The Expression of Pax6 Genes in an Eyeless Arachnid Suggests Their Ancestral Role in Arachnid Head Development

The Expression of Pax6 Genes in an Eyeless Arachnid Suggests Their Ancestral Role in Arachnid Head Development | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results The Expression of Pax6 Genes in an Eyeless Arachnid Suggests Their Ancestral Role in Arachnid Head Development Isabella Joyce , View ORCID Profile Austen A. Barnett doi: https://doi.org/10.1101/2025.01.29.635487 Isabella Joyce Department of Biology, DeSales University, Center Valley , PA, United States of America Find this author on Google Scholar Find this author on PubMed Search for this author on this site Austen A. Barnett Department of Biology, DeSales University, Center Valley , PA, United States of America Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Austen A. Barnett For correspondence: austen.barnett{at}desales.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Background Many animal lineages utilize Pax6 transcription factors during eye development. Within Arthropoda, evidence suggests that Pax6 genes are necessary for the specification of eyes in myriapods, crustaceans, and insects. However, recent data have argued that Pax6 genes lack a role in the development of the eyes in Chelicerata (=arachnids, horseshoe crabs, and sea spiders). An alternative hypothesis argues that the absence of Pax6 expression in developing chelicerate eyes could be explained by an earlier role for these genes in patterning eye precursor cells. The arachnid mite Archegozetes longisetosus lacks eyes, however it retains two Pax6 paralogs in its genome. By leveraging these aspects of A. longisetosus , we tested the hypothesis that ancestrally chelicerates did not use Pax6 genes to pattern their eyes but rather used them to pattern the central nervous system. We reasoned that if we observed comparable expression patterns of Pax6 genes in A. longisetosus in comparison to those in arachnids that have retained eyes, then this would support the hypothesis that Pax6 genes were not ancestrally used for eye specification in chelicerates. Results We followed the expression of canonical arthropod retinal determination genes to confirm that A. longisetosus does not develop vestigial eyes. We found that the expression of the Pax6 paralogs was consistent with their roles in the development of the ocular region and central nervous system. By co- staining for these genes simultaneously with the conserved head patterning gene orthodenticle , we also observed early expression patterns of these genes in the protocerebrum of early A. longisetosus embryos that are comparable to those arachnids with embryonic eyes. Conclusions Our data provide support for the hypothesis that Pax6 genes were not ancestrally used to pattern chelicerate eyes. The expression patterns of Pax6 genes in A. longisetosus were comparable to those of other arachnids that have eyes. This suggests that the retention of Pax6 genes in A. longisetosus is due to their ancestral, non-eye patterning roles. Further supporting this hypothesis is our observation that A. longisetosus does not pattern vestigial eyes. Lastly, our data suggests that the Pax6 genes, with orthodenticle , acted to specify the ancestral arachnid protocerebrum. Background Eyes have likely evolved independently multiple times within animals, underscoring their adaptive advantage in a variety of ecosystems [ 1 , 2 ]. These complex and diverse organs can be found across disparate metazoan lineages, including cnidarians, mollusks, vertebrates, and arthropods (reviewed in [ 3 ]). Ancestrally, the arthropods (=insects, crustaceans, myriapods and chelicerates) had a pair of multifaceted compound eyes, as well as multiple simple eyes called ocelli [ 4 , 5 ]. This basic theme is generally conserved across arthropods, albeit with lineage-specific modifications. Evidence suggests that the arachnid lateral eyes are homologous to the insect compound eyes, whereas the medial eyes are homologous to the ocelli [ 6 ]. This ground plan differs from that of the likely sister group of arachnids, Xiphosura (=horseshoe crabs [ 7 ] but see [ 8 , 9 ] for an alternate hypothesis). Instead, horseshoe crabs retain the basal arthropod state of housing both compound eyes and ocelli [ 4 ]. Sea spiders (=pycnogonids) also represent a non-arachnid chelicerate group, and these animals bear two pairs of median eyes, but lack lateral eyes [ 10 , 11 ]. At the other extreme of chelicerate visual systems lie groups that have lost their eyes entirely, including many members of Acariformes, the clade comprised of mites [ 12 ]. Despite the multiple occurrences of independent eye evolution across animal phyla, the development of these organs usually involves the utilization of Pax6 transcription factors [ 1 ]. Within arthropods, including chelicerates, phylogenetic evidence suggests that the last common ancestor of arthropods likely had at least two paralogous Pax6 genes (see [ 13 ]), first identified as eyeless ( ey ) [ 14 ] and twin of eyeless ( toy ) [ 15 ] in the fruit fly Drosophila melanogaster . These distinct Pax6 orthologs are used differentially across arthropod groups, obfuscating their ancestral roles in arthropod eye development. For example, in D. melanogaster, both ey and toy are expressed in the eye/antennal imaginal discs, which give rise to the adult eyes (note that Pax6 input is not necessary for the development of its larval eyes [ 16 ]). In these imaginal discs, both ey and toy have been shown to be necessary for the development of the compound eye [ 14 ], whereas only toy is necessary for specifying the ocelli [ 17 – 19 ]. In another insect, the beetle Tribolium castaneum , both Pax6 orthologs are required for the formation of the larval eyes, however RNAi of these genes showed only mild effects on the formation of the adult eyes [ 20 ]. In crustacean exemplars, the knockout of ey in Daphnia magna generated eye deformities [ 21 ]. In the decapod crustacean Exopalaemon carinicauda , the knockout of ey resulted in a range of compound eye deformities, whereas the knockout of toy had no effect on eye development [ 22 ]. Studies into the utilization of Pax6 orthologs in myriapods is thus far restricted to an expression study in the millipede Glomeris marginata, where both toy and ey are expressed in the optic lobes, the regions that give rise to eyes in other arthropods [ 23 ]. These studies are suggestive of a conserved role of Pax6 orthologs in at least some processes of eye development in the mandibulates, i.e. , the clade comprised of insects, crustaceans and myriapods. The utilization of Pax6 orthologs in chelicerates, however, is much less clear, as initially demonstrated by studies on horseshoe crabs, Current data suggest that the last common ancestor of Xiphosura underwent three rounds of whole-genome duplications [ 24 – 26 ]. These expansions of the horseshoe crab genome resulted in the retention of three ey orthologs and two toy orthologs in their genome [ 26 ]. Prior to these findings, an expression study in the Atlantic horseshoe crab Limulus polyphemus showed that a toy ortholog was not expressed in any of the eye anlagen during embryogenesis [ 27 ]. This discovery was surprising, as it was the first to suggest that a Pax6 ortholog does not contribute to eye development in a chelicerate species. This unexpected revelation was further complicated by studies into the utilization of Pax6 orthologs in the likely sister-clade to Xiphosura, the arachnids. Within arachnids, the expression of ey and toy has been studied extensively in spiders. In the spider Cupiennius salei , ey (= Cs-pax6a ) is expressed in the developing medial eyes. However, its toy ortholog (= Cs-pax6b ) is only expressed in the anlagen of the optic neuropils that become associated with the medial eyes, and not the eyes themselves [ 28 ]. In the spider Parasteatoda tepidariorum , neither its ey (= Pt-pax6.1 ) nor its toy (= Pt-pax6.2 ) orthologs are expressed in any of the developing eyes but are rather expressed in the developing neural tissue that is adjacent to the anlagen of the anterior medial eyes [ 29 , 30 ]. A recent survey of eye development genes across seven spider species also showed no evidence of Pax6 gene input into the development of their eyes [ 31 ]. Another study also showed Pax6 expression in the developing head region of the opilionid (daddy-longlegs) Phalangium opilio . This work revealed that its Pax6 paralogs are expressed in parts of the brain and its eyes. One paralog, Po-Pax6a , is expressed in the lentigenic layer of the eye at later embryonic stages, and both of its Pax6 paralogs are mainly expressed in the neural tissue of the eye folds [ 32 ]. To explain the unexpected expression patterns of Pax6 genes in spiders, it was hypothesized that the role of ey and toy in eye development may occur much earlier. This hypothesis suggests that early Pax6 expression specifies photoreceptor precursor cells prior to their integration into the eye tissue at later stages [ 29 , 33 , 34 ]. Support for this hypothesis comes from early Pax6 expression in the anterior rim of the ocular segments of the spider P. tepidariorum . In this spider, there were stripes of Pax6 gene expression in the rim of the ocular region of early germ band stage embryos [ 34 ]. The expression of these two genes is associated with stripes of orthodenticle gene expression [ 34 ], a gene that is associated with early head development in a range of arthropod taxa (see [ 35 ] for review and arguments therein). These data are congruent with an alternative hypothesis for the ancestral use of Pax6 genes in chelicerates, i.e. , that Pax6 genes ancestrally patterned components of the ocular region of the head. The ocular region (note that this is also referred to as the “ocular segment”, however the segmental composition of this region is currently debated [ 35 ]) is the anterior-most body region along the antero-posterior axis of arthropods. During arthropod embryogenesis, the ocular region gives rise to the protocerebral portion of the brain, which includes the optic neuropils (see [ 36 ]). Outside of chelicerates, there is additional evidence that arthropod Pax6 genes operate in patterning the ocular region. For example, in T. castaneum , both ey and toy are expressed in the ocular region, and act redundantly to specify the head lobes, or the bilateral outgrowths of the ocular segment that give rise to, among other structures, the larval eyes [ 37 ]. To distinguish what ancestral roles Pax6 genes had in arachnid embryonic development, we followed the expression of these genes in the mite Archegozetes longisetosus . A. longisetosus is a member of the mega-diverse arachnid clade Acariformes, of which no studies into the expression or role of Pax6 genes have so far been conducted. Furthermore, A. longisetosus has secondarily lost its eyes, a condition that has convergently occurred multiple times within Acariformes (see [ 38 ]). Paradoxically, a recent genome sequence revealed that A. longisetosus retains orthologs of genes commonly used in arthropod eye development, collectively called the Retinal Determination Gene Network (RDGN), including both Pax6 gene paralogs, ey and toy [ 38 ]. By leveraging these aspects of A. longisetosus , we reasoned that, if ancestrally Pax6 genes were used in the patterning of the ocular region rather than in the early establishment of eye components, we should see comparable early and late expression patterns of these genes between arachnids that have eyes and A. longisetosus . Alternatively, if Pax6 genes were ancestrally used in the development of arachnid eyes, we would see greatly dissimilar early and late Pax6 gene expression patterns in comparison to other studied chelicerates. By following the expression patterns of Al-ey and Al-toy , we conclude that ey likely participated in the establishment of brain compartments, specifically the optic vesicles and the mushroom bodies of the protocerebrum. Furthermore, we show that the expression dynamics of Al-toy are consistent with a role in establishing the prosomal shield, a conserved arachnid structure that migrates late in development to cover the brain. Because testing these hypotheses hinges upon A. longisetosus truly lacking vestigial or embryonic eyes, we also visualized the expression patterns of orthologs of the RDGN gene components. These results show a lack of RDGN expression in any tissue associated with eyes, which supports the hypothesis that A. longisetosus lack vestigial eyes. We also followed the expression of the conserved head patterning gene orthodenticle in A. longisetosus simultaneously with Pax6 expression. This provided further support for the role of Pax6 genes in the development of the early A. longisetosus head/anterior region. Taken together, our results support the hypothesis that Pax6 genes were likely not required for the development of the ancestral arachnid eye, but rather the morphogenesis of the arachnid protocerebrum. Methods Animal husbandry, embryo collection, and embryo fixation Mites were reared on a plaster-of-Paris/charcoal substrate in plastic jars to maintain appropriate humidity. Mites were kept in these jars in an incubator at 25°C with wood chips to promote oviposition. Mites were fed with brewer’s yeast daily. Mite embryos were collected and fixed in the same manner described in[ 39 ]. Detailed protocols are available from AAB. Gene identification and bioinformatic analyses The A. longisetosus orthologs of wingless, peropsin, rhodopsin, eyes absent, Six3, sine oculis, and atonal were identified previously in [ 38 ]. The ortholog of orthodenticle was identified in [ 40 ], and dachshund in [ 41 ]. To identify the potential A. longisetosus orthologs of ey, toy, beta-arrestin, and myosin-III , the D. melanogaster orthologs of each gene were used as queries for in a tBLASTn screen of the A. longisetosus transcriptome [ 38 ]. The resulting top hits were transcribed and subsequently aligned with selected metazoan protein sequences using MUSCLE with eight iterations [ 42 ]. These alignments were then used with PhyML [ 43 ] and the Smart Model Selection (SMS) tool [ 44 ] to construct phylogenetic trees. Branch support for these trees were also calculated using the approximate likelihood-ratio test (SH- like) [ 45 ] All trees were then edited to make publication-quality images using FigTree (v1.4.3). All phylogenetic statistics are reported in Table S1. Hybridization chain reactions and imaging For all single and double hybridization chain reactions (HCRs), we followed the protocol developed by [ 46 ]. Probes specific to each mRNA were developed using the HCR 3.0 Probe Maker software [ 47 ]. The mRNA sequences used for probe production were from the transcriptome assembled in [ 38 ]. The identifiers of all transcripts used to design probes, as well as their associated HCR amplifiers, can be found in Table S2. All resulting probes were ordered as oPools from Integrated DNA Technologies at a scale of 50 pmol per oligo. The probe sequences that were used in this study are listed in Tables S3-S17. If a transcript was too small to make the recommended 20 pairs of probes ( i.e., Al-arrestin-2 and Al-peropsin ), we increased the probe concentration two-fold as recommended by [ 46 ]. Control HCRs were performed in parallel but lacked the addition of DNA probes. All HCR buffers and HCR amplifiers were purchased from Molecular Instruments. The amplifier fluorophores were also ordered from Molecular Instruments, and included fluorophores 594, 514, and 647 for use with amplifiers B1, B2, and B3, respectively. All HCR imaging was done on a Zeiss LSM 880 at Lehigh University, Bethlehem, PA. All images were processed in FIJI (v.2.9.0/1.53t), and all figures were assembled using Adobe Illustrator CS6. Results Development and compartmentalization of the A. longisetosus brain In an effort both to establish a basis for comparable gene expression patterns between A. longisetosus and other study arachnids, and to determine if any vestiges of eye development are retained during A. longisetosus embryogenesis, we followed the embryonic development of the A. longisetosus brain. It is important to note that, unlike most emerging arthropod systems, A. longisetosus adults lay eggs at mixed developmental stages. This is because its oviducts serve as brood chambers, and therefore its clutches of eggs often contain embryos at different stages of development [ 48 ]. Consequently, the traditional “hours after egg laying” criterion cannot be used for this species. Instead, we use morphology to establish brain/head development-specific stages for the remainder of this paper, e.g. “ B rain- D evelopment S tage-1 (=BDS-1).” The arthropod brain is generally comprised of three cephalic domains. These domains, from anterior to posterior, are the proso-, proto-, and deutocerebrum [ 35 ] (however, see [ 49 ] for an alternate view). It is the region anterior to the deutocerebrum that the arthropod visual system develops from, and thus this region is often described in totality as the “ocular segment.” The “ocular segment” of chelicerates is also sometimes described as the “pre-cheliceral region” due to its location anterior to the cheliceral/deutocerebral segment. For consistency, we use the term “pre-cheliceral region” to describe the area anterior to the deutocerebral region, i.e. , the cheliceral segment, for the remainder of the paper. As is typical of arthropod brain development, the first stage of brain morphogenesis begins with the pre-cheliceral region of A. longisetosus bifurcating into two lateral lobes, often called the “optic lobes” during BDS-1 ( Fig 1A -A2). At BDS-2 ( Fig. 1B -B2), we observed the appearance of “pits” in the pre- cheliceral region ( Fig. 1B 2, arrowheads). We take these “pits” to be invaginating neural precursor cells, based on their similarity to structures found in spider head development [ 28 , 50 – 53 ]. This stage is also characterized by medial boundaries forming around each of the optic lobes, resulting in an antero- posteriorly oriented “groove” between them ( Fig. 1B 2, dotted line demarks the boundaries of this structure). Also during BDS-2, we observed a pair of anterior-medial grooves, as well as two lateral grooves on each optic lobe. Based on comparative data from the spiders Cupiennius salei [ 28 , 50 , 54 ] and Parasteatoda tepidariorum [ 30 , 51 ], and the opilionid Phalangium opilio [ 32 , 55 ], we take the two lateral grooves to be the lateral furrows, and the two anterior grooves to be the anterior furrows ( Fig. 1C -C2). Download figure Open in new tab Fig. 1 The development and compartmentalization of the A. longisetosus pre-cheliceral region . A An embryo at B rain D evelopment S tage 1 (BDS-1). A2 The same embryo shown in A , showing the paired optic lobes (OL) of the pre-cheliceral region. The dotted line demarks the boundary between the deuterocerebral region and the pre-cheliceral region. B An embryo at stage BDS-2. B2 shows the same embryo in B . The arrowheads point to “pits” in the presumptive neural tissue that are likely neural- precursor cells. Paired lateral furrows (LF) and anterior furrows (AF) are present at this stage, as well as a “groove” medially separating the optic lobes (dotted line). C An embryo at stage BDS-3. C3 A closer image of the same embryo shown in C , showing the presence of the anterior and lateral furrows, as well as the newly formed stomodaeal opening (St.). D An embryo at stage BDS-4. D2 A close-up of the head region of the same embryo shown in D . The labral halves have fused at this stage, and the labrum (Lb) has migrated posteriorly. E An embryo at stage BDS-5, and a close-up of this embryo, E2 , showing the continuous opening formed by the fusion of the anterior and lateral furrows. The arrowheads mark a continuum between the lateral and anterior furrows, and the dotted line represents the anterior boundary of the embryo that is out of focus. Also at this stage, the medial subdivisions (MS) have begun to send out extensions that will eventually subdivide these continuous tubes. F An embryo at stage BDS-6, and F2 a close-up of the head region of the same embryo. At this stage, the projections of the medial subdivisions have expanded to almost make contact with the lateral edges of the continuous tube of the lateral and anterior furrows. G An embryo at stage BDS-7. G2 shows a ventral confocal 3D projection of this embryo. At this stage, the two halves of the prosomal shield (PS) have migrated ventrally and posteriorly to cover the developing brain region. The arrows show the movement of the prosomal shield halves. G3 A confocal image of the same embryo in G-G2 showing a more dorsal Z-slice. Embryos at this stage have subdivided the continuous tubes of the lateral and anterior furrows into the paired arcuate bodies (AB), mushroom bodies (MB), and optic vesicles (OV). H A ventral image of the prelarval stage of A. longisetosus . In H2 , the embryo has been rotated so that its dorsum is shown. The brain region has migrated dorsally at this stage, and appears “upside-down” in comparison to the prior stages. The dotted line demarks the fused arcuate body. I Schema outlining the deduced morphogenetic events shown in A- H2. See text for details. All embryos shown in A-H2 are oriented with the anterior of the embryo directed towards the top of the page, however please note that in prelarvae, the brain region is inverted as it folds over the head region. I Schematics of the aforementioned stages. Note that in the prelarval stage, the brain is “upside-down” in relation to the other stages. Abbreviations are Ch, chelicerae; L1-L3, walking legs 1-3, respectively; Pp, pedipalps. Scale bars in A, B, C, D, E, F, G, H and H2 represent 50 µm. The scale bars in the remaining images represent 20 µm. All embryos shown in the confocal images were stained with DAPI. BDS-3 is characterized by the deepening of the anterior and lateral furrows into the embryo. The antero-posterior groove that subdivides each ocular lobe was maintained at this stage, and a clear stomodeal opening was present at its posterior terminus ( Fig. 1C -C2). BDS-4 follows the fusion of the labral halves, and their subsequent posterior migration (see [ 56 ] for details). At this stage, the anterior furrows were more pronounced, and visibly distinct from the lateral furrows ( Fig. 1D -D2). At BDS-5, the anterior furrows expanded posterior-laterally and grew in size. Also at this stage, the anterior furrows made a continuous opening with their adjacent lateral furrows ( Fig. 1E 2, arrowheads mark a continuum between the lateral and anterior furrows). This is noteworthy, as a similar morphogenetic movement has not been observed in either opilionids or spiders, which maintain distinct lateral and anterior furrows during brain morphogenesis [ 28 , 30 , 32 , 50 , 51 , 55 ]. Also at BDS-5, we observed a field of cells that were growing towards the middle of each anterior furrow ( Fig. 1E 2). In the spiders P. tepidariorum and C. salei , two fields of neural precursor cells, called the medial subdivisions, also appear on each of the optic lobes and partially cover the anterior furrow [ 28 , 50 , 51 , 54 ]. Due to the similarity of these structures to those in the aforementioned spiders, we take these structures to be homologous to the medial subdivisions ( Fig. 1E 2). This is notable, as it has been proposed that the medial subdivisions give rise to the optic neuropils of the median eyes of C. salei (see Discussion in [ 28 ]). At stage BSD-6, the lateral furrows have closed and have distinct boundaries ( Fig. 1F -F2). The medial subdivisions extended anteriorly to contact the opposite sides of each anterior furrow ( Fig. 1F 2). In the spiders P. tepidariorum and C. salei , a second group of neural precursor cells, called the lateral subdivisions, migrate and partially cover the lateral furrows [ 28 , 50 , 51 ]. We did not observe any comparable morphogenetic movements or structures in A. longisetosus . This is also of interest, as these lateral furrows are presumed to form the lateral eye optic neuropils in these spiders[ 28 ]. In a number of arachnids, the non-neurogenic ectoderm at the anterior rim of the head lobes migrates to cover the developing brain. This “hood-like” structure is often called the prosomal shield [ 28 , 30 , 32 , 50 , 51 , 54 , 55 ]. We observed the same structure form in A. longisetosus , and its downward migration was mostly complete by BDS-7 ( Fig. 1G -G2). Also at BDS-7, we observed that the medial subdivisions divided the anterior furrow into two distinct compartments. Based on observations of comparable stages of spider brain development, we take the anterior-most compartments to be the arcuate bodies (=central complexes) and the immediately posterior compartments to be the mushroom bodies (see [ 54 ], their Fig.4 compared to our Fig. 1G 3). By using comparable stages in spiders, we also deduce that the lateral furrow forms the homolog to the spider optic vesicle (see[ 54 ], their Fig. 4 compared to our Fig. 1G 3). We use these terms to describe these structures at this stage and in subsequent stages. In the following prelarval stage, the anterior of the brain “flipped” resulting in the anterior of the brain pointing posteriorly. This movement appears to be conserved in arachnids, as this is also seen in spiders and opilionids, as well as in an extinct chelicerate [ 8 , 32 , 54 , 57 ]. In Fig. 1H 2, we demarcate the structures by dotted lines that we understand to be the arcuate bodies to highlight this morphological movement. Taken together, the development of the A. longisetosus brain is similar to that of spiders and opilionids, albeit with lineage-specific differences. All groups develop both anterior and lateral furrows. However, these combine to form a continuous “tube” during mid-embryogenesis in A. longisetosus . These tubes are then subdivided to form specific compartments in the brain, i.e. , the optic vesicles, mushroom bodies, and the arcuate bodies. Surprisingly, the presence of structures associated with eye development in spiders are also present in A. longisetosus , despite their lack of eyes. These structures include the optic vesicles, as well as the formation of medial subdivisions that have been suggested to be the precursors of the optic neuropils of the median eyes [ 28 ]. Developmental gene expression shows no evidence of vestigial eyes in A. longisetosus Because of the similarities of A. longisetosus brain development to that of spiders, we asked if gene expression patterns could be used to detect embryonic eye primordia that may subsequently degenerate, in a similar manner that was used to discover the vestigial lateral eyes of P. opilio [ 32 ]. In all studied chelicerates, the eye anlagen originate from the non-neural ectoderm lining the peripheral rims of the head lobes [ 28 , 30 , 32 , 33 ]. As the prosomal shield migrates over the neural ectoderm, the eye anlagen presumably locate and connect to their associated optic neuropils (reviewed in [ 33 ]). Prior to the migration of the prosomal shield, the eye precursor cells can be identified by their expression of RDGN genes in the lateral, non-neural margins of the head lobes. To detect potential vestigial eye anlagen in A. longisetosus , we performed HCRs targeting its orthologs of the RDGN genes eyes-absent ( Al-eya ), sine-oculis ( Al-so ), dachshund ( Al-dac ), six3 ( Al-six3 ), and atonal ( Al-ato ). We also targeted the ortholog of all-trans retinal peropsin ( Al-peropsin ), which has been used to detect embryonic eyes in the spider P. tepidariorum [ 30 ]as well as in the opilionid P. opilio [ 32 ]. Aside from peropsin, the only remaining opsin gene that has been retained in the A. longisetosus genome is rhodopsin [ 38 ]. We therefore also used its potential expression patterns to detect possible vestigial embryonic eyes. Lastly, beta-arrestins are also important for arthropod photoreception [ 58 ], as is the myosin-III gene. Both genes have been used to mark the embryonic eyes of chelicerates [ 32 , 59 ]. We therefore targeted these genes to also identify potential vestigial embryonic eyes in A. longisetosus . It is important to note that many of the RDGN component genes are expressed simultaneously in the non-neural and the neural ectoderm in the margins of the head lobes ( e.g. , [ 32 ]). To date, there is no clear marker gene that can be used to distinguish the non-neural and neural ectoderm of the pre- cheliceral region. We were thus careful to make these distinctions to disambiguate the potential misidentification of vestigial eyes. Briefly, if gene expression was observed in the outer-most rims of the head lobes, we took this as evidence for that gene’s potential role in eye development rather that brain development. Below, we describe the resulting expression patterns of these aforementioned orthologs. Al-eyes absent expression In Drosophila , the gene eyes absent ( eya ) encodes a protein tyrosine phosphatase that acts as a transcriptional co-activator with the products of sine oculis and dachshund within the RDGN [ 60 ]. The use of eya in patterning the eyes of arthropods appears to be ancestral, as exemplified by its expression in chelicerate eyes, i.e. , spiders and opilionids [ 28 , 30 – 32 ]. Unlike many of the RDGN genes, only a singleton copy of eya has been recovered in all surveyed spider taxa [ 31 ]. In the developing head of the spiders P. tepidariorum and C. salei , eya was expressed in the non-neural margins of the head lobes of early embryos. In each lobe, eya was detected in two separate domains, i.e. , an anterior and posterior domain. As the prosomal shields migrated, eya expression was enriched in the edges of the prosomal shield, and these cells were taken to be the primordia of the eyes. Upon the completion of prosomal shield migration, eya was expressed in all eye types of P. tepidariorum [ 30 ]. However, in C. salei , eya was only expressed in the secondary eyes ( i.e., all eye types to the exclusion of the anterior median eyes) [ 28 ]. This may be specific to C. salei , as a phylogenetic survey of a wide range of spider species showed that their eya orthologs are also expressed in all eye subtypes [ 31 ]. Outside of spiders, the only other chelicerate in which the embryonic expression of eya was surveyed was in the opilionid P. opilio. Like spiders, the earliest expression pattern of Po-eya was in the lateral margins of the head lobes. Po-eya was later expressed in the developing rims of the lateral furrows, as well as in the anterior furrows. This observation is interesting, as it was reported that there was no eya expression in the lateral furrows in the spider P. tepidariorum [ 30 ] nor in C. salei [ 28 ]. With the aid of high-resolution imaging of Po-eya expression, the authors were able to distinguish between non- neural and neural Po-eya expression. Po-eya expression appeared to be expressed in both the anlagen of the median and vestigial lateral eyes in the non-neural ectoderm as well as in the adjacent neural ectoderm. As the prosomal shield migrated, Po-eya expression was observed in the developing median and vestigial lateral eyes [ 32 ]. Outside of eye and brain development, the aforementioned studies on spiders and opilionids showed eya expression in the labrum, stomodaeum, segmental clusters of the ventral central nervous system, and also in what appears to be the mesoderm of the appendages. Taken together, the data from spiders and an opilionid suggest that eya expression can be used to detect both embryonic and potentially vestigial eyes. Using HCR, we first observed the expression of Al-eyes absent ( Al-eya ) at the early stages of prosomal segmentation, when the cheliceral, pedipalpal, and the first two walking leg segments had formed ( Fig. 2A -A3). At this stage, Al-eya expression was in each of the developing prosomal segments, as well as in a lateral domain in the pre-cheliceral region (arrowhead in Fig. 2A 2). This lateral expression domain is similar to the triangular expression domains of Al-ey at this stage (see below) and may thus be involved in the formation of the same structure ( i.e. , the lateral furrows; discussed below). We also observed Al-eya in an anterior ectodermal domain, which likely demarks the “groove” that separates the head lobes at BDS-2 (see Fig. 1B -B2). Interestingly, we also observed Al-eya expression in the presumptive mesoderm of the developing prosomal segments, as well as in the growth zone of the opisthosoma ( Fig. 2A 3). This is markedly different from the ectodermal expression of Al-eya noted above. Download figure Open in new tab Fig. 2 A l-eyes absent (Al-eya), Al-sine oculis (Al-so), and Al-dachshund (Al-dac) expression. A-A3 Confocal images of Al-ato expression in an early segmental-stage embryo. A and A2 are maximum projections, whereas A3 is a Z-slice. The asterisks in A2 and A3 demark expression in the medial groove, whereas the arrowhead in A2 points to expression in the lateral pre-cheliceral region. B-B3 Confocal images of Al-eya expression in an embryo at BDS-1. The asterisks demark expression along the lines of the median groove, whereas the arrowheads demark thin lines of expression that appear at this stage. C- C2 Al-eya expression in an embryo at BDS-2. The dotted lines in the DAPI image of C delimit the median boundaries of the lateral furrows where Al-eya expression appears at their posterior regions (arrowheads). D-D3 Al-eya expression in an embryo at BDS-3. The DAPI image in D delimit the median boundaries of the lateral furrows. The asterisks demark Al-eya expression in the anterior furrows, and the arrowheads demark Al-eya expression in the medial lips of the lateral furrows. E-E3 Al-eya expression in an embryo at BDS-4. The dotted line in E2 demarks the boundary between the neural and non-neural head ectoderm. F-F3 Al-eya expression in an embryo at BDS-7. The arrowheads in F3 demark Al-eya expression underneath the prosomal shield in the mushroom bodies. G-G3 Al-so expression in an embryo at BDS-2. Magenta-outlined arrowheads in G3 demark expression in the medial grooves, and the white arrowheads demark expression in two lateral domains of the pre-cheliceral region. H-H3 Al-so expression in an embryo at BDS-3. The dotted line in H2 delimits the neural/non-neural ectodermal boundary, and the arrowheads demark expression in the lateral neural regions of the head lobes. I-I2 Al- so expression in an embryo at BDS-7. At this stage, Al-so expression is not present in the developing head and brain. J-J3 Al-dac expression at BDS-5, showing no expression in the head/brain region. K-K3 Al-dac expression in the brain of a prelarva. All abbreviations are the same as in other figures. All scale bars represent 50 µm. These expression patterns largely continued into BDS-1 ( Fig. 2B -B3), however Al-eya was also expressed in two lateral expression domains that connected the bilateral triangular domains in the pre- cheliceral region (arrowheads in Fig. 2B 2-B3) to the medial groove-lining domains (asterisks in Fig. 2B 2- B3). We take these medial groove-lining domains to be the same ectodermal domain observed in Fig. 2A 2 (asterisk). At approximately BDS-2 ( Fig. 2C -C3), the bilateral domains of Al-eya expression moved anteriorly. The previously described Al-eya expression domains were retained at this stage, however two new bilateral Al-eya expression domains appeared. These domains were restricted to the posterior margins of the lateral furrows (arrowheads in Fig. 2C 2-C3). Al-eya expression was broadly retained in these domains at BDS-3. Intriguingly, however, the two domains of Al-eya expression merged into one, lateral domain on each of the head lobes ( Fig. 2D -D3). In addition, two domains of Al-eya expression appeared on the medial “lip” each lateral furrow (arrowheads mark one pair in Fig. 2D 2). Al-eya was also expressed in the anterior furrows at this stage (asterisks in Fig. 2D 2). Embryos at BDS-4 displayed comparable Al-eya expression patterns to the previous stage ( Fig. 2E -E3). However, it was at this stage that we were able to observe that Al-eya expression was confined to the neural ectoderm of the pre- cheliceral region (dotted outline in Fig. 2E 2 demarks the presumed boundary between the neural and non-neural ectoderm). Because the eye anlagen in spiders and opilionids are derived from the non-neural ectoderm, these data support the hypothesis that A. longisetosus does not develop vestigial eyes. Additional data supporting this hypothesis came from the expression of Al-eya in BDS-7 embryos ( Fig. 2F -F3). At focal planes underneath the prosomal shield, we identified Al-eya expression in the lateral margins of the mushroom bodies (arrowheads in Figs. 2F 2-F3), as well as in the arcuate bodies (asterisks in Figs. 2F 2-F3). In these older embryos we did not detect any Al-eya expression in the non-neural prosomal shield that suggested either rudimentary or vestigial eyes. Al-sine oculis expression In Drosophila , the Six-family gene sine oculis ( so ) encodes a transcription factor that interacts with several members of the RDGN and has been shown to directly form a protein complex with Eya (reviewed in [ 61 ]). Like many of the genes in the RDGN, all surveyed spider species retain two paralogs of so in their genomes [ 28 , 31 ], however the expression of these paralogs in the development of eyes varies across spiders. In all of the spider taxa investigated in [ 31 ], so1 was shown to be expressed in all of the eye subtypes. A notable exception to this was the usage of so1 in C. salei (= Cs-six1a ), which was expressed in all of the eye subtypes to the exclusion of the anterior-median eyes [ 28 ]. The expression of the second paralog, so2 , was present in all of the eye subtypes in the majority of the species examined by [ 31 ]. However, in the two species of spiders that belong to the clade Synspermiata, so2 expression was not expressed in any of the eye anlagen. The variability of so2 usage in eye development was also demonstrated by its expression in only the anterior-lateral eyes of P. tepidariorum . Lastly, the C. salei so2 ortholog (= Cs-six1b ) was expressed in all of the eye subtypes, to the exclusion of the anterior-lateral eyes [ 28 ]. Outside of spiders, the single copy of so in the opilionid P. opilio was expressed in the medial eyes as well as in the vestigial lateral eyes [ 32 ]. Like P. opilio , A. longisetosus has only a single-copy ortholog of sine oculis ( Al-so ). We first detected its expression in BDS-2 embryos ( Fig. 2G -G3). Al-so was expressed in the presumptive mesoderm of the prosomal appendages, in a similar manner to Al-eya expression (see above). BDS-2 embryos showed Al-so expression in the medial grooves in a similar manner to Al-eya ( Fig. 2G 3, asterisks). Al-so expression was also detected in bilateral domains adjacent to the medial-groove expression domains (magenta-outlined arrowheads in Fig. G3) and also in two lateral domains (arrowheads in Fig. 2G 3). At roughly BDS-3, Al-so expression remained similar to its expression at BDS-2. We did observe, however, that the Al-so expression domains at the lateral edges observed at BDS-2 expanded to line the edge of the neural ectoderm of each head lobe, as well as the lateral lip of each lateral furrow. We did not observe Al-so expression in the presumptive non-neural ectoderm that was suggestive of any eye primordia ( Fig. 2H -H3). As development progressed into BDS-7, the expression of Al-so continued its expression in the mesoderm of the appendages ( Fig. 2I -I3). However, in the pre- cheliceral region expression of Al-so (?) widely disappeared ( Fig. 2I -I3). In summation, these data support the hypothesis that A. longisetosus embryos do not develop vestigial eyes during embryogenesis. Al-dachshund expression In Drosophila , Dm-dachshund ( Dm-dac ) interacts with other components of the RDGN, and Dm-dac mutants lack eyes [ 62 , 63 ]. As with most of the RDGN genes, spiders have two paralogs of dac . The expression of these paralogs in spider eyes seems to be clade specific; however, in each species surveyed, at least one dac paralog is expressed in an embryonic eye [ 31 ]. In P. opilio , Po-dac is expressed in the medial eyes as well as the vestigial lateral eyes. Furthermore, RNAi targeting Po-dac results in the absence of the lateral eyes, without affecting the median eyes [ 32 ]. In A. longisetosus , we performed HCRs targeting its single-copy ortholog ( Al-dac ). As previously reported, Al-dac is expressed in the medial domains of the extending embryonic limbs [ 41 ]. However, we did not observe Al-dac expression in the pre-cheliceral region at any embryonic stage (an example is shown in a BDS-5 embryo in Fig. 2J -J3; earlier embryos not shown), consistent with [ 41 ]. We did, however, observe post-embryonic expression of Al-dac in the brains of prelarvae ( Fig. 2K -K3). In these prelarvae, Al-dac was detected in regions we take to be the mushroom bodies, as well as in three small domains in the arcuate bodies ( Fig. 2K -K3). Taken together, the lack of Al-dac expression in either the neural or non-neural ectoderm of the developing pre-cheliceral region supports the hypothesis that A. longisetosus does not develop vestigial eyes. Al-Six3 expression The six-family transcription factor Six3/Optix has a highly conserved role in demarking the anterior-most region of animal embryos [ 64 ]. In arthropods, this region has been proposed to be the prosocerebrum of the brain [ 35 ]. In addition to its role of anterior head regionalization, Six3 is also involved in the formation of animal eyes ( e.g. , [ 65 ]). In Drosophila , Six3 is required for the progression of the morphogenetic furrow of the developing retinas in the eye/antennal imaginal discs [ 66 ]. Spiders have two paralogs of Six3 , and in most spider species, one or both paralogs are expressed in at least one of the eye anlagen, except for the eyes of the spiders A. geniculata and P. phalangioides [ 31 ]. In the daddy-longlegs P. opilio, Six3 is expressed in the developing median eyes [ 32 ]. In A. longisetosus , we observed Al-Six3 expression in an early germ band stage ( Fig. 3A -A3). At this stage, Al-Six3 was detected in the anterior-most region of the embryo, consistent with observations in other animal taxa [ 64 ]. At approximately BDS-3, Al-Six3 was observed in a large anterior domain that spanned and connected the anterior furrows ( Fig. 3B -B3). We also detected Al-Six3 expression in two domains within the neuroectoderm that we take to form the mushroom bodies (arrowheads in Fig. 3B 2- B3) as well as in two domains that were in the region of the lateral furrows (arrowheads in Fig. 3B 2-B3). As embryogenesis progressed to BDS-4, Al-Six3 expression was retained in the neuroectodermal anterior furrows ( Fig. 3C -C3). The mushroom-body associated expression was also retained ( Fig. 3C 2- C3, arrowheads). The expression of Al-Six3 in the lateral furrows of this stage was restricted to the lateral edges of each furrow ( Fig. 3C 2-C3, asterisks). Al-Six3 was also present in the labrum, as well as in the extending pedipalpal lobes ( Fig. 3C 2; note that these are underneath the chelicerae at this stage; see [ 41 , 56 ]). At approximately BDS-6, Al-Six3 expression remained in the neuroectoderm of the pre-cheliceral region ( Fig. 3D -D3). However, the expression of Al-Six3 in the lateral furrows was markedly reduced and appeared to be restricted to their center ( Fig. 3D 2-D3, asterisks). Additional differences in Al-Six3 expression from the previous stages include its expression in the distal tips of the first and second pairs of walking legs ( Fig 3D 2, arrowheads), as well as stronger expression in the extended pedipalpal lobes. Download figure Open in new tab Fig. 3 A l-Six3 and Al-atonal (Al-ato) expression. A-A3 Al-Six3 expression in an early germ band embryo. B-B3 Al-Six3 expression at BDS-3. The arrowheads in B2-B3 demark expression in the incipient mushroom bodies. C-C3 Al-Six3 expression at BDS-4. The arrowheads in C2-C3 demark expression in the incipient mushroom bodies, and the asterisks demark expression in the lateral furrows. D-D3 Al-Six3 expression at BDS-6. Asterisks demark expression in the lateral furrows, whereas the arrowheads demark expression in the tips of the developing legs. E-E4 Al-Six3 expression at BDS-7. The arrowheads demark potential expression in the synganglion. F-F3 Al-Six3 expression in a prelarva. Note the expression in the synganglion (Sg). F4-F6 Al-Six3 expression in the same prelarva, rotated to the dorsum to visualize expression in the brain. G-G3 Al-ato expression in an embryo at BDS-2. The arrowheads in G2 demark expression in the lateral furrows. G4-G6 An HCR of Al-ato showing lack of expression at BDS-7. All abbreviations are the same as in other figures. All scale bars represent 50 µm. Also, Al-Six3 expression in the mushroom bodies disappeared by this stage. In these aforementioned stages, we did not detect Al-Six3 transcripts in the non-neural head ectoderm. This was made further evident by Al-Six3 expression at BDS-7, at which point the prosomal shield has migrated over the neural ectoderm ( Fig. 3E -E3). At BDS-7, Al-Six3 expression was completely absent in the non-neural prosomal shield ( Fig. 3E 2). However, in Z-stacks deeper into the embryos, Al-Six3 expression was still present in the anterior furrows/ arcuate bodies, as well as in the lateral furrows/optic vesicles ( Fig. 3E 3-E4). We also observed “patches” of Al-Six3 expression in the midline of each embryo at this stage ( Fig. 3E 3-E4; arrowheads) which were absent in control experiments (not shown). In prelarvae, Al-Six3 expression persisted ( Fig. 3F -F6). Al-Six3 expression in prelarvae was observed in two “spots” of expression in each of the walking legs ( Fig. 3F 2-F3; arrowheads demarcate two such spots in a third walking leg). Additionally, Al-Six3 was ubiquitously expressed in a structure whose position and shape suggest that it is the synganglion [ 67 ]. Al-Six3 was also observed in the arcuate bodies at this stage, as well as in punctate domains in the anterior brain ( Fig 3F 4-F6). Taken together, our Al-Six3 expression data also support the hypothesis that A. longisetosus does not have vestigial eyes. Al-atonal (Al-ato) expression In Drosophila , the product of atonal ( Dm-ato ) is activated by the products of Dm-so and Dm-eya to initiate photoreceptor development (reviewed in [ 61 ]. In spiders, the ato1 paralog seems to have a conserved expression domain in the anlagen of all eye subtypes, with the only exception being the spider Segestria senoculata , a member of the Synspermiata [ 31 ]. The same study also provided evidence that supports the hypothesis that the ato2 paralog was ancestrally expressed in the primary eye primordia. We detected the earliest expression of the singleton atonal ortholog in A. longisetosus ( Al-ato ) in BDS-2 embryos ( Fig. 3G -G3). In these embryos, Al-ato was expressed in “clusters” of cells in the developing prosomal appendages, in a similar manner to both paralogs of atonal in the spider P. tepidariorum [ 29 ]. In the pre-cheliceral region, Al-ato expression was observed in two clusters of cells in each optic lobe, in regions straddling each presumptive lateral furrow within the neural ectoderm ( Fig 3G 2, arrowheads). We did not observe any Al-ato expression in the pre-cheliceral region that would be indicative of vestigial eye formation, i.e., in a similar manner to spider ato1 expression in the non-neural ectoderm of the head. In fact, Al-ato expression at this stage was most similar to the expression of the ato2 paralog of the spider P. tepidariorum [ 29 ]. Pt-ato2 was shown to be expressed in the pre-cheliceral region in two neuroectodermal clusters similar to our observations of Al-ato (see Fig 5I in[ 29 ]). We did not observe any changes in Al-ato expression throughout development, and its expression was never subsequently observed in new locations of the pre-cheliceral region (not shown). This trend ended in later BDS-7 embryos, at which all Al-ato expression ceased ( Fig. 3G 4-G6). Because spider ato1 orthologs are expressed in the eye primordia of the prosomal shield at comparable stages [ 31 ], our data provide further evidence that A. longisetosus lack vestigial eyes. Download figure Open in new tab Fig. 4 A l-eyeless ( Al-ey ) expression. A-A4 Confocal images of a double hybridization chain reaction (HCR) in an early, prosomal-segmentation stage embryo targeting both Al-wingless ( Al-wg ) and Al-ey . All images of this embryo are oriented to show its lateral side, and its anterior is directed towards the left of the page. A DAPI nuclear counterstain image of this embryo, showing the location of the pre-cheliceral region (PCR), outlined in a dotted line. A2 Al-wg is expressed in each of the developing prosomal segments at this stage, as well as in the segmental growth zone (GZ) and in a stripe of expression in the pre-cheliceral region (asterisk). A3 Al-ey is expressed in this same embryo in a triangular-shaped domain of expression, and also in paired, clusters of cells in each of the prosomal segments (asterisks). A4 A merged confocal image of this embryo showing Al-wg expression (green) concurrently with Al-ey expression (magenta). Note the co-expression of both of these genes in the pre-cheliceral region (asterisk). B-B6 An embryo at a stage approximately between BDS-1 and BDS-2. B DAPI channel. B2 Al- ey expression in the same embryo, showing the retention of Al-ey expression in triangular domains in the pre-cheliceral region, and also in paired clusters of expression in each of the prosomal segments (asterisks). B4-B6 Confocal images of the same embryo, zoomed in to highlight the morphology of the pre-cheliceral region (B4) DAPI channel. (B5) Al-ey expression in this region. (B6) Both channels merged. C-C6 Confocal images of a single embryo at BDS-2. C Nuclear counterstain with DAPI. C2 Expression of Al-ey in this embryo, showing the retention of Al-ey expression in paired domains of the pre-cheliceral region, and also the paired expression in clusters of each prosomal segment (asterisks). C3 Merged image of both the DAPI counterstain (blue) and Al-ey expression (magenta). C4-C6 Confocal images of this embryo zoomed-in to show the appearance of the anterior and lateral furrows (C4 , AF and LF, respectively). Al-ey expression “clears” from the lateral furrows at this stage (arrowheads, C5 ). C6 Merged confocal image of DAPI (blue) and Al-ey expression (magenta). D-D6 Confocal images of an embryo at stage BDS-3. D Nuclear counterstain with DAPI. D2 Confocal image of Al-ey expression in this embryo, showing the retention of Al-ey expression in the pre-cheliceral region and in the paired clusters of the prosomal segments (asterisks). D3 merged image of DAPI (blue) and Al-ey expression (magenta). D4-D6 Zoomed-in confocal images of this embryo. At this stage, the anterior and lateral furrows are more distinct (D4). D5 Al-ey expression is “ring-like” at this stage, as it surrounds the deepening lateral furrow. Two additional clusters of expression are also present at this stage (arrowheads), just posterior to each anterior furrow. D6 A 3D-projection of this embryo, showing the merged DAPI (blue) and Al-ey (magenta) channels. The embryo has been rotated (see rotated arrow for orientation) to show the absence of Al-ey expression in the lateral furrows. E-E5 Confocal images of a single embryo at stage BDS-4. E DAPI nuclear counterstain. E2 Al-ey expression in this embryo, showing the retention of Al-ey in the pre- cheliceral region and in the paired, segmental clusters of the prosomal segments (asterisks). E3 Merged image of DAPI (blue) and Al-ey expression (magenta). E4 Zoomed-in image of the same embryo showing the DAPI nuclear counterstain of the pre-cheliceral region. E5 Al-ey expression (magenta) merged with the DAPI counterstain (cyan) showing the “opening” of the Al-ey expression domains around the lateral furrows (see text for details). Arrowheads point to the Al-ey expression domains at the posterior of the anterior furrows. Dotted lines in E4-E5 mark the position of the orthogonal slices shown in E6-E8 . E6 Confocal orthogonal slice through the antero-posterior axis of this embryo. E7 Al-ey expression in the same orthogonal slice. E8 Merged image of the DAPI (cyan) and Al-ey (magenta) confocal channels in the same orthogonal slice. F-F5 Confocal images of an embryo at stage BDS-5. F DAPI nuclear counterstain of this embryo. F2 Al-ey expression in this embryo. Note that the paired domains in the prosomal segments are retained at this stage, however only those of the second and third walking legs are visible (asterisks). F3 Merged confocal image of this embryo showing the DAPI counterstain (cyan) and Al-ey expression (magenta). F4 Al-ey expression in a Z-slice towards the dorsum of the same embryo. F5 Merged confocal image of the same embryo, showing the DAPI counterstain (cyan) and Al-ey expression (magenta). In both F4 and F5 , arrowheads point to Al-ey expression in clusters associated with the anterior furrows, and the arrows point to newly-appeared clusters of Al-ey expression posterior to these. Dotted lines in F4-F5 mark the position of the orthogonal slices shown in F6-F8. F6 DAPI image of this orthogonal slice. F7 Al-ey expression in this orthogonal slice, showing the internalization of Al-ey positive cells migrating inward. F8 Merged DAPI and Al-ey channels in this orthogonal slice. G-G6 Confocal images of an embryo at late BDS-6/early BDS-7. G DAPI image of this embryo. G2 Al-ey expression in this embryo. G3 Merged DAPI and Al-ey expression channels in this embryo. Note the appearance of paired clusters of Al-ey expression in the fourth walking leg segment bearing the fourth limb buds (Lb4) and also in paired clusters in the opisthosoma (Op). Furthermore, note the appearance of a new, central cluster of cells expressing Al-ey just above the labrum (arrowheads in G2 and G3 ). G4 DAPI image of this embryo, zoomed-in to show the structure of the pre-cheliceral region at this stage. The arrowhead points to the closing boundary separating the mushroom body from the arcuate body. G5 Merged DAPI (cyan) and Al-ey (magenta) channels, showing expression in this closing boundary (arrowhead), and in the surface of the region above the mushroom body (MB). G6 Dorsal Z-slice of this embryo, showing the internalized Al-ey expression in the pre-cheliceral region (see text for details). The horizontal, dotted line demarks the region of the orthogonal slices shown in G7-G9 . G7-G9 Orthogonal confocal slices through the pre-cheliceral region showing the internalization of Al-ey positive cells. G7 DAPI channel. G8 Al-ey expression. G9 Merged DAPI channel (cyan) and Al-ey channel (magenta). H-H8 Al-ey expression persists in the prelarval stage. H DAPI stain. H2 Al-ey expression. H3 Al-ey expression in the same prelarval. The paired, segmental Al-ey expressing clusters have become more complex. The left-most clusters are highlighted in pink and outlined. H4 Z-slice showing the more dorsal cheliceral expression of Al-ey . H5 Merged DAPI (cyan) and Al-ey expression (magenta) channels. Asterisks denote artefactual cuticle staining. H6-H8 Confocal images of the same embryo, rotated to show the dorsally-migrated brain/pre-cheliceral region at this stage. H6 DAPI stain; the arcuate bodies (AB) are outlined with a dotted line. H7 Al-ey expression in this region, showing its expression in the mushroom bodies (MB) and the arcuate bodies. The left mushroom body is outlined and highlighted in pink. H8 Merged DAPI (cyan) and Al-ey expression (magenta) channels. Scale bars represent 50 µm in all images, except in F6-F8 and G4-G6 , where they represent 20 µm. All other abbreviations are the same as in other figures. Download figure Open in new tab Fig. 5 A l-twin of eyeless ( Al-toy ) is dynamically expressed throughout development. A-A4 An early blastoderm/ prosomal segmentation stage embryo. A DAPI nuclear counterstain of this embryo. A2 Al-wg expression in this embryo, showing its expression in stripes of the first four prosomal segments, in a stripe in the pre-cheliceral region (asterisk), and in the growth zone (GZ). The pre-cheliceral region (PCR) is outlined. A3 Confocal image of Al-toy expression in this embryo. Al-toy is expressed in a broad domain in the cheliceral segment and its extension into the pre-cheliceral region. Al-toy was also weakly expressed in the developing prosomal limb buds. A4 A merged confocal image showing simultaneous Al-wg (green)and Al-toy (magenta) expression. B-B4 A slightly older blastoderm stage embryo. B DAPI counterstain of this embryo. The pre-cheliceral region is outlined in a dotted line. B2 Al-toy expression in a broad anterior domain. B3 Al-ey expression in the same embryo in a triangular domain in the pre- cheliceral region. B4 Al-toy (green) and Al-ey (magenta) expression overlap at this stage. C-C4 Al-toy and Al-ey co-expression at an early, four limb bud-stage embryo. C Nuclear DAPI counterstain of this embryo. The pre-cheliceral region is outlined. C2 Al-toy is expressed in the anterior of each limb bud, and also in boundary-like domain (arrowheads). C3 This Al-toy boundary (green) “outlines” Al-ey (magenta) expression. The dotted-lined box represents the region zoomed into in C4, which shows the mutually- exclusive expression domains of both Pax6 orthologs. The arrowheads demark the Al-toy “boundary” type expression pattern. D-D4 Confocal images of an embryo just prior to BDS-1. D DAPI counterstain of this embryo with the pre-cheliceral region outlined. D2 Al-wg expression. D3 Al-toy expression in the anterior of the prosomal limb buds, and in a thin domain in the pre-cheliceral region (arrowhead). D4 Merged image of Al-wg and Al-toy expression. The arrowhead demarks the thin Al-toy expression domain in the pre-cheliceral region. E-E3 Confocal images of an embryo at stage BDS-1. E DAPI counterstain of this embryo. E2 Al-toy expression in the developing limb buds. Note the absence of expression in the pre- cheliceral region. E3 Co-stain of both Al-toy (green) and Al-ey (magenta) expression. Note that both genes are not co-expressed at this stage. F-F3 Confocal images of an embryo approximately at stage BDS-6. F DAPI counterstain of this embryo. F2 Al-toy is expressed in the lateral non-neural ectoderm, in paired clusters above the labrum (asterisks), and in lines of expression connecting these to the lateral ectoderm (arrows). Arrowheads mark the anterior pre-cheliceral non-neural ectoderm that lacks Al-toy expression. F3 Merged confocal image of the DAPI (cyan) channel and the Al-toy (magenta) channel. G- G5 Confocal images of an embryo approximately at stage BDS-7. G DAPI counterstain of this embryo. The dotted line outlines one half of the prosomal shield (PS). G2 Al-toy is expressed in migrating prosomal shield. G3 Merged confocal images of DAPI (cyan) and Al-toy (magenta). The asterisks denote expression in the Claparede’s organs. G4 DAPI counterstain of a Z-slice deeper into the embryo. G5 Merged confocal images of DAPI (cyan) and Al-toy (magenta) in this Z-slice. Al-toy is notably expressed in the medullae of the mushroom bodies (MB). Embryos in A-D4 are oriented with their anterior poles directed towards the left of the page. Embryos in the remaining images are oriented with their anterior poles directed towards the top of the page. All scale bars represent 50 µm. All other abbreviations are the same as in other figures. The expression of genes associated with downstream eye development does not support the presence of vestigial eyes in A. longisetosus To ensure that we thoroughly tested for the absence of vestigial eyes in A. longisetosus , we targeted genes downstream of the canonical arthropod RDGN genes. In the opilionid P. opilio and the spider P. tepidariorum , the opsin gene peropsin is expressed in true embryonic eyes, as well as embryonic rudiments of the eyes of the opilionid after the completion of the migration of their prosomal shields [ 30 , 32 ]. Therefore, to detect the possible presence of rudimentary eyes in A. longisetosus , we performed HCRs targeting the A. longisetosus ortholog of peropsin ( Al-peropsin ). This experiment showed no expression of Al-peropsin at any stage of embryonic development, in the brain or otherwise (not shown). Alongside Al-peropsin , Al-rhodopsin-7 was identified as the only other opsin retained in the A. longisetosus genome [ 38 ]. rhodopsin-7 genes have been implicated in circadian rhythm photoreception in various taxa (reviewed in [ 68 ]). To test for the possibility that this gene may be expressed in developing, vestigial eyes, we performed HCRs targeting this gene’s expression. We also did not detect any Al- rhodopsin-7 expression at any developmental stage in the pre-cheliceral region (not shown). Beta-arrestins are utilized in photoreceptor specification, and their expression patterns have been recently used to identify the vestigial eyes of P. opilio [ 32 ]. By scouring the published A. longisetosus transcriptome [ 38 ], we identified three candidates for beta-arrestin orthologs. To verify these potential orthologs, a phylogenetic reconstruction was performed, which placed the transcript TRINITY_GG_5120_c51_g1_i7 in the same clade as Dm-Kurtz with high support (aLRT=0.90). The transcript TRINITY_GG_4713_c203_g1_i1 was placed in a clade with Dm-Arrestin-2 (aLRT=0.99), and the transcript TRINITY_GG_3318_c59_g1_i3 was placed in a clade with Dm-Arrestin-1 (aLRT=0.99) (Fig. S2). An HCR targeting all three genes showed no expression at any BDS in the pre-cheliceral region (not shown). The expression of the myosin-III gene (known as ninaC in D. melanogaster ) is expressed in the larval and adult eyes of the horseshoe crab [ 59 ], and the paralog Po-myoIII-2 was used to detect the vestigial eyes of P. opilio [ 32 ]. By using several chelicerate and arthropod NinaC/Myosin-III proteins as queries, and by subsequently performing phylogenetic analyses of our possible hits, we were unable to detect any potential myosin-III orthologs in the A. longisetosus genome or transcriptome. This is interesting, as NinaC proteins in D. melanogaster are expressed in the photoreceptor cells, and their mutational abrogation results in photoreceptor defects [ 69 ]. Therefore, the absence of a myosin-III/ninaC in the eyeless mite is interesting for future studies into how natural selection targets photoreceptor genes in species undergoing eye loss or reduction. In summation, the expression of the RDGN genes as well as the absence of the expression of opsins and beta-arrestin together support the hypothesis that A. longisetosus lack embryonic or vestigial eyes. The identification and phylogenetic assessment of the A. longisetosus Pax6 orthologs We searched the recently published A. longisetosus genome and transcriptome [ 38 ] for putative Pax6 orthologs using tBLASTn [ 70 ] and D. melanogaster Pax6 protein sequences as queries. This approach allowed us to identify two Al-ey transcripts that we refer to as Al-ey.1 (TRINITY_GG_2648_c164_g1_i2) and Al-ey.2 (TRINITY_GG_2648_c164_g1_i1), respectively (Fig. S1A). Both transcripts mapped to the same genomic locus (tig00005243_pilon), suggesting that they are isoforms rather than paralogs. The Al- ey.1 isoform is 2,268 bps long, whereas the slightly longer Al-ey.2 isoform is 2,354 bps long. Although Al- ey.2 is longer, this difference is due to its lengthened 3’UTR. Surprisingly, Al-ey.2 has a shorter coding sequence in comparison to Al-ey.1, i.e. , 670 bps and 1,401 bps, respectively. Therefore, the predicted amino-acid sequence of Al-ey.1 has a 117 C-terminal extension that Al-ey.2 lacks. By using Splign [ 71 ] to align these transcripts to the genome, we found that the differences between these two isoforms are a result of the differential use of exons towards the 5’ end of the gene. The Al-ey.1 isoform is spliced from ten exons, whereas Al-ey.2 is spliced from nine. The 3’ end of Al-ey.1 is constructed from three exons, i.e. , exons 8-10. Al-ey.2, however, lacks exons 8 and 9, and instead uses an alternative exon upstream from exon 10. We denoted this exon as “Exon 8.5” due to its position between exons 8 and 9 (Fig. S1A). Using the above methodology, we also identified a single putative Al-toy transcript (TRINITY_GG_5245_c530_g1_i1; Fig. S1B). This 1,324 bp transcript is comprised of five exons, and maps to a distinct genomic locus (tig00005236_pilon) from that of the Al-ey transcripts. The deduced protein sequences of the Al-ey and Al-toy isoforms were then evaluated with the NCBI Conserved Domain Database tool [ 72 ]. Both isoforms contain the three characteristic domains found in Pax6 proteins; the paired domain, the octapeptide-like domain, and the homeodomain (Fig. S1A- C) (see [ 73 ]). It was recently shown that arthropod Eyeless proteins contain a diagnostic lysine at position 64 in the linker region of the paired domain. This differs from arthropod Toy sequences, which instead have an arginine at this site [ 13 , 33 ]. The amino acid sequences of both Al-ey transcripts contain this diagnostic lysine residue (Fig. S1). Furthermore, the deduced Al-Toy amino acid sequence has the characteristic arginine at that site (Fig. S1C). These results support our hypothesis that the retrieved Pax6 sequences are de facto distinct eyeless and toy orthologs. To further test the hypotheses that these transcripts represent bona fide orthologs of toy and ey , we performed a maximum-likelihood phylogenetic assessment (PhyML) [ 43 ] of the deduced amino acid sequences of these transcripts with those from other metazoan Pax6 orthologs (Fig. S1D). We also used the amino acid sequence of a putative Pax2/5/8 A. longisetosus ortholog (TRINITY_GG_4424_c37_g1_i1) as an outgroup in conjunction with other selected metazoan Pax2/5/8 orthologs. This phylogenetic interrogation placed Al-ey and Al-toy in their predicted clades to the exclusion of Al-Pax2/5 with high support ( i.e. , aLRT scores of 0.98 for Al-ey and 0.96 for Al-toy; Fig. S1A). Taken together, our results support the identity of these transcripts as distinct singleton eyeless and toy orthologs. Al-ey expression We initially performed HCRs simultaneously targeting Al-ey with the segmentation gene Al-wingless ( Al- wg ), which has been used as a marker for early segmentation stages in a variety of arthropods (reviewed in [ 74 ]). Using this methodology, we detected the earliest expression of Al-ey during the prosomal segmentation stage preceding BDS-1, at which the segments of the first four prosomal segments had been delineated ( i.e. , the cheliceral, pedipalpal and first two walking leg segments; Fig. 4A -A4). Al-ey expression was observed in two paired, triangular-shaped domains within the pre-cheliceral region ( Fig. 4A 3). Embryos of this stage have an additional domain of Al-wg expression in the pre-cheliceral region ( Fig. 4A 2, asterisk), which has also been observed in a number of chelicerates during head/brain development [ 75 ]. Our double HCRs of Al-ey and Al-wg revealed that Al-ey expression in the pre- cheliceral region encompasses this Al-wg domain ( Fig. 4A 4). At this early stage, we also observed paired clusters of expression in each of the developing prosomal segments ( Fig. 4A 3-A4; asterisks in 2A3). These expression domains of Al-ey were also observed at a later stage between BDS-1 and BDS-2 ( Fig. 2B -B6) in the nascent tissue of the ventral nerve cord ( Fig. 2B 2, asterisks). This segmental expression of Al-ey was present in all subsequent stages of A. longisetosus embryogenesis, leading up to the prelarval stage. At BDS-2, we observed the “clearing” of Al-ey expression in the newly formed lateral furrows ( Fig. 4C -C6; arrowheads in C5; note the left lateral furrow in C4 is obfuscated by the surrounding tissue). This expression pattern was maintained in BDS-3, with Al-ey expression present in a ring-like domain surrounding the deepening lateral furrows ( Fig. 4D -D6). We also detected additional expression in two clusters of cells just posterior to the anterior furrows ( Fig. 4D 5, arrowheads). At BDS-4, the ring-like domain of expression was transformed into a “C-shaped” expression pattern, leaving Al-ey expression in the ventral-most cells of the periphery of the lateral furrows ( Fig. 4E - E8). This appeared to be the result of the ring of expression observed in BDS-3 “breaking” at its dorsum. Additionally, Al-ey expression was maintained in the previously aforementioned clusters of cells posterior to the anterior furrows ( Fig. 4E 5, arrowheads). In arachnids, the process of brain development involves the internalization of neural tissues ( e.g .,[ 54 ]). Therefore, we also asked if, and when, a comparable internalization occurs in A. longisetosus through visualizing orthogonal views of Al-ey stained embryos. Using this method, we observed that at BDS-4, the bilateral Al-ey positive cells were still embedded within the surrounding tissue ( Fig. 4E 6-8). At late BDS-5, we identified Al-ey expression in clusters of cells on the ventral margins of the region of the “opened” lateral furrow, as well as the insides of this combined lateral and anterior furrows ( Fig. 4F -F8). This suggests that the Al-ey expressing cells in the periphery of the lateral furrow at BDS-3 and 4 internalized as the lateral and anterior furrows form a continuous tube. To explore this further, we also imaged Al-ey expression along an orthogonal plane in a similar region to that shown in Figs. 2E 6-8. This revealed the presence of Al-ey expressing cells surrounded by the edges of the “tube” made by the fusion of the lateral and anterior furrows ( Fig. 4F 6-8). Al-ey expression was also retained in two small domains at the posterior-lateral region of each anterior furrow ( Fig. 4F 4-F5; arrowheads). Additionally, another pair of small domains appeared at this stage posterior to the initial pair ( Fig. 4F 4-F5; arrows). At late BDS-6/early BDS-7, the Al-ey expression patterns became more complex. Al-ey expression was maintained in segmental clusters in the developing central nervous system (CNS), however additional clusters appeared in the fourth walking leg segment as well as in the opisthosoma (see [ 39 , 76 ] for explanations on the divergent posterior segmentation in this species). Within the developing brain region, Al-ey expression was mostly internalized, however some external ( i.e. , surface-level) expression did remain. Of note, a small cluster of Al-ey positive cells emerged in the center of the pre-cheliceral region, just anterior to the labrum ( Fig. 4G 2, G3 and G6, dots). External expression was also found at the closing border of the continuous anterior and lateral furrows at the site the lateral furrows’ anterior border ( Fig. 4G 4-5, arrowheads). Additionally, we observed external Al-ey expression in the region above the incipient mushroom bodies ( Fig. 4G 4-5). Further towards the dorsal Z-axis, we observed Al-ey expression in the posterior-lateral region of the arcuate bodies ( Fig. 4G 6, arrowhead). We take these to be the same Al-ey expressing cells that we observed in a similar location at BDS-5 ( Fig. 4E 5 and F5). The two clusters of Al-ey expression just posterior to these were also retained at this stage ( Fig. 4G 6, arrow; compare to Fig. 4F 4 and F5, arrows). Two additional clusters of Al-ey expression were likewise seen in the medial portion of each arcuate body ( Fig. 4G 6, asterisks). Al-ey positive cells were also found in the newly compartmentalized optic vesicles, as well as inside of the incipient mushroom body region of the closing tubes ( Fig. 4G 6). An orthogonal view along the frontal plane revealed that these Al-ey expressing cell clusters of the mushroom bodies took on a triangular shape, with their vertices pointing ventrally ( Fig. 4G 7-9). This was in contrast to the shape of these clusters in BDS-5, and may indicate a pattern of internal migration via changes in cell shape, ( e.g. , apical constriction) as the continuous lateral furrow/anterior furrow tubes close. We also detected Al-ey expression in the post-embryonic prelarval stage, where its segmental expression was maintained from earlier stages ( Fig. 4H -H8). Al-ey expression in the segmental clusters of the CNS became more complex, likely reflecting the differentiation of the neural cells expressing this gene. It is also important to note that the segmental CNS expression of the cheliceral segment moved to a more dorsal position. Al-ey was also expressed in the dorsal-most region of the brain, occupying the same space as the arcuate body, and also in larger and more anterior paired expression domains, which we take to be the mushroom bodies ( Fig. 4H 6-H8). Taken together, our results indicate that Al-ey expression is expressed in the developing lateral furrows/optic vesicles, the mushroom bodies, and the components of the anterior furrow/arcuate body during embryogenesis. Furthermore, Al-ey expression persists in the CNS of post-embryonic stages. We did not observe Al-ey expression at these stages in the tissues taken to be the precursors to the embryonic eyes, i.e. , the non-neural ectoderm of the head lobes. Al-toy expression As with Al-ey , we initially co-stained embryos for Al-toy expression simultaneously with the segmentation gene Al-wingless ( Al-wg ). We detected the earliest expression patterns of Al-toy during a similar early blastoderm stage as shown in Fig. 4A -A4, i.e. , when the first four prosomal segments had been delineated by Al-wg ( Fig. 5A -A4). Also at this stage, the aforementioned pre-cheliceral stripe of Al-wg was present. Al-toy was expressed in a broad domain that extended from the anterior to the posterior of the embryo. The anterior of this domain appeared to be restricted to the cheliceral segment, and its posterior domain broadened into the pre-cheliceral region where it covered the pre-cheliceral Al-wg stripe ( Fig. 5A 3-A4). Al-toy was also expressed weakly in the developing limb buds ( Fig. 5A 3). In spiders and daddy-longlegs, the Pax6 orthologs are often expressed at the same embryonic stage, and appear to have specific early-stage expression domains [ 28 – 32 , 34 ]. We therefore performed HCRs simultaneously targeting both Al-ey and Al-toy to resolve when both orthologs are potentially co- expressed during pre-cheliceral development. We observed the co-expression of Al-ey and Al-toy in embryos at the same stage shown in Fig. 5A ( Fig. 5B ). At this early germ-band stage, we observed Al-toy expression in the pre-cheliceral region ( Fig. 5B 2), indicative of its slight anterior migration from the cheliceral segment (see Fig. 5A -A4). Low-level Al-toy expression was also present in the developing prosomal appendages. Interestingly, the triangular Al-ey expression domain (first shown in a slightly later stage in Fig. 4A -A4) completely overlapped with this Al-toy domain ( Fig. 5B 3-4B4). This is comparable to the co-expression of both toy and ey in the spider P. tepidariorum during its stage 8.1 and 8.2 [ 34 ], and thus likely represents a conserved feature for these genes in arachnids. Subsequently, when the prosomal limb-buds have begun to grow more distinct, the overlap between the Pax6 genes decreased ( Fig. 5C -C4). The expression of Al-ey was retained in its anterior, triangular domain. However, Al-toy was expressed at the margins of the pre-cheliceral region, i.e. , at the anterior cheliceral segment boundary, and at the lateral boundaries marking the lateral edges of the presumptive ocular lobes ( Fig 5C 2 and C3; arrowheads). Co-staining with Al-ey revealed that this pre- cheliceral expression of Al-toy encompassed the triangular domains of Al-ey expression, with Al-toy expression being “cleared” from the region of Al-ey expression ( Fig. 5C 3 and C4). This is remarkably similar to the expression of toy and ey in the spider P. tepidariorum , specifically at its stage 9.1 [ 34 ]. In this species, ey is also expressed in a triangular domain at its stage 9.1. As in A. longisetosus , its toy ortholog appears to also encompass the ey expression domain [ 29 , 34 ]. The high degree of similarity of these Pax6 expression patterns in A. longisetosus compared to spiders supports our hypothesis that arachnid Pax6 orthologs are acting early in the development of the pre-cheliceral region, and not the development of the eyes. Al-toy expression was also retained in the first four prosomal limb buds; however, it was restricted to the anterior of each developing limb ( Fig. 5C -C3). This observation is interesting, in that toy expression was not observed in the developing limb buds of the spider C. salei [ 28 ]. However, in the spider P. tepidariorum , toy may be expressed in a similar manner to our observations of Al-toy . The previous expression reports of the P. tepidariorum toy ortholog primarily focused on its expression in the developing head. However in [ 29 ], there does appear to be some toy expression in the prosomal appendages (see their Fig. 5J ). Furthermore, a recent study into RDGN genes in the opilionid P. opilio did not reveal toy expression in its appendages either [ 32 ]. Therefore, the role of toy in the developing arachnid limb may be a labile feature of arachnid evolution. Nevertheless, further taxonomic sampling is needed to test these hypotheses. At the onset of BDS-1, Al-toy expression was maintained in the anterior portion of the first four pairs of limb buds, and also appeared in the third walking leg buds ( Fig. 5D -D4). We also observed Al-toy expression in a thin domain trailing from anterior to posterior from the pre-cheliceral region. Based on the position of this trailing domain, as well as its position near the pre-cheliceral Al-wg stripe ( Fig. 5D 2-D4), we take this to be the remnant of Al-toy expression seen in the pre-cheliceral region of the preceding stages ( Fig 5D 3 and 3D4; arrowheads). At late BDS-1, all pre-cheliceral expression of Al-toy was absent, however Al-toy expression in the developing limbs persisted ( Fig. 5E -E4). We noticed that the expression of Al-toy in the developing limbs was reminiscent of the segmental expression of Al-ey in the embryonic midline ( Fig. 3C -C3). We therefore asked to what extent Al-toy was co-expressed with Al-ey at this stage. Our double HCR targeting both transcripts revealed that these paralogs were not segmentally co- expressed, with Al-toy being restricted to the limb buds, and Al-ey being restricted to the CNS ( Fig. 5E 3). Al-toy expression was not detected in subsequent stages (not shown) until approximately BDS-6 ( Fig. 5F -F3). This is notable, as toy orthologs are expressed at comparable stages in spiders [ 28 – 31 ] and opilionids [ 32 ]. At BDS-6, Al-toy expression was absent from the developing appendages . Al-toy transcripts were detected, however, in the lateral boundaries surrounding the optic lobes, as well as in the dorso-lateral margins of the lateral furrows. In spiders, these lateral margins have been described as the non-neural ectoderm that eventually migrates to form the prosomal shield (e.g., [ 28 , 50 , 51 ]). Al-toy was also expressed in a pair of domains just above the labrum ( Fig. 5F 2, asterisks), and also in a line of cells connecting these domains to the lateral optic lobe domains ( Fig. 5F 2, arrows). Al-toy expression was also absent from the anterior-most region of the optic lobes ( Fig. 5F 2, arrowheads). Comparable expression patterns are not seen for toy in either spiders [ 28 – 31 ] or opilionids [ 32 ]. At approximately BDS-7, Al-toy expression was ubiquitous in the prosomal shield ( Fig. 5G -G3), confirming our hypothesis that Al-toy expression in the previous stage (i.e., Fig. 5F -F3) was in the non- neural ectoderm of the head lobes. This is striking, as neither Pax6 ortholog is expressed in the developing prosomal shield in spiders. However, in the opilionid P. opilio , its toy ortholog was expressed in the leading margin of the migrating prosomal shield ([ 32 ]; their Fig. S2). Thus, our observations may represent a lineage-specific use for toy in mites. We additionally observed Al-toy expression in the Claparede’s organs. These organs are modified coxal extensions of the second walking legs that act to aid in water uptake in A. longisetosus larvae (see [ 56 ] for notes on their development). Also, deeper into the embryo, we observed punctate expression of Al - toy in the developing brain, and also its expression in the interior medullae of the mushroom bodies ( Fig. 5G 4-G5). We did not observe Al-toy in any subsequent stages, including the prelarval stage, following BDS-7 (not shown). Together, our observations show a large degree of divergence in Al-toy expression in comparison to toy expression patterns in other studied arachnids. These differences include its expression in the developing prosomal appendages, its absence in the pre-cheliceral region following early stages, its ubiquitous expression in the late prosomal shield, and its expression in an acariform mite-specific appendicular structure, the Claparede’s organ. As was the case with Al-ey expression, we did not observe Al-toy expression in any of the eye-generating tissues. Early co-expression of the Al-Pax6 paralogs and the head-patterning gene orthodenticle The similarities in Pax6 expression between mites and spiders during the development of the pre- cheliceral region, coupled with the absence of vestigial eye primordia in A. longisetosus , suggests that these genes likely did not participate in eye development in the ancestral arachnid. However, they do suggest an ancestral role in the development of the arachnid pre-cheliceral region and its non-eye derivatives. As mentioned previously, in many chelicerates investigated, there appears to be no Pax6 ortholog expression in any of the eye primordia, with the exceptions being eyeless expression in the anterior-median eyes of C. salei [ 28 ], and the late expression of Po-Pax6a in the median eyes of P. opilio after the migration of the prosomal shield [ 32 ]. To explain the widespread absence of Pax6 expression in the embryonic spider eyes, or their associated neuropils, it was hypothesized that the cellular precursors to eye primordia or photoreceptors may be specified or triggered by the early expression of Pax6 genes prior to their differentiation in later developmental stages. Another proposed explanation is that the Pax6 genes play no role in spider eye development, but they instead play a role in the patterning other aspects of the pre-cheliceral region [ 29 , 31 , 33 ]. In a wide array of arthropod exemplars, orthodenticle orthologs are co-expressed with Pax6 genes in the protocerebral region of the brain (see [ 35 ] and arguments therein for a summary). We reasoned that, if we observed early Al-ey and/or early Al-toy co-expression with Al-otd, this would be indicative of a role for these Pax6 genes in specifying the protocerebrum instead of eyes, as A. longisetosus appears to lack eye primordia (see above). Alternatively, if Pax6 genes ancestrally specified cells destined to be associated with the eyes during the early stages of development, we should see an absence of Pax6 expression, and their potential co-expression with orthodenticle , at these early stages in the eyeless mites. We chose to directly compare our observations to those of spiders, as the earliest expression patterns for otd in opilionids has thus far been reported for stages 8 and later [ 32 ], at which the pre-cheliceral region has already been established and has become morphologically complex. It is also important here to note that the lineage leading to spiders underwent a whole-genome duplication [ 77 ], which has resulted in at least two otd orthologs in spiders [ 31 ]. In the spider P. tepidariorum , orthodenticle-1 is necessary for the development of the spider pre-cheliceral region [ 78 ]. Also, a recent single-cell RNAseq study showed that Pt-otd1 is expressed with the Pax6 orthologs in cell clusters marking the pre-cheliceral region [ 34 ]. We therefore performed HCRs simultaneously targeting the Pax6 genes and the singleton A. longisetosus orthodenticle ortholog ( Al-orthodenticle; Al-otd ) in early germ band mite embryos. Following the transition from radial to bilateral symmetry in P. tepidariorum embryos, Pt-otd1 is expressed in the posterior-most boundary of the pre-cheliceral region, adjacent to the cheliceral segment [ 78 , 79 ]. Similarly, the earliest expression of Al-otd that we observed was in early germ-band embryos, where it was expressed in a continuous domain in the embryonic anterior, as well as in the cells incipient ventral nerve cord (=VNC, Fig. 4A -A2). This anterior Al-otd domain overlapped with the bilateral Al-ey expression domain at this stage, specifically at the lateral margins of Al-otd expression (Fig. 6A3, A5 , A7 , A8, and A11). Furthermore, Al-otd was expressed in the same cells as Al-toy at this stage, however its co-expression with Al-otd was more extensive than that of Al-ey ( Fig. 6A4, A6, A9, and A12). This early expression of Al-toy extended more medial-ventrally than that of Al-ey , where it overlapped Al-otd expression in all but the medial domains of Al-otd ( Fig. 6A 4 and A6). Taken together, these early expression patterns of Al-ey, Al-toy , and Al-otd are similar to those observed in the spider P. tepidariorum at roughly its Stage 8 during the early development of the pre-cheliceral region[ 34 , 78 ]. Download figure Open in new tab Fig. 6 The co-expression of the Al-Pax6 orthologs with the head-patterning gene orthodenticle . A-A6 Al- Pax6 co-expression with Al-orthodenticle ( Al-otd ) in an early, pre-segmental embryo. The ventral portion of the embryo is shown. A DAPI counterstain of this embryo. A2 Al-otd expression in this embryo in a continuous stripe of expression in the embryonic anterior (left of the page). Also, Al-otd is weakly expressed in cells of the incipient ventral nerve cord (VNC). A3 Al-ey expression is restricted to two, lateral domains in the anterior rim of the embryo. A4 Al-toy is also expressed in two, paired domains in the anterior of the embryo. A5 Merged image of the Al-otd (green) and Al-ey (magenta) channels. A6 Merged image of the Al-otd (green) and Al-toy (magenta) channels. Dotted boxes in these images represent the fields of view shown in A8-A12. A7 Three-dimensional projection of the same embryo, rotated to show the frontal-most expression of Al-otd (green) and Al-ey (magenta; arrowheads) in the anterior. The embryo is oriented with the ventral towards the top of the page. A8-A10 Single channel confocal images of Al-ey , Al-toy , and Al-otd , respectively, in the region of the dotted boxes outlined in A5 and A6. All Confocal slice of the region of the embryo outlined in the dotted box in A5. Al2 Confocal slice of the region of the embryo outlined in the dotted box in A6. A11-A12 show that both Al-Pax6 orthologs are co-expressed with Al-otd in this region. B-B6 Al-Pax6 and Al-otd expression in an early, three prosomal segmented staged embryo. B DAPI nuclear counterstain. B2 Al-otd expression in the developing head. Expression also persists in the VNC. B3 Al-ey expression. B4 Al-toy expression. B5 Merged confocal channels of Al-otd (green) and Al-ey (magenta) expression. B6 Merged confocal channels of Al-otd (green) and Al-toy (magenta) expression. Note that this is the same embryo shown in Fig. 3B -B4. C-C6 Al-Pax6 and Al-otd expression in an early, limb-bud stage embryo. C DAPI nuclear counterstain. C2 Al-otd expression. C3 Al-ey expression. C4 Al-toy expression. C5 Merged confocal channels of Al-otd (green) and Al-ey (magenta) expression. C6 Merged confocal channels of Al-otd (green) and Al-toy (magenta) expression. D-D6 Al-ey and Al-otd expression in an embryo at approximately stage BDS-2. D DAPI nuclear counterstain. D2 Al-otd expression. D3 Merged confocal channels of Al-otd (magenta) and the DAPI counterstain (cyan). D4 The same merged image shown in D3, zoomed in on the developing pre-cheliceral region. Asterisks demark the “blocks” of Al-otd expression that surrounds the incipient stomodaeum. LF= the sites of the developing lateral furrows. D5 Image showing Al-ey expression in this embryo. D6 The same embryo, however the channels showing Al-otd expression (green) and Al-ey expression (magenta) have been merged. The dotted line outlines the co- expression of these genes in the anterior lateral furrows. E-E6 Al-ey and Al-otd expression in an embryo at approximately stage BDS-4. E DAPI nuclear counterstain. E2 Al-otd expression. The asterisks demark the horizontal lines of expression at the proximal-most boundary of each prosomal appendage. E3 Merged confocal channels of Al-otd (magenta) and the DAPI counterstain (cyan). E4 The same merged image shown in E3, zoomed in on the developing pre-cheliceral region. Arrowheads mark the “blocks” of Al-otd expression. Asterisks mark expression in the lateral margins of the anterior furrows. E5 Image showing Al-ey expression in this embryo. E6 The same embryo, however the channels showing Al-otd expression (green) and Al-ey expression (magenta) have been merged. The dotted line outlines the co- expression of these genes in the ventral-most portion of the lateral furrows. F-F11 Al-ey and Al-otd expression in an embryo at approximately stage BDS-5. F DAPI nuclear counterstain. F2 Al-otd expression. F3 Merged confocal channels of Al-otd (magenta) and the DAPI counterstain (cyan). F4-F6 Confocal slices of the same embryo, however the slices were taken more dorsally in the embryo. F4 The same merged image shown in F3, zoomed in on the developing pre-cheliceral region. Arrowheads point to Al-otd expression in the margins of the head lobes in the presumptive non-neural ectoderm. Al-otd expression in one the medial subdivisions is outlined with a dotted line. E5 Image showing Al-ey expression in this embryo. F6 The same embryo, however the channels showing Al-otd expression (green) and Al-ey expression (magenta) have been merged. F7-11 The same embryo, however the images were taken at a more “surface-level”, or ventral position of the embryo in comparison to F4-F6. F7 DAPI counterstain at this position. F8 Al-otd expression at this level, showing its expression relative to the anterior lateral furrows (ALF) and the posterior lateral furrows (PLF). MS=expression in the medial subdivision. The right-most MS is outlined in a red, dotted line. F9 Al-ey expression at this position. F10 Merged image showing Al-otd expression (green) and Al-ey expression (magenta). Note the co- expression of these genes in the lateral component of the medial subdivisions. The left one is outlined in a dotted line. F11 The same image shown in F10, however it is zoomed into one of the lateral furrows. Arrowheads point to the boundaries between Al-ey and Al-otd expression. G-G5 Al-ey and Al-otd expression in an embryo at approximately stage BDS-7. G DAPI nuclear counterstain. The dotted outline shows the position of the labrum which has been obscured by the movements of the pre-cheliceral region at this stage. G2 Al-otd expression in this embryo, showing its expression in the lateral margins of the two halves of the prosomal shield (PS). G3 Merged confocal channels of Al-otd (magenta) and the DAPI counterstain (cyan). G4 Image showing Al-ey expression in this embryo. G5 The same embryo, however the channels showing Al-otd expression (green) and Al-ey expression (magenta) have been merged. Embryos shown in B-C6 are oriented with their anterior pointing towards the left of the page, and their dorsal regions towards the top of the page. All subsequent images are oriented so that the anterior of the embryos are directed towards the top of the page. All scale bars represent 50 µm, except as follows: A8- A12, 10 µm; F4-F11, 20 µm. All other abbreviations are the same as in other figures. Download figure Open in new tab Fig. 7 Summary drawings showing the relative expression patters of the Al-Pax6 orthologs and Al-otd throughout the development of the pre-cheliceral region. See text for details. In a subsequent germ-band stage, at which the first three prosomal segments had been delineated ( i.e. , the cheliceral, pedipalpal and first two walking leg segments), Al-otd expression was retained in the incipient ventral nerve cord, as well as the pre-cheliceral region ( Fig. 6B 2). Al-ey was co- expressed with Al-otd at this stage in its lateral, triangular domains of expression ( Fig 6B 3). Al-toy was also co-expressed with Al-otd at this stage, however Al-toy ’s expression domain was much broader and fully encompassed the domain of Al-otd where it extended more posteriorly in the pre-cheliceral region ( Fig. 6B 4 and B6). As show in Fig. 3 , Al-toy expression dramatically decreases in the pre-cheliceral region following its initial expression. Approximately at the stage when this decrease begins, and when the prosomal limb buds became more distinct, Al-otd retained its expression domain in the pre-cheliceral region where it was still co-expressed with Al-ey ( Fig. 6C2 , C3, and C5). However, Al-toy was not observed to be co- expressed with Al-otd at this stage ( Fig. 6C 6). We argue that these early expression patterns are likely indicative of a role for both Pax6 genes and Al-otd acting together to specify the neural field of the incipient protocerebrum. Evidence for this includes the fact that eye development in all chelicerates studied occurs after the protocerebral region has been specified, and also that the eye precursor cells form in the non-neural cells of the peripheral head lobes. Thus, the co-expression of Pax6 genes and otd , as seen in spiders, is likely independent of their expression in the incipient eyes. Therefore, our results are more consistent with a role of Pax6 genes in specifying the protocerebrum, and that this role was inherited from the last common ancestor of spiders and mites. Late expression of Al-otd suggests the ancestral role of orthodenticle in arachnids It was also shown that both the duplicated spider otd paralogs, as well as the singleton otd ortholog in the opilionid P. opilio , are expressed in the developing brain and eyes at developmental stages following the establishment of the head [ 28 , 30 , 32 , 34 ]. We thus utilized these data to ask how the spider otd paralogs were potentially subfunctionalized, or neo-functionalized, in the lineage leading to spiders. We began these observations at BDS-2, when the morphology of the pre-cheliceral region becomes more complex (note that Al-otd is expressed broadly at BDS-1 in a manner similar to Fig. 4C 5; not shown). At this stage, we detected Al-otd expression in the pre-cheliceral region as well as its retention in the developing ventral nerve cord ( Fig. 6D -D3). Within the pre-cheliceral region, Al-otd was expressed in the anterior and posterior margins of the nascent lateral furrows. Note that otd2 is also expressed in the lateral furrows of the spider P. tepidariorum [ 30 , 31 ], as is otd in the opilionid P. opilio [ 32 ]. Al-otd was also expressed in connected domains that span the central to the lateral pre-cheliceral region ( Fig. 4D 4). We also detected Al-otd expression in two “blocks” of cells surrounding the site of the future stomodaeum ( Fig. 4D 4, asterisks) in a similar manner to otd in P. opilio [ 32 ] and otd2 in spiders [ 31 ].We likewise co-stained these embryos with Al-ey expression, and found that Al-otd and Al-ey expression overlap in the anterior of the lateral furrows (note that Al-toy expression is absent in the pre- cheliceral region at this stage). However, they do not overlap at the posterior portion of the lateral furrows ( Fig. 6D 6, the left anterior portion of the lateral furrow, ALF, is outlined). At approximately BDS-4, Al-otd expression was still present in the developing ventral nerve cord ( Fig. 6E -E2). Faint Al-otd expression was also detected in horizontal lines of expression at the proximal- most boundary of each prosomal appendage ( Fig. 6E 2, asterisks). This appendicular expression may be homologous to that of Pt-otd2 expression at later stages ( i.e. , stages 12 and 13; see Figs. 11H-I in [ 30 ]) and also to otd expression in opilionid P. opilio ( [ 32 ], their Fig. S2). Within the pre-cheliceral region, Al- otd expression was detected in the developing labrum ( Fig. 6E -E3), in a similar manner to spider otd2 orthologs [ 28 , 31 ] and to the expression of otd in P. opilio [ 32 ]. Al-otd expression was subsequently retained in the “blocks” of cells surrounding the stomodaeum ( Fig. 6E 4, arrowheads), and was also detected in the lateral margins of the anterior furrows ( Fig. 6E 4, asterisks). By co-staining for Al-ey expression, we were able to detect its co-expression with Al-otd expression in the ventral-most portion of the lateral furrows ( Fig. 6E 6, dotted outline). At BDS-5, the expression of Al-otd in the ventral nerve cord remained, and the proximal appendicular expression domains became more pronounced. Furthermore, each of these domains appeared to combine to become continuous with one another on their respective half of the embryo ( Fig. 4F -F3) similar to otd expression at stages 12-15 in P. opilio [ 32 ]. Also in a similar manner to the spider otd2 orthologs [ 28 , 31 ], and the P. opilio otd ortholog [ 32 ], we additionally detected Al-otd expression in the margins of the head lobes in the presumptive non-neural ectoderm ( Fig 6F 4, arrowheads). Also, Al- otd expression surrounded the periphery of the fused labrum ( Fig. 6F -F3). Recall that at this stage, the medial subdivisions send out “extensions” of cells that will separate the anterior furrows from the nascent mushroom bodies ( Fig. 1E -E2). We detected Al-otd expression in these medial subdivision extensions ( Fig. 6F 4; the right-most medial subdivision’s extension is outlined). Because of their proximity to the persisting “blocks” of Al-otd expression surrounding the labrum at this stage, we take the “blocks” of Al-otd expression in BDS-2 and BDS-4 to be the medial subdivisions. We also detected the co-expression of Al-ey and Al-otd within a population of cells in the lateral halves of these medial subdivisions ( Fig. 6F 8-11; one of these populations is outlined in F10). These patterns are similar to those of spider otd.2 expression [ 31 ] and to P. opilio otd expression [ 32 ], suggesting a high degree of conservation of otd expression in these tissues. Al-otd expression was then detected at late BDS-5/early BDS-6 in the ventral-most half of the lateral furrows, and its expression was the highest in their anterior and posterior poles. Because Al-ey at this stage is also expressed in the ventral-most half of the lateral furrows (see Fig. 4F -F3), we asked to what extent Al-otd and Al-ey are co-expressed in these “open” lateral furrows. By co-detecting Al-ey with Al-otd , we found that their expression is mutually exclusive in the anterior lateral furrows, with Al-otd expression located more “inwardly” in the lateral furrow that Al-ey expression ( Fig 6F 10-11; arrowheads in F11 point to the boundaries between Al-ey and Al-otd expression). These expression patterns are interesting, as they may point to a coordinate-type system to establish polarity, or a mechanism of regionalization, in the lateral furrows. Lastly, both Al-ey and Al-otd were co-expressed in the ventral- lateral cells that are adjacent to the lateral furrows ( Fig 6F 10-11; these cells are outlined in the left side of the embryo in F10). At approximately BDS-7, when the prosomal shield halves had migrated and fused, we observed Al-otd expression in the margins of the fused prosomal shield. This expression domain was continuous to the lateral appendicular expression domains of the previous stages ( Fig. 6G -G3). A similar colorimetric staining pattern was observed for otd2 orthologs in the spider species studied in [ 31 ], however the authors described these as the results of artefactual cuticle staining. Interestingly, we did not observe similar staining to that shown in Fig. 6G -G5 in our control experiments, nor in any other HCR experiments of other genes. Furthermore, P. opilio otd is expressed in a similar pattern at stages 12-15 [ 32 ]. Therefore, we take this expression pattern to be a conserved mode of otd expression amongst arachnids. Al-otd expression was also present in the ventral nerve cord and the periphery of the labrum at this stage ( Fig. 6G -G3). Within the pre-cheliceral region, we did not observe Al-otd in any of the tissues in which it was expressed in the previous stages. This was shown via the co-staining of Al-otd with Al-ey , which is expressed largely in the mushroom bodies at this stage ( Fig. 6G 4-G5). We did not observe expression of Al-otd at any subsequent pre-larval or larval stages (not shown). In terms of eye development, one P. tepidariorum otd ortholog, Pt-otd2 , is expressed late in development in the anterior median eyes [ 30 , 31 ]. In the spider C. salei , both of its otd paralogs are expressed in tissues associated with the eyes. Specifically, Cs-otxa (= otd1 ) is expressed in the vesicles of the posterior-lateral eyes, whereas Cs-otxb ( =otd2 ) is expressed in all sets of lateral eyes as well as the posterior median eye vesicles [ 28 ]. In a recent, comprehensive study of diverse spider taxa, it was shown that in all of the spider species studied, otd2 orthologs are expressed exclusively in the anterior- median eyes, with the notable exception of a lack of any eye otd expression in Pholcus phalangioides [ 31 ]. Additionally, a recent study on P. opilio revealed that its otd ortholog is expressed in all of its eye primordia [ 32 ]. We did not observe similar expression patterns of Al-otd in the aforementioned HCR experiments, further confirming the absence of vestiges of eyes in A. longisetosus during embryonic development. In summation, our results, coupled with those observed in P. opilio [ 32 ], suggest that Al-otd expression is most similar to spider otd2 ortholog expression in the pre-cheliceral region [ 31 ] Discussion The morphogenesis of the arachnid head and brain in light of A. longisetosus Modern studies into the development of the arachnid head have largely focused on the spiders P. tepidariorum (e.g., [ 51 ] and C. salei (e.g.,[ 50 , 54 ]). These studies, in conjunction with a recent studies on the development of the opilionid P. opilio [ 32 , 55 ], have revealed potential synapomorphic features of arachnid head development. These synapomorphies include the appearance of the anterior and lateral furrows, followed by the migration of the non-neural prosomal shield over the pre-cheliceral region. Despite the conservation of these features in A. longisetosus , our data show major morphogenetic divergences between mites and the aforementioned arachnids in the embryonic pre-cheliceral region. First, we observed differences in the timing of the appearance of the lateral and anterior furrows. In the aforementioned spider exemplars, the lateral furrows form first, followed by the anterior furrows [ 50 , 51 ]. This may also be the order of appearance in A. longisetosus , however with our methods, we were only able to visualize the appearance of both pairs of furrows simultaneously at BDS-2. In P. opilio , it is also unclear as in which order these furrows appear, as they also seem to appear simultaneously (see Fig. 8C in [ 8 ]). Another point of divergence between spiders and A. longisetosus can be seen in the later morphogenesis of the anterior and lateral furrows. In spiders, the division of the anterior furrows into the mushroom and arcuate bodies occurs through the migration of the medial subdivisions expanding into the anterior furrows, which results in the compartmentalization of the arcuate and mushroom bodies [ 50 , 51 , 54 ]. Our observations suggest that this aspect of brain morphogenesis is conserved between mites and spiders, with one major exception. In the aforementioned spider species, the anterior furrows do not appear to make continuous grooves with the lateral furrows at any stage. In A. longisetosus , however, the lateral and anterior furrows become continuous with one another after their initial appearances as distinct structures (see Fig. 1 ). These continuous grooves are then subsequently subdivided to form the arcuate bodies, mushroom bodies, and the optic vesicles, with the arcuate and mushroom bodies delimited by extensions of the medial subdivisions. A secondary, and as of yet unnamed, group of cells forms later to separate the posterior of the mushroom bodies from the anterior of the lateral furrows. One last point of divergence was seen in the development of the lateral furrows. In spiders, the lateral furrows are further subdivided through the expansion of another grouping of paired elevated neural tissues called the lateral subdivisions. These lateral subdivisions subdivide the lateral furrows into the lateral and medial optic vesicles [ 28 – 32 , 54 ]. Our observations of A. longisetosus brain compartmentalization did not show evidence of any lateral subdivisions. Interestingly, it has been proposed that it is these lateral vesicles give rise to the optic ganglia of the lateral eyes of spiders [ 54 ]. It is therefore tempting to attribute the absence of the lateral subdivisions in A. longisetosus to their absence of eyes. However, to test this hypothesis, the compartmentalization of the brains of acariform mites that have retained their eyes needs to be studied. Taken together, our results suggest that presence of paired anterior and lateral furrows, as well as the migration of the prosomal shield are conserved aspects of arachnid brain/pre-cheliceral region development. Despite this, the subsequent subdivisions of these furrows may be lineage-specific. An alternative explanation for our observations could be that the mode of brain compartmentalization in A. longisetosus is highly derived within Acariformes. To clarify this, more studies into the brain development of members of this hyper-diverse clade are needed. Also, given that A. longisetosus lacks eyes, the presence of the lateral furrows that give rise to the optic vesicles needs an explanation. The simplest explanation is that these compartments of the brain do not only give rise to the optic neuropils as they do in spiders, and they therefore may contribute to other important components of the brain. Alternatively, these lateral furrows could be vestiges of the optic neuropils of true acariform eyes. Cellular lineage tracing experiments, which are not yet available for this species, are needed to further explore these hypotheses. Pax6 genes and arachnid eyes The ancestral role of Pax6 gene expression in the development of chelicerate eyes has thus far puzzled researchers (reviewed in [ 33 ]). An early expression study of a single toy ortholog in the horseshoe crab Limulus polyphemus showed no clear expression in any of its eye anlagen [ 27 ]. Since this study, it has been revealed that L. polyphemus has at least five Pax6 orthologs, two of which that are likely toy orthologs and three of which that are likely eyeless orthologs [ 13 , 26 ]. It may still be shown that some of these orthologs are indeed expressed in the developing horseshoe crab eyes. However, recent data from spiders have shown no expression of any Pax6 gene in any of its eye anlagen [ 29 – 31 , 34 , 80 ], except for the expression of eyeless in the anterior-median eyes of C. salei [ 28 ]. Furthermore, a recent study into the RDGN genes of P. opilio showed that both Pax6 genes are expressed in the developing median eyes[ 32 ]. These observations have thus far neither supported nor falsified the hypothesis that Pax6 genes did not play an ancestral role the development of chelicerate eyes and may instead suggest that a lack of Pax6 expression in spider eyes may be specific to that clade. An alternative explanation has been proposed, which posits that the expression of Pax6 genes observed during early spider development act to specify the fates of eye photoreceptor cells prior to their incorporation into the eyes during subsequent morphogenetic events. Using our eyeless mite, as well as its expression patterns of the early head-patterning gene orthodenticle , we tested this hypothesis. Our data show similarities to the early expression patterns of eyeless and toy of spiders in the pre-cheliceral region. We therefore argue that these data are more consistent with an ancestral role of Pax6 genes acting with otd to specify the neural cells of the protocerebrum in arachnids. It will be interesting to see if this holds true throughout in the remaining arachnid groups, including other non-spider members of Arachnopulmonata, as well as other members of Acariformes. Al-ey expression in comparison to other arachnids Our data show both shared and derived modes of eyeless expression in arachnids. We found that early Al-ey expression is similar to that of spiders, specifically in comparison to early P. tepidariorum eyeless expression [ 30 , 31 , 34 ]. In both taxa, eyeless is expressed early in an anterior domain in the pre-cheliceral region. Furthermore, our data highlights other conserved aspects of arachnid eyeless expression. We observed its expression in the nascent mushroom bodies and the optic vesicles. eyeless expression was similarly observed in spiders [ 28 , 30 , 31 , 34 ], and may also be expressed in these structures in P. opilio [ 32 ]. By evaluating eyeless expression patterns reported for other taxa, we conclude that in spiders, in an opilionid and in A. longisetosus ( Fig. 2 ), eyeless is subsequently expressed in cells associated with the lateral furrows. Because the lateral furrows likely develop into the optic vesicles in spiders [ 54 ], the usage of these optic vesicles in arachnids lacking eyes should be a key focus of study to further our understanding of both the arachnid brain, its development, and the function of eyeless in patterning these structures. Our results cannot falsify the hypothesis that our observed Pax6 expression patterns are vestigial, i.e. , relictual features of eye development. We are currently exploring methods to abrogate gene expression in A. longisetosus , however we are thus far limited to gene expression surveys. Once methods to test for gene functions are in place, we plan to knock down both Pax6 genes to test this hypothesis directly. If Pax6 gene expression is indeed vestigial in A. longisetosus , we would expect to see no morphogenetic anomalies in Pax6 -depleted embryos. Related to this, we cannot thus far falsify the hypothesis that the lateral furrows/optic vesicles are themselves vestigial. Because we do not yet know if, or exactly what, other non-visual roles of the optic vesicles may be, more functional neural studies into these compartments of the arachnid brain are necessary before making this conclusion. Al-toy expression in comparison to other arachnids Our data also suggests both conserved and derived aspects of toy expression amongst arachnids. To date, early expression data for toy in arachnids is limited to the spider P. tepidariorum [ 34 ]. In both this spider and in A. longisetosus , toy is expressed in a broad anterior domain in the pre-cheliceral region. Following this pattern, Al-toy expression deviates dramatically from its ortholog’s expression in other arachnids. For instance, Al-toy is expressed in the developing prosomal appendages, in a manner not seen in other studied arachnids. Also, Al-toy expression disappears from the pre-cheliceral region at intermediate stages of development. This is in stark contrast to opilionids and spiders, in which toy expression persists in the pre-cheliceral region at comparable stages [ 31 , 32 ]. We also observed ubiquitous expression of Al-toy in the migrating prosomal shield at late stages of development. In the opilionid P. opilio , both Pax6 orthologs appear to be ubiquitously expressed in this structure at later stages [ 32 ]. In spiders, neither Pax6 ortholog appears to be expressed ubiquitously in the prosomal shield [ 31 ]. It is tempting to conclude that these deviations in the use of toy in A. longisetosus is due to their absence of eyes. However, since toy does not contribute to eyes in any studied arachnid, the differential use of toy in A. longisetosus necessitates future functional studies. Eyes in Acari: future directions Members of Acari (mites and ticks) display a wide degree of morphological diversity, owing to their occupancy of numerous ecological niches [ 81 , 82 ]. The monophyly of Acari is currently still contested (e.g., [ 7 , 83 , 84 ]), however it is generally agreed that Acari is comprised of two internally-monophyletic groups, the Parasitiformes ( e.g., ticks) and Acariformes (mites). Within Acariformes, the number, position, and the types of eyes present are extremely diverse, also owing to their ecological diversity (see [ 81 , 82 , 85 ]for review). Examples of this diversity include the retention of both lateral and median eyes, with varying numbers of each ( e.g. , the mite Heterochthonius gibbus has one median eye and a pair of lateral eyes [ 86 ]), or the parallel loss of all eyes independently in a number of acariform groups (reviewed in [ 81 , 82 ]). Notwithstanding, it has been hypothesized that the ground plan for acariform mites is the presence of two median eyes and two pairs of lateral eyes, a condition that was inferred in an early study [ 87 ]. However, it has been cautioned that determining the pleisiomorphic condition of acariform eyes is a complex problem considering the morphological disparity between acariform sub-clades [ 85 ]. Nonetheless, given the wide degree of eye diversity in mites, coupled with the emergence of new developmental data on the expression and function of RDGN genes in arachnids, mites are a clade of extreme interest in terms of exploring the developmental evolution of arachnid eyes. Conclusions Eye loss is extensive across mite species, however paradoxically, the eyeless mite A. longisetosus retains two Pax6 paralogs in its genome. These observations, coupled with recent data suggesting that there is no role for these genes in the development of spider and horseshoe crab eyes, allowed us to test the hypothesis that ancestrally chelicerates did not use Pax6 genes to pattern their eyes. By following the expression of genes canonically associated with arthropod eye development, we found support for the hypothesis that A. longisetosus does not develop vestigial eye tissues. Following this, we showed that both early and late Al-ey and Al-toy expression patterns are highly similar to those of studied chelicerate taxa that do have eyes. An alternative hypothesis, i.e., that chelicerates do use Pax6 genes to pattern early photoreceptor cells prior to their incorporation into the eyes, is not supported by our findings. We reasoned that, if this hypothesis were supported, we would have observed either an absence of early Pax6 gene expression, and/or highly derived Pax6 gene expression in comparison to other studied chelicerates. Our findings do not support this hypothesis but rather suggest that these genes were used in the development of the ancestral chelicerate brain/central nervous system. Our results also support the hypothesis that ancestrally, Pax6 genes worked with orthodenticle to specify the neural cells of the protocerebrum independent of orthodenticle’s role in eye specification. Lastly, our results suggest that one Pax6 ortholog, eyeless , was likely used ancestrally in chelicerates to specify the paired structures of the chelicerate brain called the “optic vesicles” as well as the mushroom bodies. Studies into the function of Pax6 genes in model, and emerging model, chelicerates are needed to further test these hypotheses. The potential roles of Pax6 genes observed in the median eyes of the spider C. salei [ 28 ], and the median eyes of the opilionid P. opilio [ 32 ] , also need further investigation to test the hypotheses that these patterns are due to lineage-specific adaptations, or due to developmental systems drift. Furthermore, studies into the use of Pax6 genes are needed in members of Pycnogonida (sea spiders), as they are the likely sister-group to the remaining chelicerate taxa. If Pax6 gene expression were also absent in their embryonic eyes, this would further support the hypothesis that Pax6 genes played no role in the ancestral development of chelicerate eyes. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials All original confocal images are available upon request to AAB. The transcriptome sequences are publicly available from [ 38 ]. All probe sequences for the hybridization chain reactions are available in Supplementary Tables S3-S17. Competing interests The authors declare no competing interests. Funding Funding for this work was provided by the DeSales University Berg Endowment, and funds from the DeSales University Center for Teaching Excellence Innovation Grant awarded to AAB. Author contributions IJ and AAB developed the study design and experiments. IJ and AAB both performed the wet-lab experiments, performed the confocal microscopy, as well as the bioinformatic analyses. Both authors wrote the original draft. All authors edited and approved the final version. Fig. S1 The architecture and phylogenetic interrogation of A. longisetosus eyeless and twin of eyeless sequences. ( A ) Schematics showing the deduced genomic architecture of Al-eyeless , as well as the architecture of its spliced transcripts. In the genomic schema, the exons are represented as grey boxes, and the dotted lines represent splicing patterns. The dotted lines on the top of the map correspond to splicing patterns of the Al-ey.1 isoform. The blue box below exons 8 and 9 represents exon 8.5, which is used in the Al-ey.2 isoform. Below the genomic schema are schematics representing the architecture of Al-ey.1 and Al-ey.2 . These show how each exon corresponds to the resulting mRNA landmarks, including the untranslated regions (UTRs), the paired domains (PD), the octapeptide-like domains (OP-Like), and the homeodomains (HD). ( B ) Schematics showing the deduced genomic architecture of Al-toy , as well as the architecture of its spliced transcript. All annotation styles and abbreviations are the same as in A . ( C ) Alignments of the deduced amino acid sequence of the paired domains of both Al-Ey and Al-Toy with selected arthropod Pax6 orthologs. The linker region bisects the paired domain into PAI and RED sub- domains. The linker region of Al-Ey, as well as that of other arthropod orthologs, contains a distinct arginine (R) at position 64 (highlighted in red). The linker region of Al-Toy and other arthropod Toy orthologs contains the diagnostic lysine (K) at this position (also highlighted in red). ( D ) The resulting tree from a maximum-likelihood phylogenetic analysis of selected arthropod Ey, Toy, and Pax2/5 proteins. The clade with green-highlighted branches represents a monophyletic Toy clade, and the clade with blue branches represents the monophyletic Ey clade. A. longisetosus Pax sequences are denoted with red labels. The node colors correspond to approximate likelihood ratio test (aLRT) scores (see legend). All labels have their GenBank accession numbers in parentheses. Fig. S2 Phylogenetic analysis of putative A. longisetosus Arrestin protein orthologs. 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