Ciliary photoreceptors in the cerebral eyes of a protostome larva Passamaneck et al.

Passamaneck et al. EvoDevo 2011, 2:6 http://www.evodevojournal.com/content/2/1/6 (1 March 2011) Passamaneck et al. EvoDevo 2011, 2:6 http://www.evodevojournal.com/content/2/1/6

RESEARCH Open Access Ciliary photoreceptors in the cerebral eyes of a protostome larva Yale J Passamaneck1*, Nina Furchheim2, Andreas Hejnol1,3, Mark Q Martindale 1, Carsten Lüter2

Abstract Background: Eyes in bilaterian metazoans have been described as being composed of either ciliary or rhabdomeric photoreceptors. Phylogenetic distribution, as well as distinct morphologies and characteristic deployment of different photopigments (ciliary vs. rhabdomeric opsins) and transduction pathways argue for the co-existence of both of these two photoreceptor types in the last common bilaterian ancestor. Both receptor types exist throughout the Bilateria, but only vertebrates are thought to use ciliary photoreceptors for directional light detection in cerebral eyes, while all other invertebrate bilaterians studied utilize rhabdomeric photoreceptors for this purpose. In protostomes, ciliary photoreceptors that express c-opsin have been described only from a non- visual deep-brain photoreceptor. Their homology with vertebrate rods and cones of the human eye has been hypothesized to represent a unique functional transition from non-visual to visual roles in the vertebrate lineage. Results: To test the hypothesis that protostome cerebral eyes employ exclusively rhabdomeric photoreceptors, we investigated the ultrastructure of the larval eyes in the brachiopod Terebratalia transversa. We show that these pigment-cup eyes consist of a lens cell and a shading pigment cell, both of which are putative photoreceptors, deploying a modified, enlarged cilium for light perception, and have axonal connections to the larval brain. Our investigation of the gene expression patterns of c-opsin, Pax6 and otx in these eyes confirms that the larval eye spots of brachiopods are cerebral eyes that deploy ciliary type photoreceptors for directional light detection. Interestingly, c-opsin is also expressed during early embryogenesis in all potential apical neural cells, becoming restricted to the anterior neuroectoderm, before expression is initiated in the photoreceptor cells of the eyes. Coincident with the expression of c-opsin in the presumptive neuroectoderm, we found that middle gastrula stage embryos display a positive photoresponse behavior, in the absence of a discrete shading pigment or axonal connections between cells. Conclusions: Our results indicate that the dichotomy in the deployment of ciliary and rhabdomeric photoreceptors for directional light detection is not as clear-cut as previously thought. Analyses of brachiopod larval eyes demonstrate that the utilization of c-opsin expressing ciliary photoreceptors in cerebral eyes is not limited to vertebrates. The presence of ciliary photoreceptor-based eyes in protostomes suggests that the transition between non-visual and visual functions of photoreceptors has been more evolutionarily labile than previously recognized, and that co-option of ciliary and rhabdomeric photoreceptor cell types for directional light detection has occurred multiple times during animal evolution. In addition, positive photoresponse behavior in gastrula stage embryos suggests that a discrete shading pigment is not requisite for directional photoreception in metazoans. Scanning photoreception of light intensities mediating cell-autonomous changes of ciliary movement may represent an ancient mechanism for regulating locomotory behavior, and is likely to have existed prior to the evolution of eye-mediated directional light detection employing axonal connections to effector cells and a discreet shading pigment.

* Correspondence: [email protected] 1Kewalo Marine Laboratory, Pacific Biosciences Research Center, University of Hawaii, 41 Ahui Street, Honolulu, HI 96813, USA Full list of author information is available at the end of the article

© 2011 Passamaneck et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Passamaneck et al. EvoDevo 2011, 2:6 Page 2 of 17 http://www.evodevojournal.com/content/2/1/6

Background expressing the r-opsin photopigment, while the deploy- Bilaterian photoreceptor cells are generally classified as ment of ciliary photoreceptors in the vertebrate eye is the having either ciliary or rhabdomeric morphologies, result of their having been substituted for rhabdomeric depending upon the origin of the elaborated membranes photoreceptors early in the evolution of the chordate that compose their light-sensitive structures [1,2]. Recent lineage [3]. phylogenetic and expression analyses of opsin photopig- Brachiopods represent an intriguing group for under- ments suggest that two non-homologous phototransduc- standing the evolution of photoreceptors, given their tion cascades characterize ciliary and rhabdomeric representation in the early fossil record of metazoans. photoreceptors in bilaterians [3-7]. Opsins code for Recent phylogenomic analyses have evidenced that bra- G-coupled protein receptors, which are localized to the chiopods are derived members of the clade Spiralia elaborated membranes of photoreceptors and participate (Lophotrochozoa), closely related to nemerteans and in the phototransduction pathway. Phylogenetic analyses annelids [11,12]. The larvae of several articulate brachio- have demonstrated that all bilaterian opsins have a pods have been described as having larval eye spots; monophyletic origin, with several classes, including cili- however, the morphology of these structures has not ary opsins (c-opsins) and rhabdomeric opsins (r-opsins), been examined in detail [13,14]. having diverged prior to the last common ancestor of To ascertain the nature of larval eyespots in articulate bilaterians [3,7]. In both deuterostomes and protostomes, brachiopods, we have conducted a detailed morphologi- ciliary opsin (c-opsin) genes have been found to be cal study of these structures in Terebratalia transversa. expressed in photoreceptors withaciliarymorphology, We have also assessed the presence of molecular compo- while rhabdomeric opsin (r-opsin) genes are expressed in nents of eye formation. Finally, we have tested for photo- photoreceptors with rhabdomeric morphology. This responsive behavior in early stage embryos to evaluate dichotomy suggests that these two distinct types of the possible function of an unexpected early domain of photoreceptors, and their associated phototransduction opsin gene expression. pathways, coexisted in the bilaterian ancestor [5-7]. Larval eyespots of protostomes are of particular interest Results in the study of photoreceptor evolution, since their struc- Ultrastructure of Terebratalia eyes ture has been proposed to resemble the bilaterian proto- The lecithotrophic larvae of the brachiopod Terebratalia type two-celled eye [3,8]. Cerebral eyes have been defined transversa have two rows of a variable number (three to as pigmented photoreceptor organs that (a) are positioned eight) of pigmented spots, which extend in a mediolateral in the anterior region of the body in a region of Otx line slightly anterior of the dorsal rim of the apical lobe. expression, (b) are connected to the anterior axonal scaf- These pigment spots, which have previously been fold and, (c) express Pax6 [3]. Based upon these criteria described as eye spots [15], are visible only in the fully the eyes of many protostome larvae and of deuterostome developed swimming larvae (Figure 1A, B; 96 hours post tornaria larvae have been identified as potentially homolo- fertilization at 8°C). To determine whether these pigment gous structures. The discovery that r-opsin orthologs are spots are associated with photoreceptors, we performed expressed in larval rhabdomeric photoreceptors of the transmission electron microscopy (TEM) on ultrathin sec- polychaete Platynereis dumerilii and in vertebrate retinal tions of the larva (Figure 1C-G). Ultrastructural analysis ganglion cells (with non-visual function), together with the demonstrated that the pigment spots are part of a simple development of these photoreceptors from ath-positive eye composed of two putative photoreceptor cells (Figure precursor cells in both animal groups, has led to the 1D, E). One cell contains an apical intracellular lens-like assumption of homology of these cell types [4]. In contrast structure, while the other cell contains the pigment gran- to this, ciliary photoreceptors in protostomes seem to be ules (Figure 1D, E). These pigment granules are arranged primarily restricted to non-visual functions (for example, in cuboidal vesicles to form a shading structure adjacent deep-brain photoreceptors in P. dumerilii), but their to the basal surface of the lens. Both cells possess enlarged photopigment represents the invertebrate ortholog of the ciliary membranes, characteristic of ciliary photoreceptor c-opsin expressed in visual rods and cones of the verte- cells, located between the lens and the shading pigment brate eye, suggesting common ancestry of these ciliary (Figure 1E, F, I; Additional File 1A-K). The receptive cilia photoreceptors [6]. Those protostome visual photorecep- of both cells have a typical 9 × 2 + 2 pattern of microtu- tors that do have a ciliary morphology (for example, the bule organization (Figure 1G; Additional file 1L). Both mantle eyes of scallops) have generally been inferred to be photoreceptor cells also contain axons extending from the non-homologous evolutionary novelties [3,9,10]. Following basal surface of the cell (Figure 1I). Serial ultrathin sec- this scenario, protostome larval cerebral eyes are predicted tions of whole larvae demonstrated that both photorecep- to deploy exclusively rhabdomeric photoreceptors tor cells of each larval eyespot have axonal connections Passamaneck et al. EvoDevo 2011, 2:6 Page 3 of 17 http://www.evodevojournal.com/content/2/1/6

ABC

20 μm 20 μm 20 μm D E

ls ls pg ls y

y pg 2.5 μm * F

1 μm

I ls

lens cell pg G H pigment lens cell cell

apical axons ganglion pigment y cell

y endoderm mesoderm axons

Figure 1 Ultrastructure of Terebratalia transversa larval eyes. (A, B) Brightfield microscopy of a Terebratalia transversa larva, with red eye spots visible in the apical lobe (black arrows). (A) Dorsal view. (B) Lateral view. (C-F) Ultrastructure of eyes in the larva of Terebratalia. (C) Longitudinal section through whole larva with eyes (black arrows) on either side of the apical lobe. (D) Two neighboring eyes with lenses (ls) in the lens cells and pigment granules (pg) in the shading pigment cells, separated by two epidermal cells. Yolk granules are present in both cells (y). (E) Detail of a pigment cell showing the enlarged membrane (asterisk) of its sensory cilium (arrow). (F) Detail of the enlarged ciliary membranes of both the lens cell (lc; black arrow) and the pigment cell (pc; black arrowhead) that fill the optical cavity. (G) Receptive cilia of both photoreceptor cells have a typical 9 × 2 + 2 microtubule pattern, exemplified by a cross section of the lens cell cilium (black arrow). (H) Reconstruction of larval eye axon tracts from serial sections. Axons from the lens cell and pigment cell extend to the apical ganglion (green), which overlays the mesoderm (pink) and endoderm (blue). (I) Reconstruction of a larval eye of Terebratalia from serial sections, consisting of a lens cell and a shading pigment cell. Notice the two enlarged sensory cilia of both cells (black arrows) and the proximal axons. Passamaneck et al. EvoDevo 2011, 2:6 Page 4 of 17 http://www.evodevojournal.com/content/2/1/6

leading to the apical concentration of nerve cells (the c-opsin, the glutamate counterion found in transmem- ganglion or ‘larval brain’), supporting the cerebral nature brane domain III of many chordate c-opsins is not con- of brachiopod larval eyes (Figure 1H). served in Terebratalia c-opsin (Figure 2). Phylogenetic reconstructions of the opsin gene family using both Orthology assignment of Terebratalia transversa ciliary Bayesian and maximum likehood methods support that opsin the gene cloned is an ortholog of other bilaterian c-opsin The ciliary nature of the Terebratalia larval photorecep- genes (Figure 3). We have, therefore, designated this gene tors was further tested by examining the expression of a Terebratalia transversa ciliary opsin (Tt-c-opsin). ciliary opsin (c-opsin)geneclonedfromTerebratalia. A 422 amino acid (aa) full-length gene product was pre- Developmental expression of Tt-c-opsin dicted from cloned cDNA sequences and aligned to pro- As detected by whole mount in-situ hybridization with a tein sequences of opsin proteins from other bilaterian 1,584 nucleotide antisense probe, Tt-c-opsin expression is taxa. The predicted Terebratalia c-opsin protein sequence observed at the middle larval stage, when the three lobes possesses seven transmembrane domains, which is charac- (apica, mantle and pedicle) of the larva are well devel- teristic of all G protein-coupled receptors, as well as a con- oped and setogenesis has commenced (Figure 4A, B). served lysine in transmembrane domain VII, which is Tt-c-opsin is expressed in two rows of punctate domains specific to opsins and forms a Schiff base with retinal to on the dorsal surface of the apical lobe (Figure 4A, B). form rhodopsin (Figure 2). In addition, the predicted pro- These expression domains are positioned anterior to the tein possesses conserved C-terminal motifs that are unique rim of the apical lobe and extend in a mediolateral line, to c-opsins (Figure 2; Additional file 2). As in Platynereis separated at the midline. As such, the larval expression of

I II ◊ III

Terebratalia c-opsin VIIVLVLFVTAIGL FFMN GSV L IIF YRKK E LRS P T N MF I I SMA FNDLMMS IL-G N P FAAVS NLH - HRW IFG DAA - C VWYGF IMFFLG LNGI YHL TA IAF D RY I Platynereis c-opsin N I CAAYLFF IACL G VSLN V L V L VLF I K DRKLRS P N N FLYV S LALGDLLVAVF -G TA FKF I I TAR - KTL LREEDGFC KWYGF ITYL GG LAAL MTLS V IAF V R CL Anopheles GRPop11 NGAAVTLFF IG FFG FFLN IFV IALMYK DVQL W TPM N IFIL NLVCSD FSVS- II G N P LTLTS AIS-HRW LYG KS I - C VAYGFFMSL LG IASIT TLTLSYV E R FC Branchiostoma opsin4 TA I A TGLA L I G LVG SMNN FVV ILLIGCHRQLRTPF N LLL LN VSVADLLVS VC - G NTLSF AS AVQ - HRW LWG RPG- C VWYGFANSL FG IVS L V TLSA LA FERYC Homo encephalopsin ERLALLLGSIG L L G VGNN L L V L VLYYK FQRLRTPTHLLL VN ISLSDLLVS L-F G VTFTF VS CLR - NGW VWD T V G - C VWDGFS G S L FG IVSIA TLTLV A Y E RY I Ciona opsin1 SFLCVYMTFVFLL SCSLN I L V IVATLK N K V LRQ P L N YII VNLAVV DLLSGFV -G GF I S IAANGA -GYFFWG KTM-C QIEG YFVSNFGVTGL LS IAV MA FERYF Geotria LWS NLTSFWMI IVV I L SLFTN G L V L VATLK F K K LRH P L N W ILVNLA IAD IGET IF -ASTVSVVNQI F -GYF I LG HPL - C VFEGFTVSVCG ITAL WSL AI I S FER WM c-opsin Rana SWS1 TLQA I FMGVVF I I G TPLN AVV L LVTVK Y K K LRQ P L N Y ILVN ISVGGL L ICIF -SNFVVF INSWQ-GYFFFG KA F - C AIEAF V G TAL G LVTGWSL AFLA FERY I Latimeria Rh1 SV LCAYMFL L I IGL FP IN FTLVTFL L K H K K LRQ P L N Y ILVNLAVA S L FMVVF -G FTVTF YS SLN-GYFVLG PMG - C AMEGFFA TL GG QVAL WS L VLV A I E RY I Xenopus green-rod opsin MS I SA FML F T I I F G FPLN LTIICTVL K Y K K LRSHLN Y ILVNLAVA NL IV ICF -G STTAF YS FSQ -MYFSLG TLA -C KIEGFTA TL GG IIGL WSL AVV A FER FL Platynereis r-opsin YAVAAWMTFFGIL GVSGN L L V VWT F L K T K S LRTAPN ML L VNLA IGD MA F S AING F P LLT IS SIN-KRW VWG KLW-RELY A F V G GIFGLMSIN TLAW IAIDR FY Mizuhopecten GqRhodopsin YIIGVYITIVG L L G IMGN TTV VY I F SNTK S LRS P S N LFVVNLAVSDL IFS AVNG F P LLTVS SFH-QKW IFG SLF -C QLYGFV G GV FGLMSIN TLTA I SIDRYV Octopus opsin YSVGIFIGVVGI I G ILGN GVV IYLF S K T K S L Q TPA N MF I I NLAMSDLSFS AING F P LKT IS AFM-KKW IFG KVA - C QLYGLLG GIFGFMSIN T MA M I SIDRYN Sepia opsin YSLG I F IG ICGI I G CTGN GIV IYLF T K T K S L Q TPA N MF I I NLAFSD FTFS LVNG F P LMT IS CF I - KKW VFG MA A - C KVYGF I G GIFGLMSIM T MSM I SIDRYN Hemigrapsus Rh1 YLLGVVYLFLG VISIAGN G L V IYLYMK SQA L K TPA N ML I VNLALSDL IMLTT-NFP PFCYNCFSGGRW MFSGTY - C EIY AA LG AITG VCSIW TLCM I SDF RYN Limulus ocellus opsin S I LGVAMI I LGI ICVLGN GMV IYLMMTTK S LRTPT N LLVVNLAFSD FCMMAF -MMP TMASNCFA - ETW ILG PFM- C EVYGMA G S L FGCA SIWSMVM I TLDRYN r-opsin Xenopus melanopsin YT IGSF IL I IG SVG IIGN M L V L YAF YRNK K LRTAPN YF I I NLA ISD FLMS AT -QAP VCF LS SLH-REW ILG DIG-C NVY A F C G A L FG ITS MMTLLA I SINRY I Homo melanopsin YTLGTV I LLVG LTG MLGN LTV IYTF CRSRSLRTPA N MF I I NLAVSD FLMS FT -QAP VFF TS SLY -KQW LFG ETG - C EFY A F C G A L FG ISS MITLTA IALDRYL Homo RGR LAVGMVLLVEALSG LSLN TTIFSL F C K TPELRTPCHL LV LSLALAD SG I S L - -NALVAATS SLL -RRW PYG SDG- C QA HGFQ G FV TA LASICSSAA IAWGRYH Branchiostoma opsin3 LIVGLYLFVIGI I G TIEN GITL ATF S K FRSLRS P TTMLL V H LA IADLGICIF -G Y P FSGAS SLR-SHW LFG GVG - C QWYGFN G MFFGMA N I GLL TCVA VDRYL Mus peropsin SV IAAYL IVAGITS ILSN VVV L GIF I K Y K E LRTPT N AV I I NLAFTD IGVS SI-G Y P MSAA S DLH -GSW KFG HAG - C QIY AGLN I FFGMV SIGLL TVA MDRYL IV V

Terebratalia c-opsin VIA K P LL SSK - I TTRVA ALA I SVCW FGA FLW SV FP V FGWS S Y VEEGEHSSCSINW -- ISQSIVDSSY IISIF IFFC FLP ILLVIFS Y FHVYMTVR T IA KSNTF Platynereis c-opsin AVLR - LG SFTGLTTRMGVAAMAF IW IYS LAFTLAP L L GWNHY I PEGL ATWC SID W --LSDETSDKSYVFA IF I FFC LVP VL I I VV S Y GL I YDKVR K VKT--A - Anopheles GRPop11 L I SRP FA AQN - RSKQGA CLAVLF IW SYS FA L TSP PFGWL GAY VNE AAN I SCSVNW - - ESQTANA TSY IIFLF IF GL I LPLAV I IYS Y INI VLEMR K NSAR - - - Branchiostoma opsin4 V VV RS - - - S EML TY KSS LGM I AF IW MY S LLW TSLP L L GWS S Y QFEGHSVGC SVNW - - VKHNVNNVSY IITL MVTC F FV P MVVVCWS Y AC I WRTV R M SEMKSA E Homo encephalopsin RVV HA - - - - RV I NFSWA WRA I TY IW LYS LAW AGA P L L GWNRY I L DV HGLGC TVD W --KSKD ANDSS FVLFLF LGC LVVPLGV I AHCYGH I LYS IR M LRCVEDL Ciona opsin1 VIC K P FG PVR - FEEKHS I FG I VITW VWS MFW NTP P L IFW DGY DTEGL GTSCAPNW - - FVKEKRERLF I I LYF VFFC VIPLAV I MICYGKL I L TLR Q IKESSA - Geotria LWS V VCK P FNLK-FDGKVG A IVL I IFSW AWS AGW CA P P I FGWS R Y W P H G L KTSCGPD VFSGSTD PGVQSYMV VL M I TC C F I PLAL I IICYLQVWLA IHTVQQQKA E c-opsin Rana SWS1 VIC K P MG TFT -FTSKHA LAVVLTTW M I GVGV SV P P F FGWS R Y I PEGL QCSCGPD W YTVGTKYHSEYY TWF IF V FFC IIPLTL I CY S Y ARLLGALR A VA AQQQE Latimeria Rh1 V VCK P MG NFR - FASSHA IMGI AFTW IMALACAAP P L V GWS R Y I PEGL QCSCGPD YY T L NPD FHNESYVMYLF LV HF LLP IIII FFTY GRL I CKVKEAA AQQQE Xenopus green-rod opsin VIC K P MNFT-FRESHG A VLGCILTW V IGLVAA I P P L L GWS R Y I PEGL QCSCGPD W Y TVNNKWNNESYVLFLF CFFC GFPLAI I VFS Y GRLLLALHAVKQQEA Q Platynereis r-opsin VITNP LG AAQTMTKKRA FI ILTIIW ANASLW ALAP F FGWGAY I PEGFQTSCTYD Y- -LTQD MNNY TY VLGMYLF G F IFP VA I I FFCYLG I VRA I FAHHA EMMA Mizuhopecten GqRhodopsin VIT K P LQASQTMTRRKVHLMI VIVW VLS ILLSIP P F FGWGAY I PEGFQTSCTFD Y - - LTKTARTRTY IVVL YLF G F LIPL III GV CYVL I IRGVR R HDQKMLT Octopus opsin VIGRP MA ASKKMSHRRA FLMI IFVW MW S IVW SVGP V F N W GAY V PEG ILTSCSFD Y- -LSTD PSTRS FILCMYFCGF ML P IIII AFCYFN I VMSV SNHEKEMAA Sepia opsin VIGRP MAASKKMSHRRA FLMI IFVW MW S TLW SIGP I FGWGAY VLEGVLCNC SFD Y- - ITRD SA TRS NIVCMYIF A F CFP ILI I FFCYFN I VMAVSNHEKEMAA Hemigrapsus Rh1 I I CNGFN GPK - L TQGKA TFMCGLAW VIS VGW SLP P F FGWGSY TLEG ILDSCSYD Y- -FTRD MNT I TY NICIF IF D F FLP ASV I VFS Y VF I VKA I FAHEAAMRA Limulus ocellus opsin VIVRGMA AA P - L THKKA TLLLLFVW IWS GGW TILP F FGWS R Y V PEGNLTSCTVD Y- -LTKD WSSA SYVI IYGLAVYFLPL ITMIYCYFF I VHAVAEHEKQLRE r-opsin Xenopus melanopsin VIT K P LQS IQWSSKKRTSQ I I VLVW MY S LMW SLAP L L GWS S Y V PEGL RISCTWD Y- -VTSTMSNRSYTMML CCCV F FIPL IV I SHCYLFMFLA IR S TGRNVQK Homo melanopsin VITRP LA TFGVASKRRA AFVLLGVW LYALAW SLP PWF FG S A Y V PEGL LTSCSWD Y- -MSFTPAVRAY TMLL CCF V F FLPLLII IYCY IFI FRA I R E TGRA LQT Homo RGR HY CTR - - - - SQLAWNSA VSLV L FVW LSS AFW AA L P L L GWGHY DY E PGTCL C TLD Y- -SKGD RNFTS FLFTMSFF N F AMPLFITITS Y SLMEQKLGKSGHLQVN Branchiostoma opsin3 VICRH - D LVDKVNYNTYGVMAA LGW LFAAFW AA L P L V GWAEY ALE PSGTAC TINF--QKND SLY ISYVTSCF VLGF VV PLAVMAFCYWQASCFVSKVLKGDIA Mus peropsin T I SCP -DVGRRMTTNTYLSMI LGAW INGLFW ALMP IIGWASY A P DPTGATC T I NW - - RNND TSFVSYTMMV I VVNF IVPLTVMFYCYYHVSRSLR L YASDCA T

VI ‡ VII

Terebratalia c-opsin ------GKDSKI--TKKNEAVE R KMAKMV IIM ITVF FGSW S PYA IVSMFVAFG DGS - - SLPAAA IAAPA IIAKSAM IWNP I I YV L M N V Q FRSGFA EMYY Platynereis c-opsin ------GGSVA KAE R ELRV M TLLM VSL F MLAW S PYAVICMLA SFG PKD - - L LHP VA TV I PAM FAKSST M YNPL IYV F M N K Q FRRSLKVLLL Anopheles GRPop11 ------VGRVNRA E R RTSV M V A V M IVAF MV AW T PYA IFAL IEQFG PPE - - L IGP GLAVLPALVAKSS ICYNP I I YV G M N T Q FRAA FWR I RR Branchiostoma opsin4 ------FGNPQNTGR LV TTM V V V M IVCF LVC W T PYTVMAL IVTFG ADH - - LVTP TASV I P SLVAKSST A YNP I I YV L M N N Q FREFLLARLL Homo encephalopsin ------QTIQ---VIKILK YE KKLAKMCFLM IFTF LVC W M PY IV ICFLVVNG HGH - - LV T P TISIVSYLFAKSNVT YNPV IYV FIRKM FRRSLLQLLL Ciona opsin1 ------LSGGTSPE GEV TKMV V V M VTAF VFC W L PYAAFA MYNVVNPEA - -Q I DYALGAAPAF FAKTAT I YNPL IYIGLN R Q FRDCVVRM I I Geotria LWS ------SETTQKAE R DSRV M V V V M IFAYIFC W G PYTFFA CFA AANPGY - - A FHP LAAALPAY FAKSAIT YNP I I YV F M N R Q FRNC I MQL FF c-opsin Rana SWS1 ------SASTQKAE KEV SRM V V V M VGS F CLC YVPYAAMA MYM I TNRNH - - GLDLRFV T I PAF F S KSACVYNP I I YTFM N K Q FRGC IMETVV Latimeria Rh1 ------SASTQKAE KEV TRM V ILM VIGF LTAW V PYASAA FW I FCNRGA - - EFTA TLMTV PAF F S KSSCLFNP I I YV LLN K Q FRNCM I TTL L Xenopus green-rod opsin ------SA TTQKAE R ETRV M V I V M VVGF LVC W L PYASFALW A VTHRGE - - LFDLRMSSVP SV F S K A S T V YNPF IYIFM N R Q FRSCMMKM I I Platynereis r-opsin --TAKRMGANTGK---ADADKK SE IQIAK VAAMT IGTF MLSW T PYAVVGV FGM I K PHS EMF I HP LLAEIP VMMAKA S ARYNP I I YALSHPKFRAE IDKHFF Mizuhopecten GqRhodopsin - - I TRSMK T EDA RA - - NNKRARSE LR ISK IAMTVTCLF IISW S PYA IIAL I A QFG PA H - -W I T P LVSELP MML AKSSSMHNPVVY ALSHPKFRKA LYQRVV Octopus opsin - -MAKRLNAKELRK- -AQAGASA E MK LA K ISMV I ITQF MLSW S PYA IIALL A QFG PA E - -WV T P YAA EL P VLFAKA S AIHNP I V Y SVSHPKFREA IQTTFF Sepia opsin - -MAKRLNAKELRK- -AQAGASA E MK LA K ISIV IVTQF LLSW S PYAVVALL A QFG PIE- -WVTP YAAQL P VMFAKA S AIHNPL IYSVSHPKFREA I A ENF F Hemigrapsus Rh1 --QAKKMNVTNLRSN-EA ETQRA E IRIAK TALV NVSLWF IC W T PYAAITIQGLLG NA E - - G I T P LLTTLPASLLAK CSCYNPFVY AISHPKFRLA I TQHLL Limulus ocellus opsin - - QA KKMNVA S L RA NA DQQKQS A E CRLAK VA MMTV G LWFMAW T PYLIIA WA GV F S SG T - - R L T P LAT IWGSVFAKANSCYNP I V Y G I SHPRYKAALYQRFF r-opsin Xenopus melanopsin ------LGSYGRQSFLSQSMK NE WKMA K IAFV IIIVF VLSW S PYACV T L I A WA G HGK - - S L T P YSKTV PAVIAKA S AIYNP I I YG I I HPKYR ET I HKTVV Homo melanopsin -FGACKGNGESL---WQRQRLQSE CKMAK IMLLV ILLF VLSW A PYSAVALV A FAG YA H - - V L T P YMSSV PAVIAKA S AIHNP I I YA I THPKYR VA I AQHL L Homo RGR ------TTLPARTLLLGW G PYA ILYL Y A VIADVT- -SISP KLQMVPALIAKMV P T I N A I N Y ALGN EMV CRG IWQCL L Branchiostoma opsin3 GDLTFPVA------ANVDWEYQNHFSKMCLAM VAA F VVAW T PYSV L FL F A A FWNPA - - D I PAWL T L L P PL IAKSSALYNP I I Y IIAN RRFRNA I CSMMM Mus peropsin AH------LHRDWADQADV TKMSV I M ILMF LLAW S PYSIVCL W A CFG NPK - - K I PP SMA I I A PL FAKSST F YNPC IYV AAHKKFRKAMLAMFF Figure 2 Alignment of deduced amino acid sequences for Terebratalia c-opsin and representative opsins from other taxa.Consensus amino acids (>50%) for the alignment of all opsins are shaded grey. Consensus amino acids only for the alignment of c-opsins are shaded green. In some cases the consensus residue for c-opsins differs than that for all opsins. C-opsins have a purple background, r-opsins have an orange background, and ourgroup opsins have a white background. A red bar highlights the conserved C-terminal motif of c-opsins. Transmembrane helices I-VII are marked with black horizontal lines. (‡) marks the position of the glutamate counterion and (◊) marks the conserved lysine that forms a Schiff base with retinal to form rhodopsin. Passamaneck et al. EvoDevo 2011, 2:6 Page 5 of 17 http://www.evodevojournal.com/content/2/1/6

Terebratalia c-opsin Platynereis c-opsin 98 100 Branchiostoma opsin4 54 93 99 Branchiostoma opsin5 - 95 100 Mus encephalopsin 100 - 100 Homo encephalopsin 72 Takifugu TMT opsin 100 Anopheles GPRop11 100 Anopheles GPRop12 Ciona opsin1 80 - 100 Oncorhyncus LWS 100 Geotria LWS

Anolis P opsin c-ops 88 Geotria SWS1 58 100 Rana SWS1 100 100 86 91 Rattus blue-cone opsin i 56 100 Cynops SWS1 ns 100 62 69 52 Geotria RhB 100 100 Latimeria Rh1 56 100 - 100 100 81 Petromyzon RhA 100 Geotria RhA 100 75 Latimeria Rh2 - 97 Gallus opsin 100 100 96 Homo rhodopsin 84 100 61100 Mus rhodopsin 100Xenopus opsin

100 Geotria SWS2 99 100 Xenopus green-rod opsin 95 Gallus blue-cone opsin 100 Danio VAL opsin 100 Petromyzon pineal opsin

100 Bos RGR 100 Mus RGR 100 51 Homo RGR 62 - 100 Branchiostoma opsin3 82 89 100 Mus peropsin - 100 Homo peropsin 100 Mizuhopecten Go-rhodopsin 72 100 Branchiostoma opsin1 98 Branchiostoma opsin2 68 Mizuhopecten Gq-rhodopsin - 98 Platynereis r-opsin 100 63 Schistosoma opsin 76 Octopus opsin 100

Toradores opsin r-ops 100 100 100 98 Sepia opsin 91 100 65 Loligo s. opsin 88 100 Loligo f. opsin i 64 ns 97 Hemigraspus Rh1 100 100 100 99 100 Hemigraspus Rh2 98 Limulus ocellus opsin

100 Xenopus melanopsin 100 100 Mus melanodopsin 98 Homo melanodopsin 100 Homo muscarinic acetylcholine receptor 100 Drosophila muscarinic acetylcholine receptor 100 Homo fifth somatostatin receptor 100 91 Drosophila allatostatin receptor 80 100 Homo NK-1 receptor 100 Drosophila tachykinin-like receptor

0.1 Figure 3 Phylogenetic analysis of bilaterian opsins places Terebratalia c-opsin with c-opsins from deuterostomes and other protostomes. Phylogram from Bayesian likelihood analysis with four independent runs of 5,000,000 generations each. Posterior probabilities are presented above branches; bootstrap support values >50% from a 1,000 replicate maximum likelihood bootstrap analysis are shown below branches. Passamaneck et al. EvoDevo 2011, 2:6 Page 6 of 17 http://www.evodevojournal.com/content/2/1/6

Dorsal view Lateral view AB Dorsal Anterior larva Posterior Middle

Ventral Blastoporal view Lateral view C D Animal

* Early gastrula *

Vegetal EF

* Middle gastrula *

Dorsal GH larva Early Anterior Posterior

Ventral Figure 4 Developmental expression of Tt-c-opsin. (A, B) AtthemiddlelarvastageTt-c-opsin is expressed in two laterally symmetrical punctate domains, just anterior of the dorsal rim of the apical lobe (black arrows), consistent with the location of the larval eye spots, as observed in (A) dorsal and (B) lateral views. A medial-anterior domain of weak expression is also observed (black arrowhead in panel A). (C, D) At the early gastrula stage broad expression of Tt-c-opsin is observed at the animal pole, opposite the blastopore (*). (E, F) By the middle gastrula stage the blastopore (*) has shifted 90° relative to the animal pole, and Tt-c-opsin expression becomes localized to a medial anterior domain, which persists through the early larva stage (G, H). (C, E, G) blastoporal views; (D, F, H) lateral views. Passamaneck et al. EvoDevo 2011, 2:6 Page 7 of 17 http://www.evodevojournal.com/content/2/1/6

Tt-c-opsin directly matches the position of the eyespots, Developmental expression of Tt-Pax6 and Tt-Otx supporting the ciliary nature of these photoreceptors. To further evaluate whether the Terebratalia larval In examining a range of embryonic stages, the first photoreceptors are bona fide cerebral eyes we analyzed onset of Tt-c-opsin expression was observed, unexpect- the expression of Pax6 and Otx orthologs. These two edly, at the early gastrula stage (Figure 4C, D). At this genes encode transcription factors that have been stage, Tt-c-opsin displays a broad domain of expression hypothesized to have conserved roles in the specification at the animal pole, the site of presumptive neurectoder- of cerebral eyes across bilaterians. For Pax6 a 433 aa full mal tissue, opposite the blastopore. By the middle gas- length gene product was predicted from cDNA trula stage the blastopore has shifted 90° relative to the sequences, and for Otx a 270 aa gene product was pre- animal pole, forming the anteroposterior and dorsoven- dicted from cDNA sequences. Phylogenetic reconstruc- tral axes. At this stage Tt-c-opsin is localized to a subset tions supported the orthology assignments, and we have of cells directly anterior of the apical tuft, which are therefore named the cloned genes Terebratalia trans- derived from the animal pole (Figure 4E, F). The med- versa Pax6 (Tt-Pax6)andTerebratalia transversa Otx ial-anterior domain of expression persists through the (Tt-Otx), respectively (Figure 5). early larval stages, when the three larval regions (apical, At the early larval stage, Tt-Pax6 is expressed in two mantle and pedicle lobes) are first distinguishable, broad triangular domains in the dorsal epidermis of the (Figure 4E, F). The medial-anterior domain of expres- presumptive apical lobe (Figure 6A, D). These two sion is distinct from larval expression in the presump- domains of expression meet in a narrow region at the tive eyes, and begins to fade once the trilobed larva has midline, and expand both anteriorly and, to a lesser extent, formed and setagenesis has commenced (Figure 4A, B). posteriorly towards the lateral regions of the embryo.

A B Drosophila eyeless 94 69 Terebratalia Otx 63 Homo Pax6 94 63 Octopus orthodenticle-like - 96 Mus Pax6 - Patella orthodenticle Saccoglossus Pax6 72 Platynereis Otx 67 Euprymna Pax6 Mus Otx2 87 55 Terebratalia Pax6 Mus Otx1 Strongylocentrotus Otx 100 60 Helobdella Pax6A 100 99 - Lineus Pax6 100 Euperipatoides otd Helobdella Pax6B Drosophila orthodenticle

Mus Pax4 56 Saccoglossus orthodenticle - 100 Artemia orthodenticle 99 Platynereis Pax6 89 82 100 Drosophila poxn Ciona Otx 96 Saccoglossus poxn Hydroides Otx Drosophila shaven 83 Drosophila orthopedia 98 74 Mus Pax2 100 Platynereis orthopedia 84 71 94 65 Patella orthopedia 58 Mus Pax5 Mus Pax8 61 Mus orthopedia Drosophila poxm Drosophila paired 100 Capitella Pax3/7 100 94 82 Mus Pax1 100 99 71 Branchiostoma Pax3/7 Mus Pax9 99 54 Mus Pax7 Mus Pax3 54 Mus Pax7 Mus Pax3 Drosophila paired Drosophila aristaless 75 61 Platynereis ARX 58 97 Capitella Pax3/7 63 Mus ARX 71 Helobdella Pax3/7 0.1 0.1 Figure 5 Phylogenetic analysis of Terebratalia Pax6 and Otx. (A) Phylogram of Terebratalia Pax6 and related proteins supporting orthology assignment. (B) Phylogram of Terebratalia Otx and related proteins supporting orthology assignment. Both phylograms are from from Bayesian likelihood analysis with four independent runs of 2,000,000 generations each. Posterior probabilities are presented above branches; bootstrap support values >50% from a 1,000 replicate maximum likelihood bootstrap analysis are shown below branches. Passamaneck et al. EvoDevo 2011, 2:6 Page 8 of 17 http://www.evodevojournal.com/content/2/1/6

length of the apical lobe posterior of the anterior end of Tt-Pax6 Tt-Otx Tt-Pax6 + Tt-Otx ABC the embryo. The placement of the overlap in Tt-Pax6 and Tt-Otx expression is, therefore, consistent with these two genes being co-expressed in the site of pre- sumptive larval eyespot formation. The domains of over- lapping Tt-Pax6 and Tt-Otx expression are in stripes of contiguous cells, rather than the punctate expression observed for Tt-c-opsin expression at the middle larva stage. By the middle larval stage, when setae have begun to DEF form, Tt-Pax6 expression was observed in the dorsal half of the apical lobe, extending from the posterior edge of the lobe to just anterior of the rim of the lobe (Figure 7A, B). The anterior portion of this expression pattern overlaps with the site of Tt-c-opsin expression (Figure 7C, D).

Embryonic photoresponse behavior Figure 6 Co-expression of Tt-Pax6 and Tt-Otx in the apical Expression of opsin genes in tissues other than pigmented lobe. (A) Dorsal view of Tt-Pax6 expression in the developing lobe eyes (for example, c-opsin expression in the Platynereis of the early larva. (B) Dorsal view of Tt-Otx expression in the adult brain [6]) has generally been viewed as related to developing lobe of the early larva. (C-F) Double label in situ with Tt- detection of non-directional light signals, such as diurnal Pax6 and Tt-Otx probes. All images are of the same embryo. (C) DIC image of double label in situ with Tt-Pax6 (red; Fast Red TR/ cycles, due to the lack of a shading pigment to block off- Naphthol AS-MX) and Tt-Otx (blue; NBT/BCIP) probes, showing axis illumination of the photoreceptor [6,16]. Terebratalia overlapping expression in two lateral stripes (black arrows). embryos hatch as ciliated blastulae, and by the middle gas- (D) Fluorescence image of Tt-Pax6 expression. (E) False color image trula stage, when anteroposterior polarity is first estab- of Tt-Otx expression, captured with brightfield illumination and a lished, their swimming is spiral with a left-handed rotation TRITC filter set to eliminate Fast Red TR/Naphthol AS-MX signal. (F) Merge of Tt-Pax6 and Tt-Otx, demonstrating overlap in two lateral about the anteroposterior axis. To evaluate whether early stripes (white arrows). Tt-c-opsin might be part of an early directional photore- ceptor, we tested for photoresponsive behavior in middle gastrula stage embryos, when Tt-c-opsin expression At the early larval stage there are five domains of Tt- becomes restricted to the medial anterior domain. Otx expression on the dorsal side of the forming apical Middle gastrula stage embryos were placed in a photo- lobe (Figure 6B, E). One medial spot of expression is taxis chamber where directional illumination could be located near the posterior edge of the apical lobe. Two introduced from one side of the chamber. Prior to the sets of laterally symmetrical stripes of expression are initiation of directional illumination the embryos located in the anterior region of the apical lobe. The showed no significant difference in distribution between more anterior pair of stripes is slightly offset from the the two sides of the chamber (Figure 8; Additional file midline, and the stripes extend in an anteroposterior 3; P = 0.22; n = 3). 20 to 25 minutes after initiation of orientation. The second pair ofstripesislocatedmore directional illumination, embryos showed a significant posteriorly, and the stripes extend at approximately a bias in distribution towards the illuminated side of the 45° angle relative to the anteroposterior axis, centered chamber (Figure 8; Additional file 4; P = 0.005; n = 3). on the medial spot of expression. These more posterior 5 to 10 minutes after extinguishment of directional illu- stripes extend to the lateral sides of the embryo and do mination the embryos had returned to an equal distribu- not contact the medial spot of expression. tion between the two sides of the chamber (Figure 8; Double labeling of Tt-Pax6 and Tt-Otx demonstrated Additional file 5; P = 0.34; n = 3). Distributions before that the two genes are co-expressed only in a narrow and after illumination were equivalent (P = 0.17), while region on the dorsal surface of the developing apical both differed significantly from the distribution during lobe at the early larval stage (Figure 6C, H). At this illumination (P =0.02andP = 0.01, respectively). stage the posterior lateral bands of Tt-Otx expression Although the behavior of three-lobed stage larvae with overlap with the anterior edges of the Tt-Pax6 expres- eyespots in culture suggests that they are also photore- sion domain (Figure 6H). The region of overlap extends sponsive, measurements in a phototaxis chamber did mediolaterally along the dorsal surface of the apical not yield a quantifiable response to directional illumina- lobe, and is located approximately one-third of the tion (data not shown). Passamaneck et al. EvoDevo 2011, 2:6 Page 9 of 17 http://www.evodevojournal.com/content/2/1/6

A Tt-c-opsin B Tt-c-opsin

Dorsal view Lateral view C Tt-Pax6 D Tt-Pax6

Dorsal view Lateral view E Tt-Pax6 F Tt-c-opsin

Tt-c-opsin Tt-c-opsin G Tt-Pax6 H

Figure 7 Overlapping experession of Tt-c-opsin and Tt-Pax6 in the apical lobe. (A) Dorsal and (B) lateral views of Tt-c-opsin expression in the apical lobe of the middle larva stage. Strong expression is observed in two lateral lines of punctate staining, matching the position of the larval eyes (black arrows). (C, D) Tt-Pax6 is expressed in a broader domain at the same stage. The anterior edge of the Tt-Pax6 expression domain overlaps with that of Tt-c-opsin (white arrows). (E-H) Two-color fluorescent in situ with Tt-Pax6 and Tt-Otx probes. All images are of the same embryo. (E) Single confocal section of Tt-Pax6 expression (white arrows). Fluorescence at the edge of the apical lobe (white arrowheads) is due to endogenous autofluorescence of the vesicular bodies. Fluorescence between the mantle and pedicel lobes (blue arrowheads) is due to probe trapping. (F) Single confocal section of Tt-c-opsin expression (white arrows). Fluorescence between the mantle and pedicel lobes (blue arrowheads) is due to probe trapping. (G) Single confocal section showing overlapping Tt-Pax6 (green) and Tt-c-opsin (purple) expression (white arrows). (H) Single confocal section of Tt-c-opsin expression (purple; white arrows) overlayed with a DIC image to show the morphological position of expression domains. Passamaneck et al. EvoDevo 2011, 2:6 Page 10 of 17 http://www.evodevojournal.com/content/2/1/6

ciliary morphology have been shown to express c-opsin 150 n=3 n=3 n=3 p=0.22 p=0.005 p=0.34 class genes and rhabdomeric photoreceptors express 120 r-opsin genes (with the exception of the scallop Mizuho- pecten, where the mantle eyes have ciliary morphology, 90

embryos Illuminated side f but have been shown to express a Go opsin [20]).

60 Dark side Based upon the occurrence of rhabdomeric photore-

Number o ceptors in the larval eyes of annelids, arthropods and 30 hemichordates, Arendt and Wittbrodt [3] have suggested

0 that while rhabdomeric and ciliary photoreceptors co-ex- Pre- Directional Post- isted in the last common ancestor of bilaterians, the use illumination illumination illumination of rhabdomeric photoreceptors is the ancestral condition, Figure 8 Bar graph of results from photoresponse behavior experiments with middle gastrula stage embryos. Distributions with ciliary photoreceptors having been co-opted for of embryos on illuminated and dark halves of the phototaxis vision within the vertebrate lineage. To date, the expres- chamber are shown for sampling periods before, during, and after sion of c-opsin class genes has been investigated in only the period of directional illumination. Middle gastrula embryos two protostomes, the annelid Platynereis [6] and the hon- preferentially moved towards the light when exposed to directional eybee Apis [16]. In both cases, expression was observed illumination. in the brain, and due to the lack of pigmentation in both these structures they have been inferred to be non-visual Discussion photoreceptors. While we observed early expression of The diversity of eyes throughout the bilaterians has led Tt-c-opsin in the presumptive neuroectoderm at gastrula to an ongoing debate regarding their evolution. Based and early larval stages of Terebratalia development, we upon the observation that photoreceptive structures did not observe expression in the apical ganglion, which generally have either ciliary or rhabdomeric morphology, may be comparable to the brain of other protostomes. Eakin [1,9] proposed two lines of photoreceptor evolu- tion, with the rhabdomeric type having evolved from the Ciliary larval eyes in Terebratalia: novelty, substitution, or ciliary type early in the protostome lineage. Vanfleteren ancestral condition? and Coomans [17] concluded that rhabdomeric photore- By ultrastructural analysis we have demonstrated that the ceptors were a subset of ciliary photoreceptors, and thus larval eyespots of the brachiopod Terebratalia are com- photoreceptors are homologous across bilaterians. In posed of two photoreceptors cells, one forming the lens contrast to both these interpretations, Salvini-Plawen cell and the other the pigmented shading cell. Both cells and Mayr [10,18] interpreted the diversity of photore- possess elaborated ciliary membranes in the intercellular ceptors as evidence of polyphyletic origins, postulating space between the lens and the pigment granules, as well that photoreceptors have evolved many times indepen- as axonal projections extending to the apical ganglion. dently in bilaterians. Supporting the ciliary nature of the larval photoreceptors, The advent of molecular genetics led to the unexpected we observed that a c-opsin gene is expressed specifically discovery that homologs of several transcription factor at the position of the eyespots in the larva. Together families are required for eye formation in both verte- these results evidence that the Terebratalia larvae pos- brates and Drosophila, including otx/orthodenticle, Six3/ sess cerebral eyes that are capable of directional light sine oculis, and Pax6/eyeless. The role of Drosophila eye- detection, and that are of a ciliary nature, based upon less and vertebrate Pax6 genes in eye development, as both morphological and molecular criteria. well as the apparent functional equivalence of numerous Given that Brachiopoda group within the protostome orthologs from diverse bilaterian taxa, prompted Gehring clade Spiralia (Lophotrochozoa) in molecular phyloge- and Ikeo [8] to propose a monophyletic origin for bilater- nies [11,12,21], our results provoke the question of ian eyes. While the homology of eyes based upon a role whether ciliary photoreceptors play a more important for Pax6 as a “master control gene” has been criticized as role in protostome eye evolution than previously an oversimplification [19] it appears that Pax6 is a key thought. Whereas polychaete trochophore larvae deploy player in eye development of most bilaterians. rhabdomeric photoreceptors (r-opsin expression) directly The potential for the phylogenetic distribution of photo- connected to locomotory cilia for directional movement receptors with ciliary and rhabdomeric morphologies to [22], the morphology of the Terebratalia eyespots be of evolutionary significance has been bolstered by the strongly suggests that brachiopods use ciliary photore- recognition that the two types of photoreceptors are char- ceptors with c-opsin expression for the same purpose. acterized by the expression of distinct classes of opsin We cannot rule out the possibility that an r-opsin genes. In all cases examined to date, photoreceptors with homolog is also expressed in the larval eyes of Passamaneck et al. EvoDevo 2011, 2:6 Page 11 of 17 http://www.evodevojournal.com/content/2/1/6

AB C Protostomia Protostomia Protostomia Platynereis Terebratalia Vertebrates Platynereis Terebratalia Vertebrates Platynereis Terebratalia Vertebrates

Cerebral eyes with Cerebral eyes with Ancetral state Substitution Novelty ciliary phtoreceptors rhabdomeric phtoreceptors Figure 9 Alternative hypothesis on the evolution of photoreceptor deployment in cerebral eyes. Schematic representation of three hypotheses accounting for the deployment of ciliary photoreceptors in the cerebral eyes of Terebratalia and vertebrates, versus rhabdomeric photoreceptors in Platynereis and other protostomes. (A) Deployment of rhabdomeric photoreceptors as the ancestral state in cerebral eyes, with the larval eyes of Terebratalia, containing ciliary photoreceptors, representing an evolutionary novelty. The deployment of ciliary photoreceptors is the result of a substitution (with ciliary photoreceptors having replaced rhabdomeric photoreceptors in the cerebral eyes) early in the chordate lineage. (B) Larval eyes in Terebratalia are homologous to the cerebral eyes in other protostomes, but ciliary photoreceptors have been substituted for rhabdomeric photoreceptors, as in the vertebrates. (C) Ciliary photoreceptors in cerebral eyes represent the ancestral condition, inherited by Terebratalia and vertebrates. Deployment of rhabdomeric photoreceptors in the cerebral eyes of Platynereis and other protostomes are the result of substitution events.

Terebratalia, as our attempts to clone such a gene with photoreceptor cells having been substituted for the degenerate primers were unsuccessful. rhabdomeric photoreceptor cells observed in the larval If manifold independent acquisition of photoreceptor eyes of other taxa; III) Larval eyes with ciliary photore- cells in Bilateria can be ruled out by the duality of the ceptors are the ancestral condition for protostomes existing phototransduction cascades and their respective and have been inherited by Terebratalia. photoreceptor cell types (rPRCs versus cPRCs) based on Visual photoreceptors with ciliary-type morphologies bilaterian opsin phylogeny [3], a functional switch from have been identified in several protostomes; however, visual to non-visual roles (or vice versa)forthetwo these organs have generally been regarded as evolution- commonly inherited photoreceptor cell types may have ary novelties due to their morphological locations (for happened several times independently in bilaterian evo- example,the branchial crown eyes in polycheates [23,24], lution. Such a scenario has already been proposed for and the mantle eyes of scallops [25]. The ciliary larval the evolution of the visual rods and cones in the verte- eyes of Terebratalia could likewise represent an evolu- brate retina as derivatives of non-visual ciliary deep- tionary novelty that has recruited a ciliary photoreceptor brain photoreceptors of invertebrates, such as those in to form a cerebral larval eye comparable to, but not the annelid Platynereis [6]. homologous with, the rhabdomeric larval eyes of other For the evolution of brachiopod larval eyes this sug- spiralians (Figure 9A). gests that brachiopods have retained ciliary photore- Although the ciliary photoreceptor cells in the larval ceptor cells from the bilaterian ancestor, and deployed eyes of Terebratalia seem not to be homologous to the these for directional light detection in their larval eye rhabdomeric photoreceptor cells in the larval eyes of spots. Given that ciliary photoreceptor cells are a ple- Platynereis and other protostomes, the possibility exists siomorphic trait, rather than being independently that there is homology at the level of the larval eye. If evolved in brachiopods, three alternative scenarios may the regulation of eye specification is distinct from that account for the ciliary nature of the larval eyes in Ter- of photoreceptor cell differentiation, then the ciliary ebratalia: I) The larval eyes of Terebratalia are evolu- photoreceptor cell may have been substituted for the tionary novelties, unrelated to the rhabdomeric rhabdomeric photoreceptor cell in a homologous larval cerebral eyes of other larvae in the clade Spiralia; II) eye (Figure 9B). In a variety of protostomes and deuter- The cerebral eyes of Terebratalia are homologous to ostomes, Pax6 and Otx (among other transcription fac- the larval eyes of other members of the clade Spiralia tor genes) have been shown to be involved in eye (for example, polychaetes and mollusks), with ciliary specification and differentiation, or to be expressed in Passamaneck et al. EvoDevo 2011, 2:6 Page 12 of 17 http://www.evodevojournal.com/content/2/1/6

cerebral eyes or their precursors (for example, Droso- associated with shading pigments for use in directional phila [26,27], Platynereis [4,28], mouse [29,30]). Expres- photoperception (that is, pigmented eyes). If this is the sion of Pax6 and Otx in the precursors of the case, then the ciliary eyes of Terebratalia and other spir- Terebratalia larval photoreceptors suggests that the alians may represent an ancestral condition, rather than genetic network underlying the formation of the larval being evolutionary novelties. eyes in Terebratalia shares common features with the network underlying the specification of rhabdomeric lar- A minimal photoreceptor mediating early photoresponse val eyes in other protostomes. In Terebratalia, ciliary behavior photoreceptor cells, and c-opsin expression, may have The photoresponse behavior of the gastrula stage embryo been co-opted to supplant the rhabdomeric photorecep- is a somewhat surprising result. This photoresponse may tor cells in homologous ancestral eyes. By analogy, onto- be attributed to one of two alternative mechanisms, genetic changes from one photoreceptor cell type to the phototaxis or photokinesis. Phototaxis, movement to or other have been observed in the larval cerebral eyes of away from light, is associated with directional photore- the gastropod mollusk Aporrhais pespelecani,inwhich ceptors, which are generally viewed as requiring an asso- the photoreceptor cell initially has a ciliary morphology, ciated shading pigment to block off-axis light [3,43]. only to later develop microvilli, taking on a mixed-type Nilsson [41] has proposed that scanning photopercep- morphology [31]. Although the expression of opsin tion, wherein movement of the photoreceptor allows genes in the Aporrhais eye is unknown, the ontogentic detection of differential light intensities, may have pro- alterations it undergoes suggest that photoreceptor cell vided a primitive mechanism detecting the directionality morphology may be decoupled from cerebral eye of light; however, relatively few examples of such a specification. photoreceptor have been described. Although photore- Finally, it should be considered that eyes with ciliary ceptors without associated shading pigments have been photoreceptors represent the ancestral state for Spiralia, described from a variety of metazoans, they have almost and possibly for Bilateria (Figure 9C). Arendt and Witt- always been attributed to having non-visual roles in mon- brodt [3] proposed that cerebral larval eyes with rhabdo- itoring ambient luminance, such as for detection of diur- meric photoreceptors represent the ancestral state for nal or lunar cycles of illumination. We propose that in Bilateria, based in part upon the occurrence of eyes with middle gastrula stage Terebratalia embryos, the yolk of this morphology in annelids, mollusks, platyhelminthes, the lecithotrophic embryo acts as a partial shading pig- crustaceans and hemichordates. As stated above, eyes ment to block off axis light, while the spiral swimming with ciliary photoreceptors in protostomes have been patterns serves to generate a scanning movement for regarded as evolutionary novelties or “phylogenetically directional sampling of illumination intensities. Alterna- young organs” [10]. However, it should be noted that tively, accumulation of embryos on the brightly illumi- cerebral larval eyes with ciliary morphology have been nated side of the chamber may represent a photokinetic described from gastropod mollusks [32-35], and cerebral response; that is, a change in movement in response to larval eyes with both ciliary and rhabdomeric photore- light, independent of photoreceptor orientation. In this ceptor cells have been described in both platyhelminthes scenario, a slowing in the speed of ciliary beating of [36,37], and the hemichordate Ptychodera flava [38]. c-opsin expressing cells in response to increased illumi- Likewise, photoreceptors with ciliary morphology, which nation could cause the distribution of embryos to shift may be cerebral eyes, have been described from the lar- towards the light source. Further experiments are vae of ectoprocts [39,40] and an entoproct [41], both required to resolve whether phototaxis of photokinesis is of which are members of the protostome clade responsible for the observed positive photoresponse Spiralia, along with brachiopods, annelids, mollusks, and behavior of the middle gastrula stage embryos. platyhelminthes [12]. Irrespective of the mechanism of the photoresponse, it While ciliary photoreceptors are not the predominant is of particular interest that the positive photoresponse form in the larval cerebral eyes of protostomes, they are behavior of the middle gastrula stage embryos occurs found in a phylogenetically diverse range of taxa. It before the onset of neuronal differentiation. At this stage should, therefore, be considered that the use of ciliary the c-opsin expressing cells and their neighbors consti- photoreceptors in eyes may be an ancestral condition tute a ciliated columnar epithelium without axonal con- for Spiralia, and possibly Bilateria. In contrast to this nections. This suggests that the photoresponsive cells hypothesis, Arendt et al. [6] proposed that localization may also serve as direct behavior effectors, either through of unpigmented ciliary receptors to the deep-brain, as alteration of ciliary beating patterns or through media- seen in Platynereis, represents the ancestral state for tion of changes in the orientation of the elongate cilia of bilaterians. However, Nilsson [42] has recently suggested the apical tuft to alter the direction of swimming in that such photoreceptors might historically been response to light. An analogous case is that of the Passamaneck et al. EvoDevo 2011, 2:6 Page 13 of 17 http://www.evodevojournal.com/content/2/1/6

parenchymellalarvaeofcertainsponges,whichshow the unpigmented middle gastrula stage embryo prior to positive and/or negative phototaxis behavior in the neural differentiation. We propose that c-opsin may facil- absence of a nervous system or gap junctions for cell-to- itate photosensitivity of a simple scanning photoreceptor, cell communication. In sponge larvae, phototactic beha- in which phototaxis is facilitated through the autono- vior is thought to be mediated by a posterior ring of pig- mous activity of cells that act as both photoreceptors and mented cells [44-46]. However, the mechanism by which ciliated behavioural effectors. This would represent a changes in ciliary behavior in response to variable illumi- very simple system similar to hypothesized intermediates nation may affect larval swimming behavior is not fully in the evolution of directional photoreception. understood [45,46]. In addition, no definitive opsin ortholog has yet been isolated from sponges, although Methods over 200 rhodopsin-related GPCR genes have been iden- Animal culture tified in the recently published genome of Amphimedon Gravid adult Terebratalia transversa were obtained at queenslandica [47]. It has been hypothesized that sponge Friday Harbor Laboratories (San Juan Island, WA, larvae may use a non-homologous mechanism for photo- USA), and in vitro fertilization was performed following reception, such as flavin [48], carotenoid [48], or cyto- established protocols from Reed [51]. Briefly, gametes chrome c oxidase [49]. A minimal photoreceptor cell has were dissected from gravid animals and ovaries were also been proposed to occur in the larva of the box jelly- macerated through 250 μm Nitex mesh (Sefar Inc, fish Tripedalia cystophora, based upon morphology [50]. Depew, NY, USA to separate oocytes. Oocytes were Pigmented cells in these larvae have rhabdomeric micro- allowed to settle in a beaker of filtered seawater, washed villi and a motor cilium, and occur in the absence of a several times with filtered seawater, and maintained in a nervous system. However, opsin expression has not been flow-through sea table until germinal vesicle breakdown shown for these cells, nor has phototactic behavior been and shedding of follicle cells were observed though a demonstrated for the larvae. stereomicroscope (approximately six to eight hours). Our results suggest that in Terebratalia middle gas- Testes were macerated in filtered seawater made alkaline trula stage embryos, c-opsin expressing cells at the ante- to pH 9.8 with 1 N sodium hydroxide, and the solution rior of the embryo may be mediating a positive was monitored on a compound microscope until sperm phototactic response in the absence of discrete shading became visibly motile (approximately 20 minutes). pigments or axonal connections between cells. As such, A total of 5 ml of sperm solution was added to oocytes the Terebratalia gastrula may utilize one of the simplest in 250 ml of filtered seawater, and washed out after one systems of directional photoperception and effector hour. Embryos were collectedatdifferentstagesupto behavior described to date in bilaterians. Additional stu- the competent larva. dies will be required to understand the details of this phototactic behavior, including the effect of changes in Transmission electron microscopy light intensity on rates of ciliary beating, and the poten- Embedded three-lobed stage larvae of Terebratalia trans- tial role of c-ospin expression in mediating this behavior. versa (Sowerby, 1846) were kindly provided by Stephen A. Stricker (Department of Biology, University of New Conclusions Mexico, Albuquerque, NM, USA) for further investiga- Using both morphological and molecular analyses we tion. Details of the fixation process have been published have provided evidence that the larval cerebral eyes of elsewhere [52]. Ultrathin serial sections of about 70 nm the brachiopod Terebratalia transversa have ciliary thickness were cut with a diamond knife on a Leica photoreceptors expressing c-opsin as a photopigment. Ultracut UCT microtome (Leica, Wetzlar, Germany), The co-expression of Pax6 and Otx in the domains subsequently stained with 1% uranyl acetate (50 minutes/ where the larval eyes will form suggests that the larval 30°C) and lead citrate (25 minutes/25°C) in a Leica EM eyes of Terebratalia share common patterning mechan- Stain and examined with LEO 912 Omega (Zeiss, Ober- ismswith the cerebral eyes of other bilaterians, and may kochen, Germany) and Philips CM 120 Biotwin (FEI, be homologous to the rhabdomeric larval eyes of other Eindhoven, The Netherlands) transmission electron spiralian taxa. These results suggest that the deployment microscopes. Photographs were taken on Kodak EM of ciliary and rhabdomeric photoreceptors for directional 4489 negative films digitised with a Silver Fast Mikrotek photodetection in the Bilateria has been more evolutiona- ScanMaker 1000 × l (Lasersoft Imaging, Kiel, Germany) rily labile than current hypotheses of eye evolution have or on Ditabis photoplates digitised on a Ditabis scanner proposed. (DITABIS, Pforzheim, Germany). Digitized photographs The timing and location of early Tt-c-opsin expression were processed and arranged using Adobe CS3 (Adobe is coincident with a positive photoresponse behavior in Systems Inc., San Jose, CA, USA). Passamaneck et al. EvoDevo 2011, 2:6 Page 14 of 17 http://www.evodevojournal.com/content/2/1/6

Gene isolation antibody (Roche Applied Science, Indianapolis, IN, Fragments of Tt-c-opsin and Tt-Pax6 were amplified by USA) at a 1:1,000 dilution in 1× Blocking Reagent degenerate PCR using as template complementary DNA (Roche Applied Science, Indianapolis, IN, USA) over- from mixed stages. Degenerate primers for semi-nested night at 4°C, and stained with TSA Plus Tetramethylr- amplification of Tt-c-opsin: WSNTAYATHATHTTYY- hodamine at a 1:100 dilution for 60 minutes at room TITTYRTITTY, forward [6]; GCNTGGWSICCITAYGC, temperature. Stained larvae were washed for 48 hours nested forward; NCKRAAYTGIKTRTTCATIMMIACR- with multiple exchanges of 1× PBS buffer, and cleared TADAT, reverse [6]. Degenerate primers for nested ampli- with 80% glycerol. Confocal imaging was performed fication of Tt-Pax6: GTNAAYCARYTNGGNGGNGT, using a Zeiss LSM 710 microscope (Carl Zeiss MicroI- forward; GTNAAYGGN MGNCCIYTICC, nested for- maging, Inc., Thornwood, NY, USA) with a 40×/1.3 NA ward; RTCNCKDATYTCCCANGCRAA, reverse; TTNG oil immersion objective. GYTTNSWNCCNCCDAT, nested reverse. Tt-Otx was initially identified from an EST clone sequenced for phylo- Phylogenetic analysis genomic analysis [11]. Full length cDNAs were obtained The deduced amino acid sequences for Terebratalia by rapid amplification of cDNA ends using the SMART c-opsin, Pax6 and Otx, along with those for representa- RACE kit (Clontech Laboratories, Inc., Mountain View, tive related proteins from other taxa, retrieved from CA, USA). The use of several degenerate primer sets tar- NCBI (html://ncbi.nlm.nih.gov/; accession numbers geted against r-opsin did not lead to the amplification of a listed below), were aligned with MUSCLE [55]. The gene fragment. resultant alignments were corrected by eye and non- conserved regions excluded from further analyses. For Whole mount in situ hybridization each dataset, the best-fit model of protein evolution was In situ hybridizations were carried out using an estab- determined with ProtTest [56] and was employed in all lished protocol [53]. NBT/BCIP stained larvae were subsequent analyses. For the c-opsin alignment, the imaged under brightfield Nomarski optics with a Zeiss WAG+I+G model was determined to be the best-fit, Axiocam HR mounted on a Zeiss Axioskop 2 mot plus. while for both Pax6 and Otx the JTT model was the Double label in situ hybridizations were imaged with a best-fit. Bayesian likelihood analysis of all datasets was Hamamatsu Orca mounted on a Zeiss Imager Z1. NBT/ performed with v3.1.2 of Mr. Bayes [57,58]. For c-opsin, BCIP staining was imaged with brightfield illumination four independent runs of 5,000,000 generations each and a 45/Texas Red filter to minimize signal from the were performed, and a burn-in of 1,000,000 generations HNPP/FastRed precipitate. HNPP/FastRed staining was was applied. For Pax6 and Otx, four independent runs imaged by fluorescence illumination with a 45/Texas of 2,000,000 generations each were performed, and a Red filter. The NBT/BCIP image was inverted and false- burn-in of 500,000 generations was applied. For all data- colored in ImageJ [54], and the NBT/BCIP and HNPP/ sets, bootstrap support values were derived from a 1,000 FastRed images where merged to determine regions of replicate maximum likelihood analysis performed in co-expression. PhyML [59].

Whole mount fluorescent in situ hybridization Accession numbers for sequences included in Two-color fluorescent in situ hybridizations were car- phylogenetic analyses ried out with TSA Plus Fluorescence Kits (PerkinElmer, opsins Waltham, MA, USA). Fixed larvae with hybridized with Anolis P opsin (AAD32622.1); Anopheles GPRop11 Digoxigenin-11-UTP (Roche Applied Science, Indiana- (XP_312503.3); Anopheles GPRop12 (XP_312502.2); Bos polis, IN, USA) labelled Tt-c-opsin and Fluorescein-12- RGR (NP_786969.1); Branchiostoma opsin1 (BAC7 UTP (Roche Applied Science, Indianapolis, IN, USA) 6019.1); Branchiostoma opsin2 (BAC76020.1); Branchios- labelled Tt-Pax6 probes at 2.5 ng/μleach,for48hours toma opsin3 (BAC76023.1); Branchiostoma opsin4 at 60°C. Larvae were incubated overnight with Anti- (BAC76021.1); Branchiostoma opsin5 (BAC76022.1); digoxigenin-POD antibody (Roche Applied Science, Ciona opsin1 (BAB68391.1); Cynops SWS1 (BAB79499); Indianapolis, IN, USA) at a 1:1,000 dilution in 1× Block- Danio VAL_opsin (NP_571661.1); Drosophila allatosta- ing Reagent (Roche Applied Science, Indianapolis, IN, tin_receptor (AAF05299.1); Drosophila muscarinic acetyl- USA) overnight at 4°C, and stained with TSA Plus Cya- choline receptor (AAA28676.1); Drosophila Tachykinin- nine5ata1:100dilutionfor60minutesatroomtem- like receptor (NP_524304.2); Gallus blue-cone opsin perature. Following staining, peroxidase activity was (NP_990848); Gallus opsin (P22328); Geotria LWS extinguished by incubation in 2.7% hydrogen peroxide (AAR14680); Geotria RhA (AAR14682); Geotria RhB in 1× PBS buffer for 90 minutes. Subsequently, larvae (AAR14683); Geotria SWS1 (AAR14684); Geotria SWS2 were incubated overnight with Anti-fluorescein-POD (AAR14681); Hemigrapsus Rh1 (Q25157); Hemigrapsus Passamaneck et al. EvoDevo 2011, 2:6 Page 15 of 17 http://www.evodevojournal.com/content/2/1/6

Rh2 (Q25158); Homo encephalopsin (NP_055137.1); 8360.1); Strongylocentrotus Otx (NP_999753.2); Terebrata- Homo fifth somatostatin receptor (BAA04107.1); Homo lia Otx (HQ679622) melanopsin (NP_150598.1); Homo muscarinic acetylcho- line receptor (CAA68560.1); Homo NK-1 receptor Photoresponse behavior assay (AAA59933.1); Homo peropsin (NP_006574.1); Homo Middle gastrula stage embryos were placed in a phototaxis RGR (P47804); Homo rhodopsin (NP_000530.1); Latimeria chamber with a 1.7 cm diameter. The chamber was Rh1 (AAD30520.1); Latimeria Rh2 (AAD30519); Limulus mounted on a stereo microscope with a red filter over the ocellus opsin (P35361); Loligo f. opsin (P24603); Loligo s. transmitted light base. The chamber was agitated prior to opsin (Q17094); Mizuhopecten Go-rhodopsin (O15974.1); the initiation of transmitted illumination to evenly distri- Mizuhopecten GqRhodopsin (O15973); Mus encephalop- bute the embryos. Embryos were exposed to transmitted sin (NP_034228.1); Mus melanopsin (NP_038915.1); Mus red-light illumination for five minutes prior to initiation of peropsin (NP_033128.1); Mus RGR (NP_067315.1); Mus directional illumination. Embryos were then exposed to rhodopsin (NP_663358); Octopus opsin (P09241); Oncor- lateral directed illumination from a cold white-light source hyncus LWS (AAP58346); Petromyzon RhA (Q98980); Pet- for 30 minutes, followed by an additional 5 minutes with romyzon pineal opsin (O4249); Platynereis c-opsin only transmitted red-light illumination. Time-lapse images (AAV63834.1); Platynereis r-opsin (CAC86665.1); Rana were required during the entire experiment at a rate of SWS1 (BAA96828); Rattus blue-cone opsin (NP_112277); two frames per second. Distribution and movement of Schistosoma opsin (AAF73286.1); Sepia opsin (O16005); embryos was measured with ImageJ [52] and Volocity Takifugu TMT opsin (AAL83430.1); Terebratalia c-opsin (PerkinElmer, Waltham, MA, USA) software. Embryos (HQ679623); Todarodes opsin (P31356); Xenopus within1mmofthesidesofthechamberwereexcluded green-rod opsin (AAO38746); Xenopus melanopsin measurements to avoid possible edge effects. (AAC41235.1); Xenopus opsin (P29403) Pax6 Additional material Capitella Pax3/7 (ABC68267.1); Drosophila eyeless (NP_524628.2); Drosophila paired (NP_523556.1); Droso- Additional file 1: Presumed light-perceptive cilium of the pigment phila poxm (NP_001036687.1); Drosophila poxn cell in a larval eye of Terebratalia. (A-I) Series of aligned sections to illustrate the ciliary membrane forming the stack of membranes (m) in (NP_476686.1); Drosophila shaven (NP_524633.3); the optical cavity enclosed by the lens (ls) and the pigment granules Euprymna Pax6 (AAM74161.1); Helobdella Pax3/7 (pg). (J) Close-up of the membrane stack (m) showing the invagination (ABI17942.1); Helobdella Pax6A (ABN09915.2); Helob- of the ciliary membrane to enlarge its surface (arrow). (K) Cross-section of the same cilium showing its 9 × 2 + 2 microtubule pattern. Scale bars: della Pax6B (ABN09916.2); 0.5 μm. Homo Pax6 (NP_000271.1); Lineus Pax6 (CAA64847.1); Additional file 2: Alignment of deduced amino acid sequences for Mus Pax1 (NP_032806.2); Mus Pax2 (NP_035167.3); Mus C-terminus of Terebratalia c-opsin and representative c-opsins from Pax3 (NP_032807.3); Mus Pax4 (NP_035168.1); Mus Pax5 other taxa. Alignment of the of Terebratalia c-opsin C-terminus to the C- termini of other c-opsins. The conserved C-terminus domain is required (NP_032808.1); Mus Pax6 (NP_038655.1); Mus Pax7 for localization of c-opsin proteins to the ciliary compartment, through (NP_035169.1); Mus Pax8 (NP_035170.1); Mus Pax9 binding to the light chain dynein Tctex-1 [60]. (NP_035171.1); Platynereis Pax6 (CAJ40659.1); Saccoglos- Additional file 3: Middle gastrula swimming prior to directional sus Pax6 (NP_001158383.1); Saccoglossus poxn (NP_ illumination. Time lapse imaging of middle gastrula stage embryos swimming in the phototaxis chamber prior to the initiation of directional 001158393.1); Terebratalia Pax6 (HQ679621) illumination. Embryos are evenly distributed throughout the chamber. Otx Frame rate is 5× faster than real-time. Artemia orthodenticle (ACQ90718.1); Branchiostoma Additional file 4: Middle gastrula swimming with directional Pax3/7 (ABK54280.1); Capitella Pax3/7 (ABC68267.1); illumination. Time lapse imaging of middle gastrula stage embryos swimming in the phototaxis chamber 20 minutes after the initiation of Ciona Otx (NP_001027662.2); Drosophila aristaless directional illumination. Embryos are clustered on the left side of the (NP_722629.1); Drosophila orthodenticle (CAA41732.1); chamber, closest to the source of directional illumination. Frame rate is Drosophila orthopedia (NP_001097388.2); Drosophila 5× faster than real-time. paired (NP_523556.1); Euperipatoides otd (ABY60730.1); Additional file 5: Middle gastrula swimming after directional illumination. Time lapse imaging of middle gastrula stage embryos Hydroides Otx (ABK76302.1); Mus ARX (NP_031518.2); swimming in the phototaxis chamber 10 minutes after the cessation of Mus orthopedia (NP_035151.1); Mus Otx1 (NP_035153.1); directional illumination. Embryos have returned to an even distribution Mus Otx2 (NP_659090.1); Mus Pax3 (NP_032807.3); Mus throughout the chamber. Frame rate is 5× faster than real-time. Pax7 (NP_035169.1); Octopus orthodenticle-like (AAZ99 218.1); Patella orthodenticle (AAM33144.1); Patella orthopedia (AAM33145.1); Platynereis ARX (ADG26 Abbreviations 723.1); Platynereis orthopedia (ABR68849.1); Platynereis BCIP: 5-Bromo-4-chloro-3-indolyl phosphate; cPRC: ciliary photoreceptor cell; ’ Otx (CAC19028.1); Saccoglossus orthodenticle (NP_00115 DIC: differential interference contrast; HNPP: 2-hydroxy-3-naphtoic acid-2 - Passamaneck et al. EvoDevo 2011, 2:6 Page 16 of 17 http://www.evodevojournal.com/content/2/1/6

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