Evolution of compound eye morphology underlies differences in vision between closely related Drosophila species

Background: Insects have evolved complex visual systems and display an astonishing range of adaptations for diverse ecological niches. Species of Drosophila melanogaster subgroup exhibit extensive intra- and interspecific differences in compound eye size. These differences provide an excellent opportunity to better understand variation in insect eye structure and the impact on vision. Here we further explored the difference in eye size between D. mauritiana and its sibling species D. simulans. Results: We confirmed that D. mauritiana have rapidly evolved larger eyes as a result of more and wider ommatidia than D. simulans since they recently diverged approximately 240,000 years ago. The functional impact of eye size, and specifically ommatidia size, is often only estimated based on the rigid surface morphology of the compound eye. Therefore, we used 3D synchrotron radiation tomography to measure optical parameters in 3D, predict optical capacity, and compare the modelled vision to in vivo optomotor responses. Our optical models predicted higher contrast sensitivity for D. mauritiana, which we verified by presenting sinusoidal gratings to tethered flies in a flight arena. Similarly, we confirmed the higher spatial acuity predicted for Drosophila simulans with smaller ommatidia and found evidence for higher temporal resolution. Conclusions: Our study demonstrates that even subtle differences in ommatidia size between closely related Drosophila species can impact the vision of these insects. Therefore, further comparative studies of intra- and interspecific variation in eye morphology and the consequences for vision among other Drosophila species, other dipterans and other insects are needed to better understand compound eye structure–function and how the diversification of eye size, shape, and function has helped insects to adapt to the vast range of ecological niches

Evolution of compound eye morphology underlies differences in vision between closely related Drosophila species

Background. Insects have evolved complex visual systems and display an astonishing range of adaptations for diverse ecological niches. Species of Drosophila melanogaster subgroup exhibit extensive intra- and interspecific differences in compound eye size. These differences provide an excellent opportunity to better understand variation in insect eye structure and the impact on vision. Here we further explored the difference in eye size between D. mauritiana and its sibling species D. simulans. Results. We confirmed that D. mauritiana have rapidly evolved larger eyes as a result of more and wider ommatidia than D. simulans since they recently diverged approximately 240,000 years ago. The functional impact of eye size, and specifically ommatidia size, is often only estimated based on the rigid surface morphology of the compound eye. Therefore, we used 3D synchrotron radiation tomography to measure optical parameters in 3D, predict optical capacity, and compare the modelled vision to in vivo optomotor responses. Our optical models predicted higher contrast sensitivity for D. mauritiana, which we verified by presenting sinusoidal gratings to tethered flies in a flight arena. Similarly, we confirmed the higher spatial acuity predicted for Drosophila simulans with smaller ommatidia and found evidence for higher temporal resolution. Conclusions. Our study demonstrates that even subtle differences in ommatidia size between closely related Drosophila species can impact the vision of these insects. Therefore, further comparative studies of intra- and interspecific variation in eye morphology and the consequences for vision among other Drosophila species, other dipterans and other insects are needed to better understand compound eye structure–function and how the diversification of eye size, shape, and function has helped insects to adapt to the vast range of ecological niches

Characterising the function and evolution of enhancers in Drosophila

Enhancers are important modular cis-regulatory elements that precisely control the spatial and temporal expression of most genes. Therefore understanding how enhancers work and evolve is crucial for understanding the regulation of most developmental processes in animals. In my PhD project, I investigated three important aspects of enhancers to further our understanding of these cis-regulatory elements: enhancer identification, enhancer functionality and enhancer evolution. I identified a novel post-embryonic enhancer of the Hox gene Ubx, which provides new insights into how Hox genes are integrated into post-embryonic GRNs that determine fine-scale adult morphology. To help understand the cis-regulatory logic of enhancers, I focussed on a functionally related set of genes in a well characterised gene regulatory network. I identified a common pattern of motifs in candidate enhancers, which evidences that this approach may be a powerful for identifying key transcription factor binding sites in enhancers and de novo enhancer prediction. Finally, I described the evolutionary turnover of binding sites and intervening sequences among natural variants of the Drosophila hb P2 enhancer. This suggests that by studying the mutations gained through evolution we can learn more about flexibility and constraints in enhancers and improve on our current knowledge. I then generated tools for the detailed functional comparison of these natural variants, and observed qualitative differences in their function between species. These tools can now be applied to quantify these differences and shows this approach has great potential to better understand the function and evolution other developmental enhancers. Taken together my investigation has broadly contributed to our knowledge of enhancer organisation and functionality and provides a very useful platform for future analyses

Characterisation of the role and regulation of Ultrabithorax in sculpting fine-scale leg morphology

Hox genes are expressed during embryogenesis and determine the regional identity of animal bodies along the antero-posterior axis. However, they also function post-embryonically to sculpt fine-scale morphology. To better understand how Hox genes are integrated into post-embryonic gene regulatory networks, we further analysed the role and regulation of Ultrabithorax (Ubx) during leg development in Drosophila melanogaster. Ubx regulates several aspects of bristle and trichome patterning on the femurs of the second (T2) and third (T3) leg pairs. We found that repression of trichomes in the proximal posterior region of the T2 femur by Ubx is likely mediated by activation of the expression of microRNA-92a and microRNA-92b by this Hox protein. Furthermore, we identified a novel enhancer of Ubx that recapitulates the temporal and regional activity of this gene in T2 and T3 legs. We then used transcription factor (TF) binding motif analysis in regions of accessible chromatin in T2 leg cells to predict and functionally test TFs that may regulate the Ubx leg enhancer. We also tested the role of the Ubx co-factors Homothorax (Hth) and Extradenticle (Exd) in T2 and T3 femurs. We found several TFs that may act upstream or in concert with Ubx to modulate trichome patterning along the proximo-distal axis of developing femurs and that the repression of trichomes also requires Hth and Exd. Taken together our results provide insights into how Ubx is integrated into a post-embryonic gene regulatory network to determine fine-scale leg morphology

Leg trichome patterns in <i>miR-92a</i>/<i>miR-92b</i> mutants.

<p>(A) Flies mutant for both <i>miR-92a</i> and <i>miR-92b</i> gain trichomes in the naked valley. (B) Most trichomes on the posterior T2 femur are repressed in <i>svb</i>^<i>PL107</i> flies. (C) No trichomes are gained upon loss of <i>miR-92a</i> and <i>miR-92b</i> in a <i>svb</i>^<i>PL107</i> background.</p

The GRN controlling formation of trichomes on larval and leg epidermis differs between these developmental contexts.

<p>(A) Simplified GRN for larval trichome development (see [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007375#pgen.1007375.ref022" target="_blank">22</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007375#pgen.1007375.ref029" target="_blank">29</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007375#pgen.1007375.ref081" target="_blank">81</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007375#pgen.1007375.ref082" target="_blank">82</a>]). (B) GRN for leg trichome development. Magenta colour indicates interactions found only during leg trichome development. Dashed lines indicate likely interactions. Expression of <i>svb</i> is controlled by several upstream transcription factors and signalling pathways some of which are not active during leg trichome development. The question mark indicates that there are likely to be other unknown activators of <i>svb</i> in legs. Activation of Svb protein requires proteolytic cleavage involving small peptides encoded by <i>tal</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007375#pgen.1007375.ref030" target="_blank">30</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007375#pgen.1007375.ref032" target="_blank">32</a>]. Active Svb then regulates the expression of at least 163 target genes in embryos [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007375#pgen.1007375.ref029" target="_blank">29</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007375#pgen.1007375.ref033" target="_blank">33</a>], the expression of 135 of which is detectable in legs. The products of these downstream genes are involved in actin bundling, cuticle segregation, or changes to the matrix, which lead to the actual formation of trichomes. SoxN and Svb activate each other and act partially redundantly on downstream targets in larvae [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007375#pgen.1007375.ref034" target="_blank">34</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007375#pgen.1007375.ref036" target="_blank">36</a>] and this interaction probably also occurs in legs based on expression data. <i>miR-92a</i> is only expressed in naked leg cells where it represses <i>sha</i> and probably <i>CG14395</i> and thereby acts as a short circuit for <i>svb</i>. Its expression is likely controlled by <i>Ubx</i>. (C, D) Trichomes on the ventral side of the larval cuticle form stereotypic bands (denticle belts) separated by trichome-free cuticle. (E, F) A trichome-free region on the posterior of the T2 femur differs in size between different <i>D</i>. <i>melanogaster</i> strains. Shown are OregonR (E) and <i>e</i>^<i>4</i>,<i>wo</i>^<i>1</i>,<i>ro</i>^<i>1</i> (F).</p

Enhancers of <i>svb</i>.

<p>(A) Overview of the chromatin accessibility profile (ATAC-seq) at the <i>ovo</i>/<i>svb</i> locus. Indicated are: the deficiency used (dotted line), known larval <i>svb</i> enhancers (black boxes), and tested putative enhancers (grey boxes: no expression in pupal legs, green/orange boxes: expression in pupal legs). Region VT057077 (orange) is able to drive expression during trichome formation (see B-D). The bottom panel shows expressed variants of genes at the locus (black) and genes/variants not expressed (grey). Boxes represent exons, lines represent introns. (B) VT057077 has a naked valley of intermediate size. (C) Expression of <i>sha</i>-ΔUTR under VT057077 control induces trichome formation in the naked valley. (D, D’) Driving <i>miR-92a</i> with VT057077 represses trichome formation on the anterior and posterior of the second leg femur. Small patches of trichomes can sometimes still be observed (arrowhead).</p

Ectopic trichome formation on naked cuticle.

<p>Driving <i>sha</i>-ΔUTR (A) under control of <i>wg</i>-GAL4 does not lead to ectopic trichome formation on otherwise naked larval cuticle. Driving <i>svb</i> (B) or its constitutively active variant <i>ovoB</i> (C) is sufficient to activate trichome development, but expressing only the Svb activator <i>tal</i> (D) is not [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007375#pgen.1007375.ref032" target="_blank">32</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007375#pgen.1007375.ref038" target="_blank">38</a>]. GFP was co-expressed in each case to indicate the <i>wingless</i> (<i>wg</i>) expression domain (A’-D’). Ectopic activation of <i>sha</i>-ΔUTR in the proximal femur (E) is able to induce trichome formation, but ectopic <i>svb</i> (F) is not. Driving either <i>ovoB</i> (G) or the activator <i>tal</i> (H) leads to ectopic trichome development. Expression of <i>ovoB</i> has additional effects on leg development (e.g. a bending of the proximal femur), while expression of <i>tal</i> also leads to the development of ectopic bristles on the femur (arrowheads in H).</p

The size of the naked valley differs between and within species and is dependent on <i>miR-92a</i> expression.

<p>Reduction of <i>miR-92a</i> expression in <i>D</i>. <i>melanogaster</i> T2 legs has led to a derived (d) smaller naked valley in some populations while the ancestral state (a) is thought to be a large naked valley like in other <i>D</i>. <i>melanogaster</i> group species and other species (e.g. <i>D</i>. <i>pseudoobscura</i>). The absence of a naked valley in <i>D</i>. <i>virilis</i> is possibly due to absence of <i>miR-92a</i> expression, while the presence of small naked valleys in other species of the <i>virilis</i> group (e.g. <i>D</i>. <i>americana</i>) could be explained by a gain of microRNA expression. The coloured bars represent the spatial expression of each gene in the femur with lighter orange indicating where <i>sha</i> expression is post-transcriptionally repressed by <i>miR-92a</i>.</p

The house spider genome reveals an ancient whole-genome duplication during arachnid evolution.

BackgroundThe duplication of genes can occur through various mechanisms and is thought to make a major contribution to the evolutionary diversification of organisms. There is increasing evidence for a large-scale duplication of genes in some chelicerate lineages including two rounds of whole genome duplication (WGD) in horseshoe crabs. To investigate this further, we sequenced and analyzed the genome of the common house spider Parasteatoda tepidariorum.ResultsWe found pervasive duplication of both coding and non-coding genes in this spider, including two clusters of Hox genes. Analysis of synteny conservation across the P. tepidariorum genome suggests that there has been an ancient WGD in spiders. Comparison with the genomes of other chelicerates, including that of the newly sequenced bark scorpion Centruroides sculpturatus, suggests that this event occurred in the common ancestor of spiders and scorpions, and is probably independent of the WGDs in horseshoe crabs. Furthermore, characterization of the sequence and expression of the Hox paralogs in P. tepidariorum suggests that many have been subject to neo-functionalization and/or sub-functionalization since their duplication.ConclusionsOur results reveal that spiders and scorpions are likely the descendants of a polyploid ancestor that lived more than 450 MYA. Given the extensive morphological diversity and ecological adaptations found among these animals, rivaling those of vertebrates, our study of the ancient WGD event in Arachnopulmonata provides a new comparative platform to explore common and divergent evolutionary outcomes of polyploidization events across eukaryotes