Perlecan is expressed in the regenerating oral region and tentacles at stage 2.5 through stage 4.

<p>Animals were bisected midway through the mesentery region. Representative animals are pictured 48 hours post-bisection in (physal end-A, oral end-B). Regeneration of the oral structures and tentacles was followed in the area of the red box in (A). <i>Perl</i> activation is visible during regenerative stage 2.5–3 in the tentacle bud and oral structures (C). The developing cellular boundary between ectoderm and gastroderm is visible in the primordial tentacle (arrowhead, D) and is distinct from the developed mesoglea in the regenerated oral pole of the scapus (arrow, D). <i>Perl</i> remains elevated in the tentacles and oral structures during the intermediate stage of tentacle regeneration (stage 4, E, F). <i>Perl</i> expression recedes in the tentacles as they develop mesoglea (arrowheads, F) and remains where the mesoglea is not yet fully visible (F, arrows). Prior to completion of regeneration, <i>perl</i> expression continues in the oral ectoderm (asterisks, F). Scale bars, A,B-500μm C,E-100 μm D,F-40μm.</p

Molecular phylogenetic analysis by maximum likelihood method.

<p>The evolutionary history of perlecan was inferred by using the Maximum Likelihood method. Branch lengths are calculated according to nucleic acid substitutions per site. The eumetazoans are delineated by a dotted line.</p

Comparison of <i>H</i>. <i>sapiens</i> perlecan domain IV and <i>T</i>. <i>adhaerens</i> perl 1 and perl 2 domain IV.

<p>The primary structure of domain IV Ig modules were aligned, including all sequences between disulfide-bonded cysteines in each Ig module. The sequences were aligned and residues conserved in 25% or more of these Ig sequences were highlighted. The central Ig modules of <i>H</i>. <i>sapiens</i> domain IV, distinct from the outer Ig modules, are shown separately. Residues of the central Ig modules that are conserved amongst <i>H</i>. <i>sapiens</i> domain IV Ig modules, but not when compared alongside those of <i>T</i>. <i>adhaerens</i>, are outlined with a red dashed line. Ig sequences originating from <i>T</i>. <i>adhaerens</i> perl 1 and perl 2 are shown separately.</p

Perlecan is expressed in the ectoderm of the scapus, irregularly in the mesentery, and irregularly in the inner cell layer of the tentacles.

<p>An adult <i>N</i>. <i>vectensis</i> (A) was tested for <i>perl</i> mRNA expression. The ectoderm of the body wall has consistent <i>perl</i> mRNA signal in the ectoderm, whereas the gastroderm is mostly negative for <i>perl</i> mRNA. The acellular mesoglea is clearly visible in the physus and tentacle (arrowheads, B, D). The mesentery expresses <i>perl</i> mRNA in a sporadic fashion, with pigmented cells not expressing <i>perl</i> (C). Tentacle cells only express <i>perl</i> sporadically in the gastroderm (arrows, D,E). The oral end of the scapus does not express the <i>perl</i> transcript (A, arrowhead, E). Scale bars, A—0.5mm, B-E—40 μm.</p

Predicted perlecan orthologues or constituents in model organisms.

<p>The presumptive pre-perlecan constituents of <i>A</i>. <i>queenslandica</i> (A) and perlecan proteins of <i>T</i>. <i>adhaerens</i>, <i>N</i>. <i>vectensis</i>, <i>C</i>. <i>elegans</i>, and <i>H</i>. <i>sapiens</i> are shown in schematic form to demonstrate structural differences. Several of the scarce matches for perlecan modules in the <i>M</i>. <i>brevicollis</i> and <i>A</i>. <i>queenslandica</i> genomes are shown in schematic form. The number of unique matches for individual or paired perlecan folding modules in the <i>M</i>. <i>leidyi</i> transcriptome is shown in subscript next to each schematic module. The double line between <i>T</i>. <i>adhaerens perl 1</i> and <i>T</i>. <i>adhaerens perl 2</i> indicates that these two genes, although only thirteen kb apart on the chromosome, are separately transcribed. Blue rectangles represent parts of the <i>T</i>. <i>adhaerens</i> and <i>N</i>. <i>vectensis</i> transcripts amplified by RACE PCR and sequenced; all other parts of the protein were predicted by tBLASTn-alignment. Schematics are not to scale; gene lengths are denoted at the right end of each diagram. The red dashed line encompasses the “perlecan” clade of the animal kingdom.Surprisingly, the easily recognizable <i>perl</i> gene was found in the placozoan <i>T</i>. <i>adhaerens</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124578#pone.0124578.g001" target="_blank">Fig 1</a>). All five protein domains are included in the <i>T</i>. <i>adhaerens perl</i> gene, in the identical order to that of <i>HSPG2</i> in humans. <i>T</i>. <i>adhaerens perl</i> domain I includes a SEA module, which, until now, was thought to be found exclusively in perlecan of mammals and birds. The <i>T</i>. <i>adhaerens perl</i> SEA module includes the G-SVV motif, critical to the autocleavage of these modules in other proteins. Whereas the SEA module of perlecan in mammals and birds does not contain this autocleavage motif, we speculate that this ancestral perlecan molecule may have undergone autocleavage while in the secretory pathway. <i>T</i>. <i>adhaerens perl</i> domain II is structured exactly as that of human <i>HSPG2</i>, with three LDL receptor-like repeats followed by one immunoglobulin (Ig) domain. Domain III of <i>T</i>. <i>adhaerens</i> perl is also similar to that of <i>H</i>. <i>sapiens</i> perlecan/<i>HSPG2</i>, however in comparison, <i>T</i>. <i>adhaerens</i> perl includes one additional set of Laminin B- and Laminin EGF-like modules. Domain IV of <i>T</i>. <i>adhaerens</i> perl encodes roughly 36 total Ig modules, making it substantially larger than the domain IV of human perlecan, which only includes 21 Ig modules. Domain V of <i>T</i>. <i>adhaerens</i> perl encodes both the laminin G and EGF-like modules similar to other perlecan proteins.</p