A protocadherin mediates cell-cell adhesion and integrity of the oral placode in the tunicate Ciona

A protocadherin mediates cell-cell adhesion and integrity of the oral placode in the tunicate Ciona | 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 A protocadherin mediates cell-cell adhesion and integrity of the oral placode in the tunicate Ciona Sriikhar Vedurupaka , Bita Jadali , Christopher J. Johnson , View ORCID Profile Alberto Stolfi , Sydney Popsuj doi: https://doi.org/10.1101/2025.07.11.664433 Sriikhar Vedurupaka 1 School of Biological Sciences, Georgia Institute of Technology , Atlanta, GA, 30332, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Bita Jadali 1 School of Biological Sciences, Georgia Institute of Technology , Atlanta, GA, 30332, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Christopher J. Johnson 1 School of Biological Sciences, Georgia Institute of Technology , Atlanta, GA, 30332, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Alberto Stolfi 1 School of Biological Sciences, Georgia Institute of Technology , Atlanta, GA, 30332, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Alberto Stolfi For correspondence: spopsuj1{at}swarthmore.edu alberto.stolfi{at}biosci.gatech.edu Sydney Popsuj 1 School of Biological Sciences, Georgia Institute of Technology , Atlanta, GA, 30332, USA 2 Biology Department, Swarthmore College , Swarthmore, PA, 19081, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: spopsuj1{at}swarthmore.edu alberto.stolfi{at}biosci.gatech.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract In chordate embryos, placodes are ectodermal thickenings around the borders of the neural plate that give rise to various sensory organs and cell types. While generally thought to be a vertebrate-specific innovation, homologous placodes are proposed to exist in non-vertebrate chordates as well. In Ciona robusta, a solitary tunicate, the adult mouth (the oral siphon) is derived from one such “cranial-like” placode in the larva, which we term the oral siphon placode (OSP). At embryonic and larval stages, the OSP consists of a small rosette of cells that forms from the neuropore at the anteriormost extent of neural tube closure. While the morphogenesis of the OSP and its physical separation from other surface ectoderm structures have been described in detail, how this is regulated at the molecular level is currently unknown. Here we show the involvement of protocadherin-mediated cell-cell adhesion in the segregation and structural cohesiveness of the OSP. Protocadherin.e ( Pcdhe.e ) is expressed specifically in the OSP but not in other surface ectoderm cells. CRISPR/Cas9-mediated disruption of Pcdh.e in these cells results in loss of OSP structural integrity and ability to physically separate from other structures derived from the same cell lineage. Overexpression of Pcdh.e throughout the anterior surface ectoderm results in similar loss of a physically separate and distinct OSP territory. Furthermore, we show that Pcdh.e expession in the OSP depends on oral placode-specific transcription factors such as Six1/2 and Pitx. Our results suggest that OSP integrity and morphogenesis require precise regulation of a homotypic cell-cell adhesion molecule, which might reflect a conserved mechanism for placode formation in chordates. Introduction Tunicates are the closest living relatives to vertebrates, and their simple embryos and compact genomes make them attractive model organisms to study chordate evolution and vertebrate origins ( Fodor et al., 2021 ). The oral siphon of tunicate Ciona robusta is widely accepted as homologous to the vertebrate mouth and originates from the incomplete closure of the anterior neuropore ( Veeman et al., 2010 ). As the neural plate rolls up from posterior to anterior in a coordinated “zippering” process ( Hashimoto et al., 2015 ), the anterior end of the neural tube remains open as a small rosette of cells that form a thickening of the surface ectoderm called the stomodeum, or oral siphon placode (OSP)( Veeman et al., 2010 ). After metamorphosis, this placode will give rise to the exterior opening of mouth, also called the oral or incurrent siphon, of the post-metamorphic juvenile and later adult. Interestingly, the oral siphon of adult tunicates possess hair cell-like sensory cells ( Manni et al., 2004a ). Thus, the OSP might be similar to cranial placodes that give rise to various sensory cells and ganglia in vertebrates ( Anselmi et al., 2024 ; Fritzsch and Glover, 2024 ; Manni et al., 2004b ). Like vertebrate cranial placodes, the OSP is a thickening of the surface ectoderm arising from the anterior border the neural plate, forming a small rosette of tightly clustered cells that is distinct from the surrounding epidermis ( Figure 1A )( Manni et al., 2005 ; Veeman et al., 2010 ). However, how these cells form a distinctive cluster, to the exclusion of surrounding surface ectoderm, is not known. It has been shown that homotypic cell adhesion molecules are crucial for neural tube closure ( Hashimoto and Munro, 2019 ; Smith et al., 2021 ), but how such molecules affect OSP development has not been fully investigated. Interestingly, several conserved placodal regulatory genes such as Six1/2 ( Schlosser, 2006 ; Schlosser et al., 2008 ) have been described in the cells which produce the OSP within larval development. These expressional patterns pushed us to further investigate candidate genes associated with the maitenance of vertebrate placodes in Ciona such as protocadherins, cell adhesion molecules that represent the largest group within the cadherin superfamily ( Morishita and Yagi, 2007 ). Download figure Open in new tab Figure 1. A) Ciona robusta (intestinalis type A) embryo at Hotta Stage 25 (late tailbud III, ∼13 hours post-fertilization at 20°C), showing cell outlines and the oral siphon placode (OSP) rosette revealed by Phalloidin-AlexFluor546 staining. B ) In situ mRNA hybridization revealing Protocadherin.e ( Pcdh.e ) expression in the presumptive OSP, as well as in the Motor Ganglion Interneuron 2 (MGIN2) cells as previously reported ( Gibboney et al. 2020 ). C ) Summary diagram of the separation of Six1/2+, future Protocadherin.e (Pcdh.e)- and Pitx- expressing oral siphon placode cells (red outline) from other Foxc+ cells (blue), mainly those contributing to the papillae. D ) Protein domain analysis diagram of Protocadherin.e (Pcdh.e) from SMART ( Letunic et al. 2021 ), showing presence of a signal peptide (red block), 6 extracellular cadherin repeats (CA) and a transmembrane (TM) domain, similar to the organization of vertebrate protocadherin-family proteins. In Ciona, protocadherins are represented by five different genes with dynamic expression patterns during embryogenesis, as part of a larger classical cadherin and cadherin-related genes ( Noda and Satoh, 2008 ). One of these, which we termed Protocadherin.e ( Pcdh.e, KyotoHoya gene ID KH.C9.518, previously referred to as Ci- δ -protocadherin-5 ), was shown to be expressed in the Ciona OSP ( Figure 1B ) as the cells begin to coalesce and separate from other surface ectoderm cells ( Figure 1C )( Gibboney et al., 2020 ; Noda and Satoh, 2008 ). In vertebrates, placode rosette formation is driven in part by the formation of homotypic cadherin-dependent apical adherens junctions ( Breau and Schneider-Maunoury, 2015 ). Due to the highly localized, specific expression of Pcdh.e in the OSP, we hypothesized that this adhesion protein-encoding gene ( Figure 1D ) plays an analogous role in mediating homotypic adhesion between the cells in the OSP, allowing them to set themselves aside as a self-organizing rosette. Here we show through a combination of Pcdh.e overexpression and CRISPR/Cas9-mediated disruption that the proper segregation of OSP progenitors into the characteristic rosette of cells likely depends on homotypic Pcdh.e-mediated adhesion, linking its highly-localized expression to the formation of this important, potentially conserved sensory placode and oral opening in a non-vertebrate chordate. Materials and methods Ciona handling, fixation, staining, and imaging Adult Ciona robusta specimens were collected in California, around San Diego (M-REP) or Los Angeles/Orange County (Marinus Scientific). Dechorionated zygotes were generated and electroporated as described ( Christiaen et al., 2009a , b ). Embryos were raised in filtered artificial sea water at 20°C to desired Hotta stages ( Hotta et al., 2020 ; Hotta et al., 2007 ), then fixed and prepared onto microscope slides using MEM-FA solution (3.7% formaldehyde, 0.1 M MOPS pH 7.4, 0.5 M NaC1, 1 mM EGTA, 2 mM MgSO4, 0.1% Trition-X100), rinsed in 1X PBS, 0.4% Triton-X100, 50 mM NH4Cl for autofluorescence quenching, and a final 1X PBS, 0.1% Triton-X100 wash. Phalloidin-AlexFluor546 (Thermo Fisher) staining was carried out using a 1:50 dilution, as previously described ( Lowe et al., 2021 ). Imaging was accomplished using Leica DMI8 or DMIL LED inverted epifluorescence and scanning point confocal microscopes. For Pcdh.e reporter quantification, larvae were imaged on a Leica DMI8 using constant exposure for GFP and mCherry channel images, and mean fluorescence intensity in regions of interest encompassing the oral siphon primordia regions were recorded. GFP-to-mCherry mean fluorescence intensity ratios were calculated for each individual region of interest. One outlier (ratio > 2.0) in the control and one in the Pitx CRISPR condition were removed. Statistical data was plotted and analyzed in GraphPad Prism. Previously published and new plasmid sequences can be found in the Supplemental Sequences File. CRISPR/Cas9 sgRNA design and validation Single-chain guide RNAs (sgRNAs) were designed using CRISPOR ( http://crispor.tefor.net/ ) and expression vectors were constructed as previously described ( Gandhi et al., 2017 ; Gandhi et al., 2018 ) or custom synthesized and cloned (Twist Bioscience). 75 µg of each individual sgRNA vector was validated in vivo as previously described ( Popsuj et al., 2024 ), by co-electroporation with 25 µg of a ubiquitous ( Sasakura et al., 2010 ) Eef1a -1955/-1>Cas9 or Eef1a -1955/- 1>Cas9::Geminin plasmid (per 700 µl of total electroporation volume). Genomic DNA was isolated using QiaAMP Micro extraction kit (QIAGEN), targeted regions amplified by PCR using AccuPrime Pfx (Thermo Fisher), and PCR products purified using the QiaQuick PCR Purification kit (QIAGEN) following the published protocol ( Johnson et al., 2023 ). Amplicons were sequenced using Illumina-based amplicon sequencing (Amplicon-EZ; Azenta) and efficiency was determined by indel plotting as automatically generated by Azenta. In total, four sgRNAs were created and validated targeting Pcdh.e and two were selected for further use: sgRNAs 2.100 and 3.38 targeting exons 2 and 3, respectively. For Six1/2 CRISPR, three sgRNAs were created and validated targeting the first exon, and one was selected for further use: sgRNA 1.358. For Pitx CRISPR, two were created and validated targeting the 2 nd constitutive exon, and both were used: sgRNAs 2.126 and 1.186. The predicted protein domain organization Pcdh.e was analyzed by SMART ( Letunic et al., 2021 ). Tissue-specific CRISPR/Cas9 and overexpression of Protocadherin.e Pcdh.e was specifically disrupted by CRISPR/Cas9 in the animal pole lineages using 25 µg FOG>Cas9::Geminin-N ter ( Pennati et al., 2024 ; Song et al., 2022 ) co-electroporated with 45 µg of U6>Pcdh.e.2.100 sgRNA plasmid, 45 µg of U6>Pcdh.e.3.38 sgRNA plasmid, 50 µg of Six1/2>CD4::GFP , and 25 µg of Six1/2>H2B::mCherry . Results were compared to co-electroporation with 90 µg a standard “negative control” sgRNA (not targeting any known C. robusta genomic sequence) vector instead, as previously published ( Stolfi et al., 2014 ). The Six1/2 promoter used was based on that previously cloned and published ( Abitua et al., 2015 ). The FOG ( Friend of GATA, e.g. Zfpm , KyotoHoya ID KH.C10.536) promoter has been published and is extensively used to drive Cas9 in animal pole lineages ( Pennati et al., 2024 ; Rothbächer et al., 2007 ). Distances and lengths were measured in Leica LAS X software, and statistically analyzed and plotted in GraphPad Prism. We manually excluded outliers >100 µm (fewer than 10 individuals in any one condition) as these could not be confidently distinguished from cases of leaky reporter expression in more anterior cells, or other non-specific developmental defects. To overexpress Pcdh.e in the anterior neural plate (common progenitors of the OSP + papilla territory), we used the published Foxc promoter ( Wagner and Levine, 2012 ), co-electroporating 90 µg of Foxc>Pcdh.e, 25 µg of Foxc>H2B::mCherry, and 50 µg of Foxc>Unc-76::GFP . This was compared to a negative control without Foxc>Pcdh.e. New and previously published plasmid sequences can be found in the Supplemental Sequences File. Tissue-specific CRISPR/Cas9 of Six1/2 and Pitx Six1/2 and Pitx genes were specifically disrupted by CRISPR/Cas9 in the animal pole lineages using 30 µg FOG>Cas9::Geminin-N ter co-electroporated with 90 µg of U6>Six1/2.1.358 , 45 µg each of U6>Pitx.2.126 and U6>Pitx.2.186, or 90 µg of “negative control” sgRNA plasmids. All were co-electroporated with 80 µg of Pcdh.e -4608/-1447 + bpFOG>Unc-76::GFP and 25 µg of FOG>H2B::mCherry reporter plasmids. New and previously published plasmid sequences can be found in the Supplemental Sequences File. Results Tissue-specific disruption of Pcdh.e by CRISPR/Cas9 Pcdh.e expression was previously shown by in situ mRNA hybridization to be expressed in cells of the OSP at tailbud stages ( Gibboney et al., 2020 ; Noda and Satoh, 2008 )( Figure 1B ). We therefore decided to test its function in the OSP using tissue-specific CRISPR/Cas9-mediated mutagenesis as previously described ( Stolfi et al., 2014 ). We targeted the Pcdh.e gene using a combination of two validated sgRNAs predicted to target exons 2 and 3 ( Figure 2A ). Quantification of sgRNA-induced indels by next-generation sequencing revealed an efficacy of at least ∼20% and ∼30%, respectively ( Figure 2B ). To restrict Cas9 activity to the lineage leading to the OSP progenitors, we used the Friend of GATA (FOG) promoter to drive its expression ( FOG>Cas9). Embryos were co-electroporated with sgRNA and Cas9 expression plasmids, and with Six1/2>CD4::GFP and Six1/2>H2B::mCherry reporter plasmids to visualize the OSP. Hatched larvae were fixed and imaged at 20 hours post-fertilization (hpf), raised at 20°C. Pcdh.e CRISPR larvae were compared to negative control larvae electroporated instead with the standard U6>Control sgRNA plasmid that was designed to not target any C. robusta sequence and has been routinely used in the field ( Stolfi et al., 2014 ). Download figure Open in new tab Figure 2. A) Diagram of the Pcdh.e locus indicating single-chain guide RNA (sgRNA) target sites in exons 2 and 3. B) Indel plots generated by Azenta/Genewiz E-Z amplicon next-generation sequencing, showing the presence of CRISR-mediated indels (red arrows) in the Pcdh.e gene upon validation of either the 2.100 or 3.38 sgRNA (see text for details). The percentage of reads is indicated on y axis, position in basepairs in the amplicon indicated on x axis. Red asterisks denote naturally occurring indels. C ) Results of tissue-specific CRISPR/Cas9-mediated disruption of Protocadherin.e ( Pcdh.e ) in F0. Top: larva electroporated with FOG>Cas9::Geminin N-ter , a negative control sgRNA, Six1/2>H2B::mCherry, and Six1/2>CD4::GFP. The Six1/2 promoter drives oral siphon placode (OSP)-specific expression of H2B::mCherry (magenta nuclei) and CD4::GFP (green cell membranes). The FOG promoter drives expression in animal pole lineages, including the lineage that gives rise to the OSP. Bottom: larva electroporated with the same components as at top, but with Pcdh.e- targeting sgRNAs (2.100 and 3.38) instead. Dashed line indicates aberrant OSP cell spreading anteriorly towards the papillae. D ) Quantification of the anterior-posterior length of the Six1/2 reporter-expressing OSP cell territory in each imaged individual, compared between negative control and Pcdh.e CRISPR larvae. CRISPR was performed and analyzed in duplicate. **** indicates p H2B::mCherry (magenta) and Foxc>GFP (green), revealing normal separation of the oral siphon placode (OSP) territory and the papillae. Bottom: overexpression of Protocadherin.e (Pcdh.e) throughout the Foxc+ territory results in failure of OSP and papillae cells to properly segregate (dashed bracket). F ) Scoring the percentage of Foxc>GFP+ larvae with proper separation of OSP and papilla territories, showing substantial decrease in the Pcdh.e overexpression condition. GFP tagged at N-terminus with Unc-76 tag (see supplemental sequences file). CRISPR-mediated disruption of Pcdh.e appeared to result in loss of OSP rosette cohesion and separation from other surface ectoderm cells, compared to negative control larvae ( Figure 2C ). We decided to quantify this by measuring the maximum length between anterior and posterior edges of the OSP, as visualized by Six1/2>CD4::GFP and Six1/2>H2B::mCherry reporter expression labeling cell membranes and nuclei, respectively. We measured a statistically significant expansion of the OSP upon Pcdh.e CRISPR, and this effect was replicated in a duplicate experiment ( Figure 2D ). These results suggested Pcdh.e is necessary for maintenance of a cohesive, compact OSP territory in developing Ciona larvae. Ectopic expression of Pcdh.e prevents proper OSP separation Because we hypothesized that Pcdh.e is an adhesion molecule that maintains OSP integrity through homotypic cell-cell adhesion, we decided to overexpress Pcdh.e throughout the entire anterior neural plate using the Foxc promoter ( Foxc>Pcdh.e )( Wagner and Levine, 2012 ). Foxc+ cells of the anterior neural plate will divide at the late gastrula stage to give rise to distinct OSP and papilla progenitors ( Liu and Satou, 2019 ; Nicol and Meinertzhagen, 1988a , b ; Wagner and Levine, 2012 ). Committed OSP and papilla progenitor cells are initially adjacent to one another at early tailbud stages, however slowly separate into two physically disjointed pools of Foxc+ cells separated by cells from other lineages not expressing Foxc ( Veeman et al., 2010 ). As we expected, Pcdh.e overexpression by electroporation with Foxc>Pcdh.e plasmid resulted in a single Foxc+ territory spread out over the entire anterior portion of the dorsal surface ectoderm ( Figure 2E,F ). This suggested that the forced expression of Pcdh.e in normally Pcdh.e-negative papilla progenitors causes these cells to improperly adhere to (or prevents them from de-adhering from) Pcdh.e+ OSP cells, resulting in the failure of the papilla and OSP to separate. Our results suggest that Pcdh.e mis-expression in surface ectoderm is sufficient to cause aberrant cell-cell adhesion in the Ciona larva and prevent normal separation between different placode-like structures. Six1/2 and Pitx transcription factors positively regulate Pcdh.e in the OSP To further explore the regulatory pathways contributing to Pcdh.e expression in the OSP, we next investigated potential upstream transcription factors also important in vertebrate placode formation, Six1/2 and Pitx. Although their orthologs are conserved regulators of anterior placodes and placode-like structures in chordates ( Schlosser et al., 2014 ), their roles in OSP formation in Ciona have never been investigated. Six1/2 is expressed in the row of neural plate blastomeres that eventually give rise to the OSP ( Imai et al., 2004 ; Mazet et al., 2005 ), while Pitx is expressed in the OSP at tailbud stages, ( Boorman and Shimeld, 2002 ; Christiaen et al., 2002 ). To disrupt these genes out, we designed and validated sgRNAs targeting them for CRISPR knockout ( Figure 3A ). We obtained one sgRNA targeting Six1/2 at ∼43% mutagenesis efficacy, and two targeting Pitx, both measured to be at least 50% effective ( Figure 3A ). To assay Pcdh.e expression in CRISPR larvae, we tested different fragments 5’ to the first exon ( Figure 3B ). We found that a distal cis -regulatory sequence ( Pcdh.e -4608/-1447 relative to the start codon) was able to drive reporter gene expression in the OSP. A more proximal fragment ( Pcdh.e -1500/- 1) was not active in Foxc+ OSP cells, but rather was expressed in adjacent cells just posterior to the OSP, likely the anterior apical trunk epidermal neurons (aATENs) based on their position ( Figure 3B )( Abitua et al., 2015 ). Animal pole-specific CRISPR-mediated disruption of Six1/2 or Pitx resulted in modest and/or inconsistent loss of expression of the OSP-specific Pcdh.e reporter constructed using the -4608/-1447 fragment ( Pcdh.e[OSP]>GFP )( Figure 3C,D ). To better quantify this, mean fluorescence intensity of GFP expression was measured, normalized, and compared between CRISPR and negative control larvae ( Figure 3C,E ). This showed a significant reduction of Pcdh.e[OSP]>GFP expression in the OSP in either Six1/2 or Pitx CRISPR larvae. These data show that, although neither Six1/2 nor Pitx might be absolutely required, they appear to contribute positively to Pcdh.e expression in the OSP. Download figure Open in new tab Figure 3. A ) Top: Diagram of Six1/2 and Pitx loci, indicating the targets of the chosen, validated for CRISPR-mediated mutagensis. Bottom: Indel plots generated by Azenta/Genewiz E-Z amplicon next-generation sequencing as in Figure 2 , showing the presence of CRISR-mediated indels (red arrows). Red asterisks denote naturally occurring indels. B ) Left: diagram indicating distinct cis -regulatory sequences upstream of the Pcdh.e gene that drive reporter expression either in the OSP (-4608/-1447, henceforth referred to as Pcdh.e[OSP]>GFP ), or in adjacent, more posterior cells that may correspond to anterior apical trunk epidermal neurons (aATENs, -1500/-1). Right: representative images of Unc-76::GFP reporter plasmids constructed using the indicated cis -regulatory elements, showing expression (green) in or near the OSP (dashed outline, labeled by Foxc>H2B::mCherry expression in magenta). See Supplemental Sequences file for detailed sequence information. C ) Example of animal pole lineage-specific CRISPR-mediated knockout of Six1/2, showing reduction of Pcdh.e[OSP]>GFP expression (green) in the OSP. Lineage marked by expression of FOG>H2B::mCherry (magenta). D ) Scoring of Pcdh.e>GFP expression frequency in electroporated larvae, comparing Six1/2 CRISPR and Pitx CRISPR conditions to negative controls. Sample size of at least 37 larvae (ranging up to 100) for each experiment, performed in duplicate. One duplicate of the “negative control” condition is shared between Six1/2 and Pitx experiments, due to performing this set of electroporations in parallel. E ) Quantification of Pcdh.e>GFP/FOG>H2B::mCherry fluorescence ratio in the OSPs of CRISPR and negative control larvae, showing significant reduction of Pcdh.e reporter expression upon disruption of Six1/2 (**** p < 0.0001) and Pitx (** p = 0.0072), by two-tailed Mann-Whitney test. Discussion Here, using tissue-specific CRISPR/ Cas-9 -mediated mutagenesis and gene overexpression, we present evidence that Protocadherin.e (Pcdh.e) is expressed in the oral placode of the Ciona larva and is involved in maintaining its structural integrity as a separate and cohesive group of surface ectoderm cells. CRISPR/Cas9-mediated mutagenesis of Pcdh.e in the ectoderm resulted in less cohesive OSP territory, as measured by its spreading over the surface of the larva. This is what one would expect if Pcdh.e is required for OSP cells to adhere closely with one another, more so than to surrounding cells. In contrast, overexpression of Pcdh.e in the common progenitors of the OSP and papillae caused these two territories, which normally segregate from one another, to remain as a single cohesive unit. We further link OSP-expressed transcription factors Six1/2 and Pitx to the transcriptional activity of a Pcdh.e reporter construct. Taken together, our results support a model in which differential expression of adhesion molecules in defined subsets of surface ectoderm cells allows for their segregation and self-organization into distinct territories, such as the OSP. In vertebrates, classical cadherin adhesion molecules play crucial roles in the separation of placodes and the formation of cellular rosettes similar to that seen with the OSP of Ciona ( Breau and Schneider-Maunoury, 2015 ). For instance, classical cadherins were found to be important for the separation and invagination of the lens placode in mouse ( Pontoriero et al., 2009 ). Furthermore, Cadherin2-rich apical junctions are important for rosette formation in zebrafish lateral line primordia ( Revenu et al., 2014 ). Our results suggest that regulation of cadherin superfamily-dependent cell-cell adhesion might be a conserved feature of placode development that predates the vertebrate-tunicate split. In Ciona, there are two apparent “classical” vertebrate-type cadherin genes ( Sasakura et al., 2003 ), both of which are expressed in parts of the neural tube and surface ectoderm ( Noda and Satoh, 2008 ). However, a higher diversity of expression patterns of the related protocadherins is seen during embryogenesis ( Noda and Satoh, 2008 ). It was previously shown that delaminating tail neurons downregulate a distinct a protocadherin gene ( Pcdh.c) expressed in the surrounding surface ectoderm, and Pcdh.c mis-expression in these cells was sufficient to impair their delamination ( Stolfi et al., 2015 ). This further suggests that multiple protocadherins can mediate crucial cellular adhesion and sorting functions in Ciona . We propose that the dynamic expression of protocadherin genes observed in the Ciona embryo might underlie many cell sorting and morphogenetic events, perhaps in concert with or complementing the function of classical cadherins. While Pcdh.e might not be the only such adhesion molecule to maintain the OSP as a distinct and cohesive rosette, our data suggest it is a major factor in this morphogenetic process. In turn, the OSP is known to give rise to the future mouth (oral or incurrent siphon) of the post-metamorphic juvenile, later the adult. Additional structures and cell types arise from this region, for instance the velum and oral tentacles which contain putative mechanosensory hair cell-like cells ( Anselmi et al., 2024 ). Furthermore, other tissues are patterned around the mouth, such as the oral siphon muscles ( Berrill, 1947 ; Chiba et al., 2004 ). Future studies will be required to see if OSP integrity, potentially mediated by Pcdh.e-dependent cell-cell adhesion, is required for proper specification of these post-metamorphic tissues and structures. The authors declare no conflicts of interest Acknowledgments We thank Susanne Gibboney, Lindsey Cohen and Wesley Bartlett for technical assistance, and other members of the lab for advice. We thank Gwynna Fuller, Ankita Thawani, and Andy Groves for helpful feedback. SV was funded by a PURA scholarship award from the Georgia Institute of Technology. SP was funded by ARCS Foundation and Mortar Board fellowships. This work was funded by grant R01HD104825 from NIH/NICHD, grant R35GM158421 from NIH/NIGMS, and grant 1940743 from NSF IOS. Funder Information Declared National Institute of General Medical Sciences , R35GM158421 Eunice Kennedy Shriver National Institute of Child Health and Human Development , R01HD104825 Division of Integrative Organismal Systems , 1940743 References ↵ Abitua , P.B. , Gainous , T.B. , Kaczmarczyk , A.N. , Winchell , C.J. , Hudson , C. , Kamata , K. , Nakagawa , M. , Tsuda , M. , Kusakabe , T.G. , Levine , M ., 2015 . 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