4D). We asked if facet number variation between Canton-S and Hikone-AS also originate from changes in the subdivision of the EAD into eye versus non-eye territories. We compared the subdivision of early L3 EADs between the two strains. While the size of the entire EAD was unchanged, the eye field was proportionally larger in Canton-S than in Hikone-AS (Figures 2E -2E""). These data suggest that, despite 17 -30 million years of separated evolution between the two species groups (Obbard et al., 2012), ommatidia number variation between D. mel. and D. pse. and between two D. mel. strains, shares a common developmental logic.

A single nucleotide in a Ct binding site distinguishes the Canton-S and Hikone-AS ey regulatory sequences

Does the difference in EAD subdivision between the Canton-S and Hikone-AS also result from a differential temporal regulation of ey? To answer this question, we cloned and aligned the ey cis-regulatory sequence from the Canton-S and Hikone-AS strains (Figure 4D). In contrast to the significant divergence observed between D. mel. and D. pse., Hikone-AS and Canton-S intron sequences were nearly identical and differed only by a single nucleotide over the entire 3.2 kb intronic region, a G>A substitution at position chr4: 710326. In silico analyses revealed that this single nucleotide variant (SNV) is located in a Ct binding site, distinct from the three sites previously described in the ey cis-regulatory sequence (Figure 4E) [START_REF] Wang | Segregation of eye and antenna fates maintained by mutual antagonism in Drosophila[END_REF].

Interestingly, the two variants are predicted to display different affinities for the repressor, in a manner that anti-correlates with the number of ommatidia: the A-allele presents a lower affinity score (4.56) and is associated with larger eyes (Canton-S) as compared to the G-allele (Hikone-AS; predicted affinity score 5.22) (Figure 4E" and Figure S4). We thus performed electrophoretic mobility shift assays using a tagged recombinant Ct protein (Figure 4F), and found that Ct has the ability to bind to this sequence. Next, we tested the affinities of the two sequences by performing a competition assay in which unlabeled A-probes or unlabeled G-probes competed with the labeled G-probe. While unlabeled G-probes effectively suppressed the shift, the unlabeled A-probes did not, providing biochemical evidence that the two variants have different affinities for Ct. Put together, these results suggest that the strength of ey repression by Ct, a selector TF for antennal fate and a repressor of ey expression, influence eye size [START_REF] Wang | Segregation of eye and antenna fates maintained by mutual antagonism in Drosophila[END_REF][START_REF] Weasner | Competition among gene regulatory networks imposes order within the eye-antennal disc of Drosophila[END_REF]. Consistent with this, RNAi knock-down of ct expression during EAD development was sufficient to increase facet number in the adult eye (Figure 4G, Figure S5). We note however that this did not consistently result in antagonistic trends in face and antennal width, possibly due to pleiotropic effects of ct loss-of-function on head development (Figure S5). These findings raise two questions: first, is the G to A substitution in the ey cis-regulatory sequence sufficient to cause temporal changes in its activity; and second, if so, might such changes be caused by alterations in the regulation of the ey enhancer by Ct? To tackle these two questions, we used the same strategy described above for comparing the D. pse. and D. mel. enhancers using GFP and mCherry reporters. We first compared the activities of the Canton-S (A-allele) and the Hikone-AS (G-allele) of the ey 3.5 cis-regulatory sequences. At early/mid L2, during EAD subdivision, mCherry and GFP co-expression differed between the alleles such that the A-carrying variant (Canton-S; larger eyes) showed further posterior retraction of GFP expression than the G-carrying variant (Hikone-AS; smaller eyes) (Figure 4H-4H"). In contrast, at early L3, after EAD subdivision is completed, the two alleles drove similar expression of GFP and mCherry (Figure 4I-4I").

Could this differential temporal retraction of the ey enhancer be caused by changes in ey repression by Ct? To test this, we created two new synthetic ey enhancers, based on in silico predictions (Figure 4E"). The first, which we call the NoCt variant, is predicted to abolish Ct binding to the site harboring the G/A SNP (predicted affinity score <3). The second, which we call ConsensusCt, creates a Ct consensus-binding motif at that position (predicted affinity score 6.62). Remarkably, the ConsensusCt variant behaved similarly to the G-allele, while the NoCt variant mimicked the A-allele in that it caused faster posterior retraction of ey enhancer activity (Figure 4H-I"). This further suggests that the Canton-S A-allele may constitute a lower affinity site for the Ct repressor as compared to the Hikone-AS G-allele.

Thus far, we showed that the changes in EAD subdivision between Hikone-AS and Canton-S and between D. mel. and D. pse. are both driven by differential temporal dynamics of the posterior retraction of ey expression. Within the D. mel. this is associated with a SNV in ey eye enhancer, which introduces subtle changes in ey regulation, by affecting its repression by the antennal selector TF Ct.

A common SNP in D. mel. natural populations is associated with facet number variation

Because Hikone-AS and Canton-S flies have been in artificial lab culture conditions for decades, we asked if either of these two alleles is found in natural fruit fly populations. By investigating allele frequency patterns in whole-genome data of worldwide population samples, we found that most natural populations from Europe, North America, Asia and Australia are polymorphic at this position. Thus, neither of the two alleles corresponds to a de novo mutation and variation at this position corresponds to a relatively frequent SNP (Figures 5A andA"; Table S2). Populations from Sub-Saharan Africa are mostly fixed for the G-allele suggesting that the A-allele is a derived variant that appeared after D. mel. left Africa and colonized the rest of world. In line with this hypothesis, we found statistical evidence (FET test, p=0.02) that the few African populations carrying the A-variant are more likely to be admixed with European genetic variation than the ones with the putatively ancestral G-allele (Table S3). Moreover, the frequency of the A-variant decreased from West to East in European populations (Figure 5A" and 5B). The slope of the longitudinal frequency cline of the ey SNP deviated significantly from that of 21,008 genome-wide SNPs in short introns that presumable evolved neutrally (Figure 5C), suggesting that the clinal pattern is not solely the result of neutral evolution or demography (see also Figure S7).

Causal effect of the SNP on eye size

We further noted that natural populations from North-East America, where the Canton-S strain originated, are highly polymorphic for the ey SNP (Figure 5A, Table S2). By comparing Canton-S flies from three laboratories, we discovered that, while our Canton-S lab isolate (henceforth Canton-S BH ) carries the A-allele, two other strains from two different laboratories in Paris, France (T. Préat) and Florida, USA (R. Davis) were homozygous for the G-allele, similar to Hikone-AS. This strongly suggests that the original Canton-S population was polymorphic and that the two alleles were eventually segregated during the separate maintenance of different laboratory stocks [START_REF] Colomb | Sub-strains of Drosophila Canton-S differ markedly in their locomotor behavior[END_REF]. This provided a unique opportunity to quantify the contribution of the G/A SNP to eye size in a relatively homogenous background. By comparing ommatidia numbers between the three stocks, we observed that Canton S BH flies have significantly more facets than its two siblings (Figure 6A, Table 1), a difference that anticorrelates with their face and antennal width (Figure S6). These data suggest that the A-variant may be sufficient to drive larger facet numbers, possibly at the expenses of other head structures.

To test this idea directly, we used CRISPR/Cas9 to introduce the A-allele in a G-homozygous stock. We recovered one transformant male carrying the A-allele and controlled that it bore no other mutation in the ey regulatory intron. Comparing ommatidia numbers between engineered G>A flies and the G-carrying control revealed an increase in eye size associated with the G>A substitution (Figure 6B, Table 1). It recapitulated up to 49 % of the difference between Hikone-AS and Canton-S BH and up to 86% of the variation observed between Canton-S BH and Canton-S RD and Canton-S TP , respectively. Three other A-homozygous lines deriving from the same transformant male were established to account for subtle differences in the genetic background of the flies. The three stocks followed the same trend, i.e. an increased facet number when compared to the G-homozygous controls, reaching statistical significance in two out of the three (Figure S6). This result suggests that the ey cis-regulatory SNP is causal to facet number variation. Next, we combined the two alleles with Df(4)J2, a large deficiency covering the entire ey locus. These flies recapitulated the phenotypes of the corresponding homozygous alleles, indicating that an undetected lesion outside of the ey locus did not cause the effect on eye size attributed to the A-allele (Figure 6C). In addition, in both cases, the antennae show a trend towards decreasing in size, but this trend did not reach statistical significance (Figure S6), likely due to the small size of the difference.

DISCUSSION

In 1987, Montgomery Slatkin proposed a mathematical model [START_REF] Slatkin | Quantitative Genetics of Heterochrony[END_REF], which he referred to as "unrealistically simple", predicting that mutations modifying the time at which "traits developing from the same tissue" begin to grow independently changes the relative size of the traits. Size trade-offs between head sensory organs represent precisely the types of traits referred to in Slatkin"s model. However, whether visual-olfactory sensory trade-offs follow a "Slatkin model" and if so, what the genetic basis of such a model are, remained unexplored.

In this study, we find that differential subdivision of the head primordium into eye and non-eye progenitor fields constitutes a developmental mechanism for creating different proportions of head structures in fruit flies, including trade-offs between the olfactory antennae and the eyes. We further demonstrate that this is associated with differential temporal regulation of the expression of the conserved eye selector transcription factor, Ey/Pax6. We propose a model (Figure 6D to 6F) whereby early in development, the homogenous expression of ey, which promotes its proliferation [START_REF] Zhu | Drosophila Pax6 promotes development of the entire eye-antennal disc, thereby ensuring proper adult head formation[END_REF], causes homogenous growth throughout the entire EAD. Later, the progressive retraction of ey expression from the anterior antennal compartment creates an asymmetry in growth rate. Modulating the velocity of ey retraction through mutations affecting the bistable switch between GRNs governing antennal vs eye identity, changes the relative time during which the anterior and posterior compartments grow at different rates resulting in their different proportions. This provides direct biological evidence for mathematical models linking heterochrony in development to changes in adult traits [START_REF] Cowley | Development and Quantitative Genetics of Correlation Structure Among Body Parts of Drosophila melanogaster[END_REF][START_REF] Riska | Some Models for Development, Growth, and Morphometric Correlation[END_REF][START_REF] Slatkin | Quantitative Genetics of Heterochrony[END_REF]. Our observation of similar changes in ey temporal regulation between and within species further suggests that the temporal mechanism we identify may represent a "preferred route" to relative variation in sensory organ size because it results in no dramatic "pleiotropic" effects associated with changes to growth, patterning or specification. We speculate that such variation of the temporal regulation of EAD partitioning could be caused by a variety of molecular changes acting either in cis or in trans on the GRNs governing eye and antennal compartment identities.

In this work we uncovered an example of such a molecular change to the regulation of these GRNs. Specifically, within D. mel., a naturally occurring SNP in the eye-enhancer of ey is sufficient to modulate the velocity of the posterior retraction of the enhancer activity and to vary facet numbers in the adult eye. The SNP, a G/A substitution, is located in a binding site for ey repressor Ct. In silico predictions and EMSA experiments indicate that the G/A substitution modifies the affinity of Ct for the binding site. This suggests that different levels of ey repression by Ct are responsible for changing the dynamics of ey regulation and ultimately for causing morphological variation, a lower affinity of the binding site for Ct resulting into faster enhancer retraction and larger eyes and vice versa. This view is further supported by our findings that (i) synthetic mutations predicted to support or reduce Ct binding mimic the effect of the SNP on the velocity of the enhancer retraction; and that (ii) knocking-down Ct expression increase compound eye size. Interestingly, the highly divergent ey enhancers from D. mel. and D. pse. display similar differences in regulation, i.e. a faster retraction of the enhancer of the "larger eye" species. However, what feature of the two enhancer sequences causes their different temporal regulation is not known. A recent study identified a single nucleotide insertion that influences photoreceptor specification and ultimately color preference in Drosophila by modulating the affinity for a TF named Klumpfuss [START_REF] Anderson | Natural variation in stochastic photoreceptor specification and color preference in Drosophila[END_REF]. Together with our work, this suggests a general role for variation in suboptimal TF binding sites in sensory evolution [START_REF] Crocker | The Soft Touch: Low-Affinity Transcription Factor Binding Sites in Development and Evolution[END_REF].

In vertebrates, antagonistic relationships between GRNs and signaling pathways that promote different sensory identities also regulate the subdivision of the multipotent sensory placode [START_REF] Grocott | The peripheral sensory nervous system in the vertebrate head: a gene regulatory perspective[END_REF][START_REF] Singh | The molecular basis of craniofacial placode development[END_REF]. The anterior placodal region, which gives rise to the lens and olfactory placodes, expresses pax6. In the absence of pax6, both lens and olfactory placodes fail to thicken and to develop properly [START_REF] Ashery-Padan | Pax6 activity in the lens primordium is required for lens formation and for correct placement of a single retina in the eye[END_REF][START_REF] Collinson | Different roles for Pax6 in the optic vesicle and facial epithelium mediate early morphogenesis of the murine eye[END_REF][START_REF] Quinn | Multiple functions for Pax6 in mouse eye and nasal development[END_REF]. Interestingly, the temporal regulation of the pax6 placode enhancer is altered by manipulating suboptimal binding sites for one of its activators [START_REF] Rowan | Precise temporal control of the eye regulatory gene Pax6 via enhancer-binding site affinity[END_REF]. In addition, the pax6 ectodermal enhancer shows evidence of accelerated evolution in subterranean mammalian species [START_REF] Partha | Subterranean mammals show convergent regression in ocular genes and enhancers, along with adaptation to tunneling[END_REF]. This is consistent with our model whereby naturally occurring mutations that alter pax6 regulation, either in cis or in trans, constitute a common genetic origin of the trade-offs between visual and olfactory organ size.

In "The Origin of Species", Charles Darwin referred to the evolution of the eye as a challenge to his theory [START_REF] Darwin | On the origin of species by means of natural selection; or, The preservation of favored races in the struggle for life[END_REF]. He also discussed the importance of correlation between body parts concluding that it was "most imperfectly understood". During the last decades, the common origin of animal eyes and their evolution over long evolutionary distances has been abundantly documented [START_REF] Gehring | The evolution of vision[END_REF]. However, the developmental mechanisms by which small-scale variation in eye size or shape can take place without disrupting its organization and function remain largely elusive [START_REF] Dyer | Developmental sources of conservation and variation in the evolution of the primate eye[END_REF]. We have demonstrated that a single nucleotide change in a core regulator of eye development is sufficient to generate reciprocal sensory organ size variation, potentially providing a quick route to behavioural changes, and perhaps adaptation. As predicted by Darwin, adaptive variation in head derived structures, including the eye, can be produced by the accumulation of modest morphological changes, which our data suggest may be caused by a small number of genetic variants affecting the temporal regulation of core regulatory networks. (E) Schematics of the D. mel. ey eye enhancer (ey 3.5 ) showing the localization of the G>A substitution (chr4: 710326) and of three published Ct binding-sites (C, blue lines) [START_REF] Wang | Segregation of eye and antenna fates maintained by mutual antagonism in Drosophila[END_REF].

(E") The dashed red rectangle delineates the low-affinity putative Ct binding site overlapping the position of the G>A substitution (chr4: 710326) [START_REF] Zhu | FlyFactorSurvey: a database of Drosophila transcription factor binding specificities determined using the bacterial one-hybrid system[END_REF]. For each natural or synthetic enhancer allele, the best score of the Ct binding site as predicted by Cluster-Buster [START_REF] Frith | Cluster-Buster: Finding dense clusters of motifs in DNA sequences[END_REF] is plotted. See also Figure S5 and Table S2.

(F) Electrophoretic Mobility Shift assay. The Cut-FLAG expressing nuclear extract elicits a bandshift when incubated with the G-Probe (black arrow). Excess unlabeled G-probe, but not Aprobe effectively competes with the binding, suggesting a higher affinity of Ct for the former.

Incubation with an anti-FLAG induces a supershift (empty arrowhead). Grey arrow: non-specific binding.

(G) Ommatidia number variation following RNAi-mediated knock-down of ct. S2 andS3 for details). The exact geographic location for each sample is indicated by a black dot. (E and F) Our model, inspired from Slatkin [START_REF] Slatkin | Quantitative Genetics of Heterochrony[END_REF], proposes that different temporal dynamics of the posterior retraction of ey, a promotor of EAD proliferation, by changing the relative duration of uniform versus heterogeneous growth, modifies the proportion between the antennal and eye compartments. This could be caused by genetic changes affecting the dynamics of the bistable switch between GRNe and GRNa, like in the case of the ey G>A substitution. S2).

a Flies reared at 21°C. G>A-1 (this study); ey3.5 G>A-2 (this study); ey3.5 G>A-3 (this study); ey3.5 G>A-4 (this study).

STAR Methods

LEAD CONTACT AND MATERIALS AVAILABILITY

EXPERIMENTAL MODEL AND SUBJECT DETAILS

SPECIES

Drosophila ananassae

Isofemale D. ananassae stocks were cultured on standard cornmeal diet food at 21°C.

Morphological measures on adult

Drosophila virilis

The isofemale D. virilis stock was cultured on standard cornmeal diet food at 21°C.

Morphological measures on adult flies were performed on females only. Stock origin:

Gikongoro, Rwanda (DSSC 15010-1051.118).

DEVELOPMENTAL STAGES

For selecting specific developmental stages, embryos were collected on grape fruit plates complemented with yeast paste changed every 2 hours. Freshly hatched L1 larvae were collected every two hours and transferred to corn meal food vials in a density-controlled fashion (20 larvae / vial). Staging was performed at 25°C. Correspondence of developmental stages between D.

mel. and D. pse. was determined based on developmental transitionslarval hatching, L2 to L3 molt, pupa formation -and morphological featuresembryo morphology, rows of differentiated photoreceptors in the EAD, size of the EAD.

DENSITY-CONTROLLED CULTURE CONDITIONS

Morphological measurements were performed on flies raised in density-controlled conditions:

batches of 20 young females and males (2-5 days old) were put together and cultured at 25°C.

They were transferred in fresh vials every 24 hrs. For each vial, the individuals eclosing during the first two days of eclosion only were used for measurements.

METHOD DETAILS

GENERATION OF REAGENTS

Constructs

Enhancer reporter constructs were generated using the Gateway Recombination Cloning pEntry-ey 3.5CSBH and pEntry-ey 3.5Hik :

forward:GGGGACAAGTTTGTACAAAAAAGCAGGCTGGACTAGGCGGTATTGCT and reverse:GGGGACCACTTTGTACAAGAAAGCTGGGTTTTGCTCACACATCCATTTG. The entry vectors with mutated forms of ey3.5 enhancer, pEntry-ey 3.5NoCt and pEntry-ey 3.5consensusCt were generated by modifying the pEntry-ey 3.5Hik using primers carrying the corresponding mutations. These primers were (mutated nucleotides are in capital letters):

pEntry-ey 3.5NoCt :

forward: caataaaatggttggCaGtttttcgaactttcg reverse: cgaaagttcgaaaaaCtGccaaccattttattg pEntry-ey 3.5consensusCt :

forward: taaaatggttTgaactttttcgaactttcg reverse: gaaaaagttcAaaccattttattgttttc Enhancer inserts were next transferred using Gateway recombination into mCherry-and GFPexpressing enhancer reporter vectors amenable to phiC31 integration -mediated transgenesis [START_REF] Aerts | Robust target gene discovery through transcriptome perturbations and genomewide enhancer predictions in Drosophila uncovers a regulatory basis for sensory specification[END_REF][START_REF] Quan | Post-translational Control of the Temporal Dynamics of Transcription Factor Activity Regulates Neurogenesis[END_REF].

pAWF-Cut: ct cDNA was kindly provided by I. Lohmann (U. Heidelberg). The full length cDNA was cloned, without its ATG, into a Gateway pEntry vector using the pEntr-D-TOPO kit (ThermoFischer Scientific K2400-20) following provider specifications. It was transferred using

Gateway recombination into the pAFW vector (DGRC#1111), resulting in the addition of 3 x Flag tag coding sequence upstream ct cDNA.

pU6gRNA ey : the following complementary phospho-oligomers were used to generate a double strand DNA sequence encoding the ey eye-enhancer guide RNA (gRNA): forward: phospho-CTTCGTCGAAAACAATAAAATGGT; reverse: phospho-AAACACCATTTTATTGTTTTCGAC. After hybridization, the resulting double-strand DNA was cloned into the pU6-BBS1-chiRNA plasmid (Addgene #45946) [START_REF] Gratz | Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease[END_REF].

Enhancer-reporter lines

Transformant flies carrying enhancer reporter constructs were generated by BestGeneInc. All constructs were integrated at the Attp2 landing site using phiC31 recombination.

CRISPR/Cas9 engineering

For editing the ey eye enhancer, we injected SNP G homozygous D. mel. Act5-Cas9 embryos [START_REF] Port | Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila[END_REF] with two constructs respectively encoding the guide RNA (pU6gRNA ey ) and the SNP A -carrying ey eye enhancer sequence (pEntry-ey 3.5CSBH ), each of them at a concentration of 500 ng/l [START_REF] Port | Systematic Evaluation of Drosophila CRISPR Tools Reveals Safe and Robust Alternatives to Autonomous Gene Drives in Basic Research[END_REF]. Candidates were screened using allele-specific PCR. We isolated one CRISPR modified male from which we established four CRISPR SNP A lines. In parallel, a control line was established by mating non-injected Act-Cas9 flies following the same scheme their injected siblings. Sequencing the ey eye enhancer from the transformed SNP A and of the non-injected SNP G control stocks confirmed that they were differing only by this single nucleotide.

Allele-specific PCR

SNP A and SNP G alleles were detected by allele-specific PCR using a common reverse primer (Ey-R3: AGAAATATCACATGGCCGAG) and one of two specific forward primers differing by the 3" most nucleotide (either A or G) and including a mismatch (underlined) to increase binding specificity (Ey-SNP G -F: GGAATCGAAAACAATAAAATGGCTGG; Ey-SNP A -F: GGAATCGAAAACAATAAAATGGCTGA).

Cut-FLAG fusion protein expressing S2 cells

S2 cells cultivated at 25°C in Schneider"s medium supplemented with 10% fetal calf serum were co-transfected with 2g of pAWF-Cut and 0,2g of pCoBlast vector (ThermoFisher Scientific) using Effecten transfection reagent (Qiagen) according to the manufacturer's instructions (1/10 DNA-Effecten ratio). Blasticidin selection (10g/l) was applied three days after transfection.

After one week of selection, cells were harvested, and expression of Cut-FLAG fusion protein was checked by western-blot using an anti-FLAG antibody (Sigma-Aldrich Cat# F3165). For longterm culture, cells were maintained in 2g/l blasticidin.

IMAGING AND IMAGE QUANTIFICATION

Image Processing

Except mentioned otherwise, all image processing was performed using Image J (versions 1.45 to 1.48) [START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF].

Scanning electron microscopy

Whole flies were fixed overnight at 4°C in a 1:1 mix of 4% formaldehyde in phosphate buffer pH 7.2 and 100 % ethanol and dehydrated successively in graded ethanol series, hexamethyldisilazane (HMDS) and in a dessicator. Fly heads were mounted on specimen studs using silver paint in two distinct orientations: dorsal head up (for whole head imaging) and lateral (for ommatidia counts and measures). Samples were subsequently coated with platinium and images acquired in LEI mode with a JEOL JSM 7401F microscope at magnifications ranging from 120 (heads overviews) to 1900 times (ommatidia width) [START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF].

Transmitted light and confocal microscopy

Preparations of adult heads for the acquisition of light microscopy images were acquired from non-fixed, freshly cut adult heads glued laterally on glass slides. Images were acquired using a camera DFC295 (Leica) mounted on a DMRXA (Leica) microscope, operated via the opensource software Micro-Manager [START_REF] Edelstein | Advanced methods of microscope control using muManager software[END_REF]. Fluorescent preparations of embryos and imaginal discs were acquired using a Nikon A1R Eclipse Ti, a Leica TCS SP5 II or a Leica SP8 confocal microscope operated by the accompanying company software.

Measuring adult eye, and antennae

All head and eye measurements were performed on female flies using ImageJ [START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF]. Adult Eye:Face ratio was expressed as E(Figure 1). Ommatidia width was measured on high magnification SEM images as the distance between one interommatidial bristle and the opposite angle of the facet. For each sample, measures of six adjacent ommatidia localized at the center of the eye were taken. To limit underestimation of the ommatidia width due to perspective projection distortion, samples were carefully oriented prior to image acquisition.

Ommatidia numbers

Ommatidia numbers were manually counted on SEM images using the Image J plugin "Cell counter". We also developed an alternative method based on the approximation of the compound eye to an ellipse. With this method, the ommatidia number is calculated as the surface of an ellipse whose large and small axes correspond to the numbers of ommatidia along the compound eye anterior-posterior and dorso-ventral axes ( with a and b as the lengths of the large and small ellipse axes; Fig. S4). This method accommodates lower resolution images and does not require the use of SEM. Bland-Altman method [START_REF] Bland | Statistical methods for assessing agreement between two methods of clinical measurement[END_REF] was used to compare the outcome of the two methods applied on a common set of SEM images.

The ellipse method results in an overestimation of approximately 20 ommatidia as compared to the manual counting (bias mean = 20. 29; SD = 11. 70). Importantly, this difference is independent of ommatidia number (Figure S2). Facet number estimation of Cut RNAi and CRISPR flies (Figures 4,6,S5 and S6) were performed blind regarding to the genotype.

Measuring embryonic and larval EADs

Numbers of Ey-positive embryonic eye-antennal disc cells were counted manually. To measure the surface of the larval eye-antennal disc and eye progenitor field, regions of interest were selected manually using the Image J freehand selection tool. The number of mitotic pH3-positive cells was automatically counted using the Dead-Easy Mito-Glia ImageJ Plugin [START_REF] Forero | DeadEasy Mito-Glia: automatic counting of mitotic cells and glial cells in Drosophila[END_REF]. The mitotic index was calculated as the number of mitotic cells per surface of the Eyapositive eye progenitor field.

mCherry and GFP colocalization

Protocol for pixel-based quantifications of mCherry and GFP colocalization was adapted from [START_REF] Oliva | Regulation of Drosophila Brain Wiring by Neuropil Interactions via a Slit-Robo-RPTP Signaling Complex[END_REF]. We used Fiji/ImageJ2/ImgLib2 [START_REF] Pietzsch | ImgLib2--generic image processing in Java[END_REF][START_REF] Rueden | ImageJ2: ImageJ for the next generation of scientific image data[END_REF][START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF] macro implemented in Jython. Raw images were imported using BioFormats library [START_REF] Linkert | Metadata matters: access to image data in the real world[END_REF]. EADs were manually segmented in each stack by the user. Stack threshold levels for each channel were calculated using preselected auto-thresholding algorithms available in Fiji (Huang for both channels). Determined threshold levels were used to calculate Mander's overlap coefficient using Fiji implementation of the colocalization algorithm.

The experimental set-up is modified from [START_REF] Buchner | Elementary Movement Detectors in an Insect Visual-System[END_REF]. It exploits the spontaneous tendency of fruit flies to adjust their trajectory to the surrounding landscape. Presented with rotating vertical black stripes, tethered flies spontaneously follow their movement. Narrowing the angular distance between the stripes beyond its resolving capacities makes the fly move in the Recordings of female D. mel. (Canton-S; n=9) and D. pse. (Cat; n=9) were performed simultaneously, with alternating assignments. We calculated the zero-crossing and its variance from the two average responses surrounding the zero-crossing (one positive, one negative) using linear interpolation and error propagation followed by t-test for differences between 2 means.

IMMUNOSTAININGS

Antibodies

We used the following primary antibodies: mouse anti-Ct (1:10, DHSB hybridoma supernatant 2B10, deposited by Rubin G. M.), mouse anti-Eya (1:75, DHSB hybridoma supernatant eya10H6, deposited by Benzer, S. and Bonini, N.M.), rat anti-elav (1:100, DHSB hybridoma supernatant Rat-Elav-7E8A10, deposited by Rubin G. M.), mouse anti-futsch (1:100; DHSB hybridoma supernatant 22C10 deposited by Benzer, S. and Colley, N.); rat anti-ey (1:300, received from P. Callaerts, [START_REF] Halder | Eyeless initiates the expression of both sine oculis and eyes absent during Drosophila compound eye development[END_REF]); anti-phosphorylated Histone 3 (1:1000, pSer10;

Merck Millipore #382159); sheep anti-atonal (1:1000, received from A. Jarman and P. zur Lage, [START_REF] Jarman | Role of the proneural gene, atonal, in formation of Drosophila chordotonal organs and photoreceptors[END_REF]); mouse anti-GFP 3E6 (1:1000, Invitrogen, #A11120); rabbit anti-DsRed

(1:1000, Clonetech, #632496). Secondary antibodies conjugated with Alexa 488, Alexa 555 and Alexa 647 were used at 1:200 (Molecular Probes). Nuclei were counterstained using Draq-5

(1:1000 in PBS, Abcam #ab108410). All samples were mounted in Vectashield (Vector laboratories #H-1000).

Procedure

Fixation and immunostainings were performed following standard procedures as described in [START_REF] Patel | Imaging neuronal subsets and other cell types in whole mount Drosophila embryos and larvae using antibody probes[END_REF] (embryos) and in [START_REF] Blair | Imaginal discs[END_REF] (EADs). Briefly, embryos were collected from grape juice agar plates and dechorionated with bleach (sodium hypochlorite 3%). They were fixed in a 1/1 mixture of n-heptane and 3.7% formaldehyde in PBS and devitellinized with methanol. Larval and pupal mouth complexes including the EADs, brain and pharynx were dissected in cold PBS and fixed in 3.7% formaldehyde in PBS for 20 minutes, then washed in PBT (embryos: PBS tween 0,1%; L1 and L2 EADs: PBS Triton-X 0,1%; L3 EADs: PBS Triton-X 0,3%) for 2 hours. Blocking was performed 1 hour at room temperature in blocking solution (5% normal goat serum in PBT) and incubation with primary antibodies diluted in blocking solution overnight at 4°C. After 2 hours of washes, tissues were incubated 2 hours at room temperature with the secondary antibodies diluted in blocking solution. After two hours of washes in PBT, tissues were rinsed in PBS, incubated with Draq5 (1/500 in PBS) 1 hour at room temperature or overnight at 4°C and mounted in Vectashield (Vector laboratories cat#H-1000).

DAPI stainings were performed by adding DAPI in the mounting medium (final concentration 1,5 m).

IN SILICO ANALYSIS

Genome Assemblies

All D. mel. genome positions refer to BDGP release 6 assembly (GCA_000001215.4) and D.

pse. genome positions refer to genome release Dpse_3.0, Baylor College of Medicine (GCA_000001765.2).

Alignment of D.mel. D. pse. ey enhancer

Pairwise alignment of D. pse. and D. mel. intronic ey eye enhancers was performed with BLASTn (McGinnis and Madden, 2004)(blast.ncbi.nlm.nih.gov) as described in [START_REF] Swanson | Rapid evolutionary rewiring of a structurally constrained eye enhancer[END_REF]. Namely, the following parameters were selected: "somewhat similar sequences" and the lower complexity filter was off. The sequences used for the alignment were: for D. pse.

Ct binding site predictions

To predict possible Ct binding sites, the SNP surrounding region (500up and down) was scored with Cluster-Buster [START_REF] Frith | Cluster-Buster: Finding dense clusters of motifs in DNA sequences[END_REF] (https://github.com/weng-lab/cluster-buster version, options -c0 -m3 -f5 -G0) using the 3 available Drosophila Ct PWMs (MA0128.1 from JASPAR [START_REF] Mathelier | JASPAR 2014: an extensively expanded and updated open-access database of transcription factor binding profiles[END_REF], Ct_Cell_FBgn0004198 and Ct_SOLEXA_FBgn0004198 from FlyFactorSurvey [START_REF] Zhu | FlyFactorSurvey: a database of Drosophila transcription factor binding specificities determined using the bacterial one-hybrid system[END_REF] for the 5 different alleles (Reference, Hikone-AS, Canton-S, NoCt, ConsensusCt). Predicted Binding sites overlapping our SNP are shown with either their corresponding score in Figure 4 for the best scoring PWM (Ct_SOLEXA_FBgn0004198), and in Figure S5 for all PWMs. Predictions for the entire region are shown in Table S2.

Population genetics

We compiled whole genome sequencing data from multiple geographic samples collected in Africa, Europe, North America, Asia and Australia to investigate worldwide allele frequency patterns of the eyeless SNP at position Chr 4: 710326 in natural populations. This dataset consisted of single individual sequencing data [START_REF] Campo | Whole-genome sequencing of two North American Drosophila melanogaster populations reveals genetic differentiation and positive selection[END_REF][START_REF] Grenier | Global diversity lines -a fivecontinent reference panel of sequenced Drosophila melanogaster strains[END_REF][START_REF] Lack | The Drosophila genome nexus: a population genomic resource of 623 Drosophila melanogaster genomes, including 197 from a single ancestral range population[END_REF][START_REF] Lack | A Thousand Fly Genomes: An Expanded Drosophila Genome Nexus[END_REF][START_REF] Langley | Genomic variation in natural populations of Drosophila melanogaster[END_REF][START_REF] Pool | Population Genomics of sub-saharan Drosophila melanogaster: African diversity and non-African admixture[END_REF] and Pool-Seq data from various sources [START_REF] Bastide | A genome-wide, fine-scale map of natural pigmentation variation in Drosophila melanogaster[END_REF][START_REF] Bergland | Genomic evidence of rapid and stable adaptive oscillations over seasonal time scales in Drosophila[END_REF][START_REF] Kapun | Genomic Evidence for Adaptive Inversion Clines in Drosophila melanogaster[END_REF][START_REF] Orozco-Terwengel | Adaptation of Drosophila to a novel laboratory environment reveals temporally heterogeneous trajectories of selected alleles[END_REF][START_REF] Reinhardt | Parallel geographic variation in Drosophila melanogaster[END_REF] (Table S2). For single individuals, we obtained genotypes of the focal SNP from the Drosophila Genome Nexus website (DGN; http://www.johnpool.net/genomes.html) and estimated allele frequencies based on the number of chromosomes carrying the A-variant for populations with at least ten sequenced individuals. For

Pool-Seq datasets, we re-mapped quality-filtered raw data as described in [START_REF] Kapun | Genomic Evidence for Adaptive Inversion Clines in Drosophila melanogaster[END_REF] and estimated allele frequencies based on read counts of the A-variant relative to the total coverage. To increase sequence coverage in Queensland and Tasmania, we merged libraries of multiple collections at the corresponding locations [START_REF] Reinhardt | Parallel geographic variation in Drosophila melanogaster[END_REF]. We further used a collection of Pool-Seq data from 48 population samples collected across Europe by the DrosEU consortium [START_REF] Kapun | Genomic analysis of European Drosophila populations reveals longitudinal structure and continent-wide selection[END_REF] (accession number: PRJNA388788) for an in-depth analysis of spatial distribution of the A-variant. Specifically, we tested for clinal distribution along the latitudinal and longitudinal axes by means of generalized linear models (GLMs) in R based on allele counts to account for the biallelic nature of the focal SNP. We further contrasted the clinality of 4: 710,326 to 21,008 putatively neutral genome-wide SNPs located in short introns (<60bp) and in distance to chromosomal inversions [START_REF] Clemente | Unconstrained evolution in short introns? -an analysis of genome-wide polymorphism and divergence data from Drosophila[END_REF][START_REF] Parsch | On the utility of short intron sequences as a reference for the detection of positive and negative selection in Drosophila[END_REF]. To test if the observed p-value from a GLM at the focal SNP deviates from neutral expectation we empirically assessed significance. We therefore generated empirical cumulative density functions (ECDF) based on the -log10 transformed p-values of all neutral SNPs and calculated the area under the ECDF confined by the -log10 p-value of the focal SNP and the upper tail of the distribution by integration. This area corresponds to the percentile of neutral SNPs with p-values equal or smaller than the focal SNP and thus summarizes the significance of clinality for 4:710,326 relative to genome-wide neutral estimates. We further characterized chromosome-wide patterns of genetic variation by estimating the population genetics statistics π and Tajima"s D for all 48 samples from the DrosEU dataset using Pool-Gen [START_REF] Kapun | Genomic analysis of European Drosophila populations reveals longitudinal structure and continent-wide selection[END_REF] with implemented corrections for Pool-Seq data [START_REF] Futschik | The next generation of molecular markers from massively parallel sequencing of pooled DNA samples[END_REF][START_REF] Kofler | PoPoolation: a toolbox for population genetic analysis of next generation sequencing data from pooled individuals[END_REF]. At last, we tested whether very rare occurrences of the A-variant in Sub-Saharan Africa may be due to admixture with non-African genetic variation. We therefore used admixture probability estimates from [START_REF] Lack | The Drosophila genome nexus: a population genomic resource of 623 Drosophila melanogaster genomes, including 197 from a single ancestral range population[END_REF] (see Table S2) to classify African lines as admixed (>10% of the autosomes admixed) or non-admixed (≤ 10% of the autosomes admixed) and compared genotype counts for admixed and non-admixed lines by means of Fisher exact tests (FET) in R.

ELECTROPHORETIC MOBILITY SHIFT ASSAYS

Nuclear extracts of stably transfected S2 cells expressing the Cut-FLAG fusion protein were prepared from cells pelleted and resuspended for 15 minutes at 4°C in the membrane lysis buffer (Tris-HCl pH7,4 20mM, NaCl 10mM, MgCl2 3mM, DTT 0,5mM, NP40 1%, protease inhibitors). Cell extracts were centrifuged 5 minutes at 13000 RPM. Pelleted nuclei were then rinsed twice with PBS 1X and lysed for 30 minutes at 4°C in the nuclear envelope lysis buffer (Tris-HCl pH7,4 20mM, NaCl 100mM, MgCl2 3mM, EDTA 1mM, glycerol 10%, DTT 0,5mM, SDS 0,1%, sodium deoxycholate 0,5%, Triton X-100 1%, protease inhibitors). After centrifugation for 5 minutes at 13000 RPM, the supernatant containing the nuclear extract was recovered and stored at -80°C.

Cold Electrophoretic Mobility Shift Assays (EMSA) were performed with 8 or 16g of nuclear extract using the LightShift Chemiluminescent EMSA kit (ThermoFisher Scientific) according to the manufacturer's instructions, with a modified binding buffer (Tris-HCl pH7,5 10mM, KCl 50mM, DTT 1mM, MgCl2 5mM, EDTA 1mM, Ficoll 400 5%, BSA 0,1g/l, NP40 0,05%, poly dI-dC 50ng/l or Tris-HCl pH7,5 10mM, KCl 100mM, DTT 1mM, MgCl2 5mM, EDTA 1mM, NP40 0,05%, poly dI-dC 50ng/l) 5"-biotinylated and unlabeled probes corresponding to the G and A-alleles of the ey enhancer were generated. To this goal, labeled or non-labeled complementary oligonucleotides were synthesized and hybridized. Oligonucleotide sequences are: G-allele: 5'-ACAATAAAATGGTTGGAACTTTTTCGAACTTT-3' A-allele: 5'-ACAATAAAATGGTTGAAACTTTTTCGAACTTT-3'

The binding was performed for 20 minutes at room temperature, followed by electrophoretic migration onto native 5% polyacrylamide gel in TBE 0,5X buffer. The supershift experiment was performed adding 2g of the M2 anti-FLAG antibody (Sigma-Aldrich Cat# F3165).

QUANTIFICATION AND STATISTICAL ANALYSIS

quantification based on poor image quality or inadequate developmental stage, explaining differences in sample size between groups. Except where stated otherwise, all statistical tests and charts were performed using Graphpad Prism 7 (Graphpad software Inc.). Normality of the data were systematically assessed and statistical tests selected accordingly. Details on statistical tests, sample sizes and p values are indicated in figure legends except where mentioned otherwise.

Figure 1 .

 1 Figure 1. Natural variation in Drosophila facet number, see also Figure S1

Figure 2 .

 2 Figure 2. Developmental origin of eye size variation in D. mel. and D. pse., see also Figure

Figure 3 .

 3 Figure 3. Different temporal regulation of EAD subdivision governs the trade-off between

Figure 4 .

 4 Figure 4. Developmental and regulatory origin of intraspecific eye size variation.

  Sample sizes from left to right (n=13; n=23; n=10; n=8). Ordinary one-way ANOVA **** p<0.0001 followed by Sidak"s multiple comparisons: **** p<0.0001, * p=0.0126. See also Figure S5. (H to I") Pairwise comparison of the expression of the four D. mel. ey enhancer alleles: ey3.5 G (Hikone-AS); ey3,5 A (Canton-S); ey3.5 CC (ConsensusCt); ey3.5 NC (NoCt). (G and H) Schematics and immunostainings of EADs with ongoing (F, early/mid-L2 stage) and full (G, early L3 stage) posterior retraction of ey enhancer activity. (G" and H") Pairwise comparison of the difference in expression between GFP and mCherry, measured as the proportion of mCherry that was not colocalized with GFP, when driven by distinct combinations of ey eye enhancers during (F", early/mid-L2 stage) and after (G", early L3 stage) posterior retraction of ey enhancer activity. ey enhancer variants: ey3.5 G , G-variant (Hikone-AS); ey3.5 A , A-variant (Canton-S); ey3.5 CC , ConsensusCt-variant; ey3.5 NC , NoCtvariant. (G") Mid-L2 stage. Sample sizes: (n=16, n=24, n=20, n=12). Ordinary one-way ANOVA **** p<0.0001 followed by Dunnet"s multiple comparisons versus ey3.5 G : ****adjusted p=0.0001, n.s. adjusted p=0.9828; * adjusted p = 0.0167. (H") Early-L3 stage. Sample sizes from left to right (n=9; n=10; n=6; n=9). Ordinary one-way ANOVA n.s. p=0.9652.

Figure 5 .

 5 Figure 5. Worldwide distribution of the G>A substitution (chr4: 710326) in the genome of

  (A") Frequency distribution of the A-variant in Europe revealing the longitudinal clinal distribution of the allele. (B) Scatter plots showing allele frequencies of the A-variant along the latitudinal (left box; red regression curve) and the longitudinal axis (right box; blue regression curve) in Europe. The topright p values show the significance from generalized linear models (GLM). (C) The line plot shows the empirical cumulative density function (ECDF) from -log 10 transformed p values of GLMs with longitude as the predictor variable for 21,008 neutrally evolving intronic SNPs (black curve). The vertical red line highlights the -log 10 p value for the focal SNP (p=8.6 x 10 -13 ) which is inferior to the ones of 99.69 % of the neutral SNPs, indicating that it stronger correlation with longitude. The empirical p value (p=0.0031) is calculated from the area confined by the p value of 4: 710326 and the tail of the ECDF.

Figure 6 .

 6 Figure 6. The non-coding G>A SNP in ey enhancer causes facet number variation.

  stocks were cultured on standard cornmeal diet food at 25°C except mentioned otherwise in the corresponding figure legend. Developmental stages used in each experiment are indicated in the corresponding method details section. Morphological measures on adult flies were performed on females only. The fly strains used in this study were: Canton-S BH (Hassan lab); Canton-S TP (Préat lab, provided by P. Callaerts); Canton-S RD (Davis lab, provided by P. Callaerts); Hikone-AS (Kyoto DGRC 103421); DGRP-208 (D.m. 3, Bloomington #25174); Act5C-Cas9 (Port et al., 2014) (Bloomington # 54590); ct-Gal4(Pfeiffer et al., 2008) (Bloomington # 45603); hth NP5332 -Gal4 (DGRC Kyoto #104957); UAS-RNAi ct (VDRC #v5687); UAS-RNAi ct (VDRC #v4138); UAS-RNAi luc (Bloomington #31603); D.m.ey3.5 G GFP (this study); D.m.ey3.5 A GFP (this study); D.m.ey3.5 G mCherry (this study); D.m.ey3.5 A mCherry (this study); D.m.ey3.5 NoCt GFP (this study); D.m.ey3.5 ConsensusCt GFP (this study); ey3.5 G (this study); ey3.5

  flies were performed on females only. Stock origin: Kisangani, Congo (DSSC 14024-0371.30) and Mumbai, India (DSSC 14024-0371.31). Drosophila yakuba D. yakuba stocks were cultured on standard cornmeal diet food at 21°C. Morphological measures on adult flies were performed on females only. Stock origin: Ivory Coast (DSSC 14021-8209.0261.00) and Liberia (Reference Genome strain DSSC 14021-0261.01). Drosophila pseudoobscura Isofemale D. pseudoobscura stocks were cultured on standard cornmeal diet food at 25°C except when mentioned otherwise in the corresponding figure legend. Morphological measures on adult flies were performed on females only. Stock origin: Catalina Island, California, USA (Cat; DSSC 14011-0121.121) and Chiracahua Mountains, Arizona, USA (DSSC 14011-0121.118).

  Technology (ThermoFischer Scientific). D. pse. ey3.3 and D. mel. ey3.5 regulatory sequences were amplified respectively from D. pse. (from stock Cat; DSSC 14011-0121.121), Hikone-AS (for the G-variant) and Canton-S BH (for the A-variant) genomic DNA (extracted using Qiagen DNeasy Blood and Tissue Kit #69504) and cloned into the Gateway pDONR221 entry vector (ThermoFischer Scientific #12536017) following the provider specifications. Primers for the enhancer amplifications are: pEntry-ey 3.3Pse : forward:GGGGACAAGTTTGTACAAAAAAGCAGGCTAAGTGGTAGTGGACTAGG and reverse:GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAGAATTTTGCTAACGC;

  opposite direction, due to an interference phenomenon similar to what we perceive when looking at the wheels of a starting train. It consists of two tracking balls and of two computer screens on which moving vertical black and white stripes are displayed in a window of 90° horizontal and 74° vertical extensions. The width of the stripes (spatial wavelength ) as they move on the flat screen are adjusted such that they subtend a constant angle as seen from the fly 40 mm away of the screen. Pattern speed w is adapted to maintain the "contrast frequency"at 1 Hz. A positive optomotor response indicates the tendency of the flies to follow the direction of the movement of the stripes. Reduction of  below the resolving power of the eye causes an inversion of the apparent direction of the movement of the stripes due to the geometrical interference between the fly"s vertical columns of ommatidia and the vertical stripes and is accompanied by the inversion of the fly response towards negative values. The  value at which the response of the fly is inverted (zero-crossing angle, 2) provides a measure of spatial resolution (or visual acuity).

TABLE 1 :

 1 Ommatidia numbers. Ommatidia numbers are counted on SEM images (count) or estimated from light-microscopy images using an ellipse-based method (estimated, see Star Methods and Figure

  Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Bassem Hassan (bassem.hassan@icm-institute.org). Reagents generated during this study are available upon request from the authors.

		Ommatidia nb	Ommatidia nb
		(count)		(estimated)
		Mean	St Dev Mean	St Dev
	Canton-S	869.8 a	17.96 a 828.0	34.00
	Hikone-AS	742.4 a	20.17 a 736.0	27.09
	D. pse.	1177 a	28.02 a
	F1 CS/Hik		774.6	34.77
	ct 5687 /ct-GAL4		835.7	25.88
	ct 5687 /TM3,Sb		802.9	26.99
	ct 4138 /ct-GAL4		818.6	23.24
	ct 5687 /TM3, Sb		777.6	22.99
	Canton S BH		830.8	36.42
	Canton S TP		775.4	21.69
	Canton S RD		750.4	20.26
	ey3.5 G	713.8	29.77 745.1	43.76
	ey3.5 G>A	737.0	21.12 791		44.49
	ey3.5 Gl /Df(4)J2		822.9	34.44
	ey3.5 G>A		847.5	19.61
	/Df(4)J2

ACKNOWLEDGMENTS

This work was funded by VIB (B.A.H), the Belspo WiBrain Interuniversity Attraction Pole network and Fonds Wetenschappelijke Onderzoeks (FWO) grant G.0503.12 (B.A.H.). Work in B.A.H lab is currently funded by the Institut Hospitalier Universitaire (IHU) and the Institut du Cerveau et de la Moëlle Epinière (ICM), Paris, France. S.W. was a fellow of the FLiACT Marie Curie ITN (FP7, EU). M.K. and T.F. were supported by a Swiss National Science Foundation (SNSF) grant (PP00P3_133641) to T.F. We thank M. Lang, B. Lefèvre, S. Villain, R. Ejsmont, N. Mora, A. Soldano, N. Gompel, N. Posnien and D. Nunes for advices, discussion or/and technical help; J. Clements and P. Callaerts for sharing reagents. We acknowledge the Drosophila Species Stock Center (DSSC), the Bloomington Stock Center (NIH P40OD018537), the Kyoto Stock Center (DGRC), for sending Drosophila stocks, the Developmental Studies Hybridoma Bank (DHSN, University of Iowa) for sending antibodies and Addgene for sending plasmids. We are grateful to P. Baatsen from the Electronic Microscopy Platform for support with SEM sample preparation and imaging. Confocal imaging was performed at the VIB Light Microscopy and Imaging Network (Limone) and at the Plateforme d"imagerie cellulaire (PICPS) at the ICM. The Leica TCS Sp5 II confocal microscope and the Nikon were acquired respectively by the VIB BioImaging Core and through a Hercules type 1 grant (AKUL/09/037) to W. Annaert. B.A.H. is an Allen Distinguished Investigator and an Einstein Visiting Fellow of the Berlin Institute of Health.

Code for the macro is available on GitHub, https://github.com/rejsmont/FijiScripts/blob/master/mColoc3D.py [START_REF] Oliva | Regulation of Drosophila Brain Wiring by Neuropil Interactions via a Slit-Robo-RPTP Signaling Complex[END_REF]. The proportion of pixels expressing solely mCherry is used as a measure for GFP retraction.

BEHAVIORAL MEASURES OF VISUAL ACUITY

DATA AND CODE AVAILABILITY

Data and Code Availability Statement:

-The code for the macro used for quantification of GFP/RFP colocalization [START_REF] Oliva | Regulation of Drosophila Brain Wiring by Neuropil Interactions via a Slit-Robo-RPTP Signaling Complex[END_REF] is available on GitHub at the following link: https://github.com/rejsmont/FijiScripts/blob/master/mColoc3D.py -Raw images are available upon request from the lead contact.

KEY RESOURCES TABLE see separate Word document

DECLARATION OF INTERESTS

The authors declare no competing interests.