Reference: Biol. Bull. 227: 211–220. (December 2014) © 2014 Marine Biological Laboratory
Integrins of the Starlet Sea Anemone Nematostella vectensis QIZHI GONG, KATRINA GARVEY, CHENGHAO QIAN, ISABEL YIN, GARY WONG, AND RICHARD P. TUCKER* Department of Cell Biology and Human Anatomy, University of California, Davis, Davis, California 95616 – 8643
Abstract. Integrins are extracellular matrix receptors composed of ␣ and  subunits. Here we describe two ␣ subunits and four  subunits from the starlet sea anemone Nematostella vectensis. Phylogenetic analysis suggests that the ␣ subunits are most closely related to RGD- and LDVdependent ␣ subunits of chordates. The  subunits cluster with the previously described  integrins of the hard coral Acropora millepora. The expression of one of the ␣ subunits and three of the  subunits was confirmed by reverse transcription PCR and in situ hybridization. The ␣ subunit is primarily expressed in cells near muscles, by a subset of gastrodermal cells, and in the gonad. The three  subunits each have distinctive patterns of expression: one is concentrated in the gonad and mesenteric filament, another is found in a subset of cells in the epidermis of the oral region and in a subset of gastrodermal cells in the mesenteries, and a third is expressed widely. Changes in expression were also studied 48 h after horizontal transection by quantitative reverse transcription PCR and in situ hybridization. One of the  subunits is expressed 8-fold higher during regeneration, and its expression is observed in cells within both the epidermis and the gastrodermis at the site of regeneration. Our observations confirm that complex patterns of integrin expression were already present in basal metazoans. The integrins expressed in the gonads may play roles in mediating spermegg interactions in N. vectensis, while others may play a role in regulating proliferation during regeneration.
Introduction Integrins are dimeric extracellular matrix receptors composed of one ␣ and one  subunit (for review see Campbell and Humphries, 2011). In chordates, where these receptors are most widely studied, the ␣ subunit is composed of a head domain formed from a -propeller, integrin ␣-2 domains that form regions known as the thigh and calves (due to the knee-like bend that can form between these regions), a transmembrane helix, and a short cytoplasmic domain (Fig. 1A). A subset of ␣ integrins also has an ␣A-domain in the head region. Most  subunits are composed of an integrin  head domain followed by a series of epidermal growth factor (EGF)-like repeats, a -integrin-specific tail domain, a transmembrane region, and a -integrin-specific cytoplasmic domain. A variation on this theme is demonstrated by the 4 integrins, which have a longer cytoplasmic domain that includes a Calx  domain and a string of fibronectin type III domains (Fig. 1A). In humans there are 18 genes encoding ␣ subunits and 8 genes that encode  subunits. To date, these have been shown to form 24 different dimers, most of which can bind multiple ligands (Barczyk et al., 2010). These are typically classified as being RGD-dependent receptors, LDV-dependent receptors, laminin-collagen receptors, or leukocyte-specific receptors. The first two classes are the best understood. The RGDdependent receptors bind to the tripeptide motif arginineglycine-aspartic acid found in many extracellular matrix ligands via the cleft formed between the heads of the ␣ and  subunits. The LDV-dependent receptors bind to a leucineaspartic acid-valine (or similarly acidic) motif, and ␣ subunits with the ␣A-domain bind to laminins and collagens (reviewed by Campbell and Humphries, 2011). Ligand binding leads to conformational changes that initiate signaling cascades that are often associated with cell motility, proliferation, and differentiation (for review see Wickstro¨m
Received 2 April 2014; accepted 21 July 2014. * To whom correspondence should be addressed. E-mail: rptucker@ ucdavis.edu Abbreviations: aa, amino acid; EGF, epidermal growth factor; PBS, phosphate buffered saline. 211
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Figure 1. Stick diagrams illustrating the domain architecture of integrins. (A) Representative ␣ and  integrins from Homo sapiens. (B) The domain organization of the two ␣ integrin subunits and four  integrin subunits identified in the genome of Nematostella vectensis.
et al., 2011). In addition, some integrins mediate sperm-egg interactions both in mammals and in some invertebrates (for review see Evans, 2012). The first integrins to be cloned from early-diverging metazoans were an ␣ integrin subunit from the demosponge Geodia cydonium (Pancer et al., 1997) and  integrin subunits from the demosponge Ophlitaspongia tenuis and the scleractinian coral Acropora millepora (Brower et al., 1997). The presence of these receptors in sponges and cnidarians demonstrated their fundamental relationship to the early extracellular matrix, and the absence of integrins from the genomes of choanoflagellates, which are widely regarded to be the sister group to metazoans, led to the conclusion that they evolved together with the first animals (e.g., Rokas, 2008). However, ␣ and  integrin subunits were subsequently identified in the protist Capsaspora owczarzaki, revealing an ancient origin for these receptors predating the appearance of animals and their selective loss in choanoflagellates (Sebe´-Pedro´s et al., 2010). Knack et al.
(2008) thoroughly described the integrins of A. millepora, including the results of expression studies. They found a single ␣ integrin subunit and two  integrin subunits, one of which corresponded to the  subunit previously identified by Brower et al. (1997). Expression was confirmed by reverse transcription PCR and by whole-mount in situ hybridization with gastrulae. A potential role for integrins in fertilization in cnidarians was proposed by Iguchi et al. (2007), who found that antibodies to a  integrin subunit reduced sperm-egg binding and fertilization rates in A. millepora. Nematostella vectensis is a small, burrowing anthozoan found in estuaries along both coasts of North America and the south coast of England (Hand and Uhlinger, 1994) that can be readily maintained and manipulated in the laboratory (Hand and Uhlinger, 1992; Darling et al., 2005; Technau and Steele, 2011; Bossert et al., 2013; Layden et al., 2013; Stefanik et al., 2013). For this reason, these sea anemones have become an important model organism for studying
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evolution, development, and regeneration. Their mesoglea is remarkable in its ultrastructural similarity to the extracellular matrix of chordates (Tucker et al., 2011), and a number of homologs of chordate extracellular matrix genes have been identified, many of which are known to be integrin ligands in other organisms (Koehler et al., 2009; Ozbek et al., 2010; Tucker et al,, 2013; Tucker and Adams, 2014). In addition to their work with A. millepora, Knack et al. (2008) identified three ␣ integrin subunits and four  integrin subunits in the genome of N. vectensis, suggesting that this model organism may have a more diverse repertoire of these receptors than other early-diverging metazoans. Here we describe the integrins of N. vectensis, including their phylogenetic relationships to other integrins, their expression in the adult polyp, and changes in their level of expression during regeneration. Materials and Methods Phylogenomic analysis Putative integrin genes from the Nematostella vectensis Stephenson, 1935, genome (Putnam et al., 2007) were identified using the domain architecture analysis feature at Pfam (http://pfam.sanger.ac.uk/) to identify predicted proteins with domains unique to integrins (e.g., ␣ integrin  propeller domains and  integrin head domains). The identity of the domains and their boundaries in the predicted proteins were identified using Interpro (Hunter et al., 2011), and molecular weights were determined at ExPASy (Gasteiger et al., 2005). For phylogenetic analysis, sequences were aligned using MUSCLE (Edgar, 2004) and trees were constructed with PhyML 3.0 (maximum-likelihood) using default parameters at Phylogeny.fr (Dereeper et al., 2008). Raising and transecting Nematostella vectensis Nematostella vectensis polyps derived from the CH2 and CH6 clones of Hand and Uhlinger (1992) were maintained in microfiltered 1/3 seawater at room temperature and fed a mixture of egg yolk and Artemia nauplii. Adults used for regeneration studies were immobilized with 7.5% MgCl2 and cut with a razor blade through the center of the body column in the region where gonad-bearing mesenteries are found. Both halves were maintained in 1/3 seawater for 48 h prior to quantitative PCR studies (see below), or just the aboral half was maintained for 48 h prior to processing for in situ hybridization (see below). Molecular and histological methods RNA was isolated from either juvenile (⬃4-week-old) or adult polyps using an RNeasy Mini Kit (Qiagen) and treated with amplification grade DNase I (Invitrogen) prior to determining the concentration and quality of the RNA with a nanodrop spectrophotometer. Reverse transcription PCR
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was performed using a Super Script III One Step RT-PCR Kit (Invitrogen) as described previously (Tucker, 2010). Intron-spanning primer pairs (Eurofins MWG/Operon) used were (1) 5⬘-ccaagggcaatgaagtgtct-3⬘ and 5⬘-tccagcatgtctcctcacag-3⬘ for ␣238305 (499bp); (2) 5⬘-actctgccccgtcagaagta-3⬘ and 5⬘-gcgtaagtcccgttaccaga-3⬘ for 143492 (298bp); (3) 5⬘-tgactgccctacatgtccag-3⬘ and 5⬘-caccacacccagaatgacag-3⬘ for 80868 (301bp); and (4) 5⬘-ggatgtcgcgaagaaaacat-3⬘ and 5⬘-caaatgatccaaaccccaac-3⬘ for 91193 (304bp). All PCR products were subcloned into pCR2.1 or pCRII by TOPO TA cloning (Invitrogen) and sequenced by Davis Sequencing (Davis, CA). For in situ hybridization, adult polyps were immobilized with 7.5% MgCl2 and fixed in ice-cold 4% paraformaldehyde in 0.01 mol l⫺1 phosphate buffered saline (PBS; Sigma P-3813) overnight, then rinsed in PBS and dehydrated in a series of ethanol baths, cleared in xylene, and embedded in Paraplast. Sections 8-m thick were cut on a microtome and collected on Superfrost Plus (Fisher) presubbed microscope slides. In situ hybridization was carried out essentially as described in Tucker and Gong (2014) with the following modifications. Both sense (control) and antisense (experimental) digoxigenin-labeled riboprobes were made using pCRII clones mentioned above as templates, and Proteinase K treatment was carried out at room temperature for 3 min. After postfixation, rinsing, and dehydration, riboprobes at 1 g/ml were put on the slides that were pre-warmed to 60 °C, and hybridizations were carried out at 65 °C overnight. Quantitative reverse transcription PCR was performed using the primer pairs described above. In brief, three adult polyps, either whole or transected 48 h previously, were sheared and homogenized with a 26-gauge needle in TRIzol reagent (Life Technologies), and the total RNA was precipitated from a chloroform suspension with isopropanol. cDNAs were obtained by reverse transcription with AMV reverse transcriptase and oligo dT primers. Quantitative reverse transcription PCR experiments were done in triplicate using SYBR Green chemistry on an ABI StepOnePlus system (Life Technologies). Expression levels of each integrin were standardized to the level of 18S rRNA using primer pair 5⬘-gactcaacacggggaaactc-3⬘ and 5⬘-gcaccaccacccatagaat-3’(Reitzel and Tarrant, 2009). The amplification protocol involved a 10-min denaturation step followed by 40 cycles of 95 °C and 60 °C (30 s each). To verify the quality of each primer pair, melting curves were obtained from the first step starting from 50 °C to 95 °C. The cycle threshold (Ct) and the standard deviation were calculated for each replicate. The average Ct of the Gene of Interest (GOI) (avg. CtGOI) was normalized to the average Ct of the reference gene (avg. Ctref) for the same sample to calculate the normalized Ct for the GOI (⌬Ct ⫽ avg. CtGOI ⫺ avg. Ctref). The standard deviation of the ⌬Ct was calculated by (stdevGOI2 ⫹ stdevref2)(1/2). The calibrated value (⌬⌬Ct) for each sample was determined by ⌬⌬Ct ⫽ ⌬CtR ⫺ ⌬CtU. The
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stdev ⌬⌬Ct is the same as stdev ⌬Ct. The fold-induction for each sample relative to the calibrator ⫽ 2(⫺⌬⌬Ct). Standard deviations were accounted for within the calibrator by incorporating them into the equation as such: 2(⫺⌬⌬Ct ⫾ Stdev). Results The two ␣ and four  integrin subunits of Nematostella vectensis Domain analysis reveals two ␣ integrin subunits encoded in the genome of Nematostella vectensis. The first was given the locus tag NEMVEDRAFT_v1g196726; here this predicted protein is called ␣196726. The second ␣ integrin subunit was assigned the locus tag NEMVEDRAFT_ v1g238305 and is referred to here as ␣238305. ␣196726 (NW_001834411) is composed of 1025 amino acids (aa) and has a predicted molecular weight of 112640 Da. It is composed of six or seven (depending on the domain analysis program used)  propeller domains (aa 25– 459), an integrin ␣-2 domain (aa 443– 894), a transmembrane helix (aa 971–993) and a short cytoplasmic domain (Fig. 1B). ␣238305 (NW_001834409) is composed of 1283 aa and has a predicted molecular weight of 141245 Da. It has a head composed of a series of  propeller domains (aa 1–502), a tail composed of two integrin ␣-2 domains (aa 483–722 and aa 812–1143), a transmembrane helix (aa 1124 –1247), and a short cytoplasmic domain (Fig. 1B). Unlike ␣196726, ␣238305 does not have a predicted signal peptide, indicating that the N-terminus may be incomplete. Neither ␣ integrin subunit has an ␣A-domain, a domain that is characteristic of the laminin-collagen receptors (Campbell and Humphries, 2011). Domain analysis reveals four  integrin subunits that are similarly named here by using the numeric portion of their locus tag (Fig. 1B). 80868 (XP_001641468; EDO49405) is a 786 aa protein with a predicted molecular weight of 85470 Da. Following a signal peptide (aa 1–25), it is composed of an integrin  head domain (aa 33– 456), three EGF-like domains (aa 493– 624), an integrin  tail domain (aa 634 –715), a transmembrane helix (aa 717–739), and an integrin  cytoplasmic domain (aa 740 –786). 91193 (XP_001637894; EDO45831) lacks a signal peptide but has what appears to be a nearly complete  integrin head domain (aa 25– 437; that is, it has plexin-like folds at its N-terminus). It also has three EGF-like repeats (aa 490 – 611), an integrin  tail domain (aa 622–709), a transmembrane helix (aa 710 –732), and an integrin  cytoplasmic domain (aa 733–779). 123281 (XP_001627336; EDO35236) appears to be a complete  integrin subunit composed of 786 aa and a predicted molecular weight of 87020 Da. Following the signal peptide (aa 1–35) is a  integrin head domain (aa 34 – 455), four EGF-like repeats (aa 478 – 621), an integrin  tail domain (aa 631–715), a transmembrane helix (aa 717–739), and an integrin  cytoplasmic domain (aa 740 –786). Finally, 143492 (XP_001621822; EDO29722)
also appears to be a complete predicted protein composed of 795 aa with a predicted molecular weight of 87538 Da. An integrin  head domain (aa 34 – 451) follows a signal peptide (1–26). These are followed by three EGF-like repeats (aa 504 – 623), a  integrin tail domain (aa 634 –719), a transmembrane helix (aa 724 –746), and an integrin  cytoplasmic tail domain (aa 748 –794). Phylogenetic relationships of the Nematostella vectensis integrins Phylogenetic tree analysis of the Nematostella vectensis ␣ integrins shows that ␣196726 clusters with a clade that includes four ␣ integrins from the sponge Amphimedon queenslandica, the ␣1 integrin from the stony coral Acropora millepora, ␣PAT-2 from Caenorhabditis elegans, and the RGD-binding ␣ integrin subunits from Homo sapiens ␣2b, ␣V, and ␣8 (Fig. 2A). ␣238305 clusters with the LDV-binding ␣4 and ␣9 integrins from H. sapiens. 80868 and 123281 are related to each other and cluster with the 2 integrin of A. millepora. 91193 and 143492 are related to each other and cluster with Cn1 from A. millepora. These  subunits do not appear to be related to any of the major groups of chordate  integrins (Fig. 2B). Integrin expression in Nematostella vectensis The expression of one of the two predicted ␣ integrin subunits (␣238305) and three of the four predicted  integrin subunits (143492, 80868, and 91193) was confirmed by reverse transcriptase PCR using cDNA made from RNA isolated from juvenile polyps (Fig. 3). Multiple primer pairs as well as template made from RNA isolated from both juvenile and adult polyps were used to try to demonstrate the expression of ␣196726 and 123281, but without success. The pattern of expression of the ␣238305 subunit was determined using in situ hybridization on paraffin sections of adult polyps. At the oral end of the polyp there was scattered expression in the gastrodermis and epidermis, but there was a robust signal in large cells found in the complete mesenteries suspending the pharynx, as well as in the pharynx itself (Fig. 4A). Cross sections through the body column also revealed limited, scattered expression in the gastrodermis of the body wall, but there was a robust signal in cells adjacent to the parietal and retractor muscles, as well as in the gonadal region of the mesentery (Fig. 4B). A control section hybridized with a sense probe is shown in Figure 4C. The pattern of expression of ␣238305 is consistent with expression by gametes (it is unknown if this specimen is male or female) and in cells that make myonemes (the myofibril-like protrusions found in the pharynx, parietal muscle, and retractor muscle), but the cells expressing this integrin in the mesenteries near the pharynx are unknown. We previously identified cells with the morphology of neurons in the mesoglea of the pharynx and
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Figure 2. Phylogenetic tree analysis of Nematostella vectensis integrin subunits. (A) The ␣ integrin subunit ␣196726 clusters near the RGD-binding ␣ integrins from other species, including human ␣5, ␣8, ␣2b, and ␣V. The ␣238305 integrin clusters with the LDV-binding ␣4/␣9 integrin family. The N. vectensis integrins are highlighted with an asterisk. (B) The four  integrin subunits from N. vectensis (asterisks) are related to the 2 and Cn1 subunits of the hard coral Acropora millepora, but not to the human  integrins. Branch support (Approximate Likelihood-Ratio test) greater than 0.7 is indicated. Scale bars indicate substitutions/site.
retractor muscle; at least one cell in the mesoglea of the retractor muscle was positive for expression of ␣238305 (Fig. 4B). The expression of integrin subunits 143492, 80868, and 91193 was also studied by in situ hybridization on transverse paraffin sections through the pharynx and midbody column. All three probes revealed expression over background (Fig. 5). 143492 expression was strongest throughout the epidermis, gonad, and mesenteric filament (Fig. 5A, E). In contrast, 80868 hybridized in cells scattered in the epidermis of the oral region and in large cells found in the mesenteries of the body column (Fig. 5B, F). The 91193 probe gave an intense, uniform signal throughout the polyp (Fig. 5C, G). Changes in integrin expression during regeneration Changes in expression during regeneration (48 h after transection) of the ␣238305 subunit and two of the  integrin subunits, 143492 and 80868, were examined by quantitative PCR. The ␣238305 subunit expression increased 1.5-fold, while the expression of the 143492 and 80868 subunits increased 5-fold and 8-fold, respectively (Fig. 6A). Patterns of gene expression at this time point were also examined by in situ hybridization in longitudinal
sections through the aboral half of transected polyps (Fig. 6B). ␣238305 transcripts are found in the region where regeneration is taking place (Fig. 6C), but the pattern of expression (i.e., in scattered gastrodermal cells) is not different from the hybridization signal observed in the body wall of untransected polyps. The probe to 143492 transcripts also hybridizes in scattered gastrodermal cells in the region where regeneration is taking place, but this represents a novel site of expression (Fig. 6D). Consistent with the high level of expression observed after transection by quantitative PCR, the probe to the 80868 transcripts gives a strong hybridization signal at the site of regeneration, both in scattered gastrodermal cells and in patches within the epidermis (Fig. 6E). Discussion Integrin subunits from Nematostella vectensis have been described by others. The most detailed description comes from Knack et al. (2008), who cloned and sequenced ␣ and  integrin subunits from Acropora millepora and also identified three ␣ subunits and four  subunits from N. vectensis in silico. The four  subunits were named 1– 4, and when analyzed through phylogenetic tree construction they were found to cluster independently from the  integrins of
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Figure 3. Reverse transcription PCR using RNA isolated from juvenile Nematostella vectensis was used to demonstrate the expression of ␣238305, 1143492, 80868, and 91193. Base pair size standards are indicated to the left.
chordates. We identified the same four  subunits here, but chose to name them in such a way that they would not be confused with the 1– 4 subunits of chordates, to which they are not related (80868 corresponds to their 1, 123281 to 2, 91193 to 3, and 143492 to 4). Our phylogenetic analysis of these integrin subunits is in total agreement with theirs; our unique contributions here include the domain analysis, confirmation of expression, and patterns of expression in normal and transected adult polyps. Knack et al. (2008) found three ␣ subunits, while we found only two. During the process of searching the N. vectensis genome we originally found three candidate ␣ subunits genes, but the sequences of two were ultimately determined to be identical. Only one ␣ integrin from N. vectensis was used in phylogenetic tree construction by Knack et al. (2008). This integrin, which they named NvItg␣1, clustered with the ␣4/9 family of chordate integrins, as does our ␣238305. The integrins of N. vectensis are also mentioned in a comprehensive review of cell-cell interactions in cnidarians by Magie and Martindale (2008). They reported two ␣ integrin subunits and five  integrin subunits encoded in the N. vectensis genome, but did not include any identification numbers. Their phylogenetic analysis of the  integrin subunits, like that of Knack et al. (2008) and the results presented here, show significant divergence of the  integrin subunits after the appearance of chordates. The results of their analysis of the ␣ integrins, however, are somewhat different from ours and Knack et al. (2008). One of the ␣ integrins clusters with the RGD-binding integrins (as our analysis suggests for ␣196726), but the other clusters with the ␣A-domain containing laminin-binding integrins and not with the ␣4/9 family of LDV-binding integrins. Further
Figure 4. In situ localization of ␣238305 transcripts in sections through adult Nematostella vectensis. (A) In a section near the oral end of the polyp, a strong hybridization signal is observed in large cells (arrows) scattered throughout the gastrodermis of the mesenteries suspending the pharynx (p). There is also a strong hybridization signal in the pharynx itself (arrowheads). (B) In the gonad-bearing mesenteries ␣238305, transcripts are found in cells near the parietal muscle (pm), retractor muscle (rm), and the gonad (gc). The mesoglea between the gastrodermis (g) and epidermis (e) is indicated by an arrowhead. A single, positive cell in the mesoglea of a retractor muscle is indicated with an arrow. Mesenteric filament, mf. (C) A nearby section hybridized with a control sense probe. Scale bars ⫽ 500 m (A), 250 m (B, C).
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Figure 5. In situ localization of three  integrins in cross sections through an adult Nematostella vectensis. (A) 143492 is expressed primarily in the epidermis of the head region (arrow), but also weakly in the pharynx (p). (B) 80868 is widely expressed over background levels, but the hybridization signal is particularly intense in large, round cells found in the epidermis (arrows). (C) 91193 is expressed widely. (D) A control section showing background levels (91193 sense control probe). (E) In a cross section through the mid-column, 143492 is expressed primarily in the gonad (arrow) and the mesenteric filament (arrowhead). (F) 80868 is expressed in large cells scattered throughout the mesenteric filament (arrows). (G) There is widespread expression of 91193, including in the retractor muscle (arrowhead), mesenteric filament (arrow), and parietal muscle (double arrowhead). (H) Background levels in a section incubated with a control (91193 sense) probe. Scale bar ⫽ 250 m.
analysis may demonstrate the existence and expression of a fifth  integrin in N. vectensis, as is suggested by Magie and Martindale (2008). We were able to confirm the expression of only one ␣ integrin and three  integrins by reverse transcription PCR. This does not mean, however, that the other subunits are not expressed. It is possible that they are expressed during a narrow period of development (e.g., in the embryo or in the planula larva, neither of which were used as a source of RNA here), or that they are expressed by stressed or wounded animals but not in healthy polyps, or even that alternative primer pairs would prove successful in initiating the PCR. We did not determine the possible existence of alternative splicing of integrin subunits, which potentially adds more diversity to integrin-mediated signaling in N. vectensis. It is difficult to determine the precise cell types in the adult polyps where the integrins are expressed due to the paucity of suitable cell-type-specific markers and the tendency of single gastrodermal cells in sea anemones to play the roles of many different cell types in other organisms. However, the patterns of expression of ␣238305 are strongly suggestive of expression by cells that contribute to the formation of muscles. This would be consistent with the
known patterns of expression of other integrins in invertebrates, which are often associated with the anchoring of muscles to the extracellular matrix (e.g., Brabant and Brower, 1993). The distinctive pattern of expression of 80868 by large cells in the epidermis in the oral region and in large cells scattered throughout the mesenteries does not correspond to any particular class of neuron or cnidocyte, nor to any particular kind of mini-collagen, the intracellular matrix that reinforces the walls of the cnidocyst (Marlow et al., 2009; Zenkert et al., 2011). The upregulation of this integrin subunit at the site of regeneration in the aboral half suggests that this integrin may be expressed by the mitotically active stem cells that are abundant in this region during regeneration (Passamaneck and Martindale, 2012). Several of the integrin subunits are expressed in the gonads, including ␣238305 and 143492. It is interesting to note that 143492 is the apparent homolog of Cn1 from A. millepora, and that antibodies to Cn1 inhibit sperm-egg interactions in A. millepora (Iguchi et al., 2007). Moreover, ␣238305 is related to the ␣4/␣9 family of integrins, and ␣9 integrins mediate sperm-egg adhesion in mammals (Eto et al., 2002; Desiderio et al., 2010). In mammals, the spermbound ligands for the ␣9 integrin receptors found on the
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Figure 6. Changes in integrin expression 48 h after transection of Nematostella vectensis. (A) Quantitative PCR reveals increases in the expression of ␣238305, 143492, and 80868 during regeneration. Standard deviation is indicated by the bars. See text for details. (B) Diagrams indicating the location and plane of the transection and the general orientation of the sections shown in C–E. The oral and aboral ends of the polyps are indicated, and the area undergoing regeneration in the aboral half is indicated with an asterisk. Physa, ph; tentacles, t. (C) The ␣238305 probe hybridizes in cells scattered throughout the gastrodermis (arrowheads) in the region undergoing regeneration. The inset shows the whole aboral half of the polyp transected longitudinally and the regenerating region marked with an asterisk. Mesenteries, m. (D) Small cells scattered throughout the gastrodermis in the region undergoing regeneration are also positive for 143492 expression (arrowheads). (E) 80686 is expressed in cells found in both the epidermis (arrows) and gastrodermis (arrowhead) in the region undergoing regeneration. Scale bars ⫽ 1 mm (insets), 500 m (C–E).
surface of oocytes belong to the ADAM family of transmembrane peptidases, and homologs of ADAMs were recently identified in N. vectensis (Tucker and Adams, 2014). Thus, there is potential for ancient origins for this fundamental pair of sperm-egg binding partners. Note that while the polyp that was sectioned for studies of  integrin subunit expression was a female (oocytes were visible in some sections), the sex of the polyp sectioned for in situ hybridization with the probe to ␣238305 could not be determined.
Our phylogenetic tree analysis suggests that the two ␣ integrins of N. vectensis would recognize RGD and/or LDV-like motifs in their extracellular matrix ligands. The classic RGD- and LDV-dependent integrin ligand, fibronectin, is found only in craniates (Tucker and Chiquet-Ehrismann, 2009). A more likely ligand would be a member of the thrombospondin gene family, as these extracellular matrix molecules are expressed in N. vectensis and contain the same RGD motifs as their homologs in tetrapods (Tucker et
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al., 2013). Given the diversity of subtypes and patterns of integrin expression in N. vectensis, it seems likely that there are many more RGD or LDV-type integrin ligands present. Integrins are also receptors for laminins and collagens, both of which are found in N. vectensis (for review see Tucker and Adams, 2014). Future studies should be directed to understanding which integrin subunits dimerize as well as to identifying the actual ligands, both in the extracellular matrix and on the sperm surface, for these dimers. The observation that ␣238305 and 143492 have overlapping patterns of expression both in normal and in regenerating polyps leads us to predict that they may form a functional integrin dimer. However, the current level of our morphological analysis makes us reluctant to speculate further regarding potential functional dimers. This could be studied in the future using double-labeling techniques. Conclusions The starlet sea anemone Nematostella vectensis has at least two ␣ integrin subunits and four  integrin subunits available to dimerize and bind to the extracellular matrix or mediate sperm-egg interactions. The ␣ integrins are related to the RGD- and LDV-binding integrins of other animals. One, ␣238305, is expressed near the pharynx, parietal muscle, and retractor muscle, suggesting a role in the adhesion of myonemes to the extracellular matrix. This subunit is also expressed in the gonad and may play a role during fertilization. The  subunits have distinctive patterns of expression, which is suggestive of diverse roles for the integrins. One of the  subunits, 80686, is upregulated 8-fold during regeneration and is expressed by both gastrodermal and epidermal cells at the site of regeneration, which leads us to suggest that this subunit may play a role related to the significant cell proliferation taking place at this site. Acknowledgments We thank Carol Vines and Gary Cherr of the University of California, Davis, Bodega Marine Laboratory for their generous contribution to our project of Nematostella vectensis from Cadet Hand and Kevin Uhlinger’s CH2 and CH6 stocks, as well as for their advice and support. We also thank Jason Estep for his technical assistance with the in situ hybridization. Literature Cited Barczyk, M., S. Carracedo, and D. Gullberg. 2010. Integrins. Cell Tissue Res. 339: 269 –280. Bossert, P. E., M. P. Dunn, and G. H. Thomsen. 2013. A staging system for the regeneration of a polyp from the aboral physa of the anthozoan cnidarian Nematostella vectensis. Dev. Dyn. 242: 1320 – 1331. Brabant, M. C., and D. L. Brower. 1993. PS2 integrin requirement in Drosophila embryo and wing morphogenesis. Dev Biol. 147: 49 –59. Brower, D. L., S. M. Brower, D. C. Hayward, and E. E. Ball. 1997.
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Pancer, Z., M. Kruse, I. Mu¨ller, and W. E. Mu¨ller. 1997. On the origin of Metazoan adhesion receptors: cloning of integrin alpha subunit from the sponge Geodia cydonium. Mol. Biol. Evol. 14: 391–398. Passamaneck, Y. J., and M. Q. Martindale. 2012. Cell proliferation is necessary for the regeneration of oral structures in the anthozoan cnidarian Nematostella vectensis. BMC Dev. Biol. 12: 34. Putnam, N. H., M. Srivastava, U. Hellsten, B. Dirks, J. Chapman, A. Salamov, A. Terry, H. Shapiro, E. Lindquist, V.V. Kapitonov et al. 2007. Sea anemone genome reveals ancestral eumetazoan gene repertoire and genomic organization. Science 317: 86 –94. Reitzel, A. M., and A. M. Tarrant. 2009. Nuclear receptor complement of the cnidarian Nematostella vectensis: phylogenetic relationships and developmental expression patterns. BMC Evol. Biol. 9: 230. Rokas, A. 2008. The molecular origins of multicellular transitions. Curr. Opin. Genet. Dev. 18: 472– 478. Sebe´-Pedro´s, A., A. J. Roger, F. B. Lang, N. King, and I. Ruiz-Trillo. 2010. Ancient origin of the integrin-mediated adhesion and signaling machinery. Proc. Natl. Acad. Sci. USA 107: 10142–10147. Stefanik, D. J., L. E. Friedman, and J. R. Finnerty. 2013. Collecting, rearing, spawning and inducing regeneration of the starlet sea anemone, Nematostella vectensis. Nat. Protoc. 8: 916 –923. Technau, U., and R. E. Steele. 2011. Evolutionary crossroads in developmental biology: Cnidaria. Development 138: 1447–1458.
Tucker, R.P. 2010. Expression of usherin in the anthozoan Nematostella vectensis. Biol. Bull. 218: 105–112. Tucker, R. P., and J. C. Adams. 2014. Adhesion networks of cnidarians: a postgenomic view. Int. Rev. Cell Mol. Biol. 308: 323–377. Tucker, R. P., and R. Chiquet-Ehrismann. 2009. Evidence for the evolution of tenascin and fibronectin early in the chordate lineage. Int. J. Biochem. Cell Biol. 41: 424 – 434. Tucker, R. P., and Q. Gong. 2014. Immunohistochemistry and in situ hybridization in the developing chicken brain. Methods Mol. Biol. 1082: 217–233. Tucker, R. P., B. Shibata, and T. N. Blankenship. 2011. Ultrastructure of the mesoglea of the sea anemone Nematostella vectensis (Edwardsiidae). Invertebr. Biol. 130: 11–24. Tucker, R. P., J. F. Hess, Q. Gong, K. Garvey, B. Shibata, and J. C. Adams. 2013. A thrombospondin in the anthozoan Nematostella vectensis is associated with the nervous system and upregulated during regeneration. Biol. Open 2: 217–226. Wickstro¨m, S. A., K. Radovanac, and R. Fa¨ssler. 2011. Genetic analyses of integrin signaling. Cold Spring Harb. Perspect. Biol. doi: 10.1101/cshperspect.a005116. ¨ zbek. 2011. MorZenkert, C., T. Takahashi, M. O. Diesner, and S. O phological and molecular analysis of the Nematostella vectensis cnidom. PLoS One 6: e22725.
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Integrins of the Starlet Sea Anemone Nematostella vectensis QIZHI GONG, KATRINA GARVEY, CHENGHAO QIAN, ISABEL YIN, GARY WONG, AND RICHARD P. TUCKER* Department of Cell Biology and Human Anatomy, University of California, Davis, Davis, California 95616 – 8643
Abstract. Integrins are extracellular matrix receptors composed of ␣ and  subunits. Here we describe two ␣ subunits and four  subunits from the starlet sea anemone Nematostella vectensis. Phylogenetic analysis suggests that the ␣ subunits are most closely related to RGD- and LDVdependent ␣ subunits of chordates. The  subunits cluster with the previously described  integrins of the hard coral Acropora millepora. The expression of one of the ␣ subunits and three of the  subunits was confirmed by reverse transcription PCR and in situ hybridization. The ␣ subunit is primarily expressed in cells near muscles, by a subset of gastrodermal cells, and in the gonad. The three  subunits each have distinctive patterns of expression: one is concentrated in the gonad and mesenteric filament, another is found in a subset of cells in the epidermis of the oral region and in a subset of gastrodermal cells in the mesenteries, and a third is expressed widely. Changes in expression were also studied 48 h after horizontal transection by quantitative reverse transcription PCR and in situ hybridization. One of the  subunits is expressed 8-fold higher during regeneration, and its expression is observed in cells within both the epidermis and the gastrodermis at the site of regeneration. Our observations confirm that complex patterns of integrin expression were already present in basal metazoans. The integrins expressed in the gonads may play roles in mediating spermegg interactions in N. vectensis, while others may play a role in regulating proliferation during regeneration.
Introduction Integrins are dimeric extracellular matrix receptors composed of one ␣ and one  subunit (for review see Campbell and Humphries, 2011). In chordates, where these receptors are most widely studied, the ␣ subunit is composed of a head domain formed from a -propeller, integrin ␣-2 domains that form regions known as the thigh and calves (due to the knee-like bend that can form between these regions), a transmembrane helix, and a short cytoplasmic domain (Fig. 1A). A subset of ␣ integrins also has an ␣A-domain in the head region. Most  subunits are composed of an integrin  head domain followed by a series of epidermal growth factor (EGF)-like repeats, a -integrin-specific tail domain, a transmembrane region, and a -integrin-specific cytoplasmic domain. A variation on this theme is demonstrated by the 4 integrins, which have a longer cytoplasmic domain that includes a Calx  domain and a string of fibronectin type III domains (Fig. 1A). In humans there are 18 genes encoding ␣ subunits and 8 genes that encode  subunits. To date, these have been shown to form 24 different dimers, most of which can bind multiple ligands (Barczyk et al., 2010). These are typically classified as being RGD-dependent receptors, LDV-dependent receptors, laminin-collagen receptors, or leukocyte-specific receptors. The first two classes are the best understood. The RGDdependent receptors bind to the tripeptide motif arginineglycine-aspartic acid found in many extracellular matrix ligands via the cleft formed between the heads of the ␣ and  subunits. The LDV-dependent receptors bind to a leucineaspartic acid-valine (or similarly acidic) motif, and ␣ subunits with the ␣A-domain bind to laminins and collagens (reviewed by Campbell and Humphries, 2011). Ligand binding leads to conformational changes that initiate signaling cascades that are often associated with cell motility, proliferation, and differentiation (for review see Wickstro¨m
Received 2 April 2014; accepted 21 July 2014. * To whom correspondence should be addressed. E-mail: rptucker@ ucdavis.edu Abbreviations: aa, amino acid; EGF, epidermal growth factor; PBS, phosphate buffered saline. 211
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Figure 1. Stick diagrams illustrating the domain architecture of integrins. (A) Representative ␣ and  integrins from Homo sapiens. (B) The domain organization of the two ␣ integrin subunits and four  integrin subunits identified in the genome of Nematostella vectensis.
et al., 2011). In addition, some integrins mediate sperm-egg interactions both in mammals and in some invertebrates (for review see Evans, 2012). The first integrins to be cloned from early-diverging metazoans were an ␣ integrin subunit from the demosponge Geodia cydonium (Pancer et al., 1997) and  integrin subunits from the demosponge Ophlitaspongia tenuis and the scleractinian coral Acropora millepora (Brower et al., 1997). The presence of these receptors in sponges and cnidarians demonstrated their fundamental relationship to the early extracellular matrix, and the absence of integrins from the genomes of choanoflagellates, which are widely regarded to be the sister group to metazoans, led to the conclusion that they evolved together with the first animals (e.g., Rokas, 2008). However, ␣ and  integrin subunits were subsequently identified in the protist Capsaspora owczarzaki, revealing an ancient origin for these receptors predating the appearance of animals and their selective loss in choanoflagellates (Sebe´-Pedro´s et al., 2010). Knack et al.
(2008) thoroughly described the integrins of A. millepora, including the results of expression studies. They found a single ␣ integrin subunit and two  integrin subunits, one of which corresponded to the  subunit previously identified by Brower et al. (1997). Expression was confirmed by reverse transcription PCR and by whole-mount in situ hybridization with gastrulae. A potential role for integrins in fertilization in cnidarians was proposed by Iguchi et al. (2007), who found that antibodies to a  integrin subunit reduced sperm-egg binding and fertilization rates in A. millepora. Nematostella vectensis is a small, burrowing anthozoan found in estuaries along both coasts of North America and the south coast of England (Hand and Uhlinger, 1994) that can be readily maintained and manipulated in the laboratory (Hand and Uhlinger, 1992; Darling et al., 2005; Technau and Steele, 2011; Bossert et al., 2013; Layden et al., 2013; Stefanik et al., 2013). For this reason, these sea anemones have become an important model organism for studying
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evolution, development, and regeneration. Their mesoglea is remarkable in its ultrastructural similarity to the extracellular matrix of chordates (Tucker et al., 2011), and a number of homologs of chordate extracellular matrix genes have been identified, many of which are known to be integrin ligands in other organisms (Koehler et al., 2009; Ozbek et al., 2010; Tucker et al,, 2013; Tucker and Adams, 2014). In addition to their work with A. millepora, Knack et al. (2008) identified three ␣ integrin subunits and four  integrin subunits in the genome of N. vectensis, suggesting that this model organism may have a more diverse repertoire of these receptors than other early-diverging metazoans. Here we describe the integrins of N. vectensis, including their phylogenetic relationships to other integrins, their expression in the adult polyp, and changes in their level of expression during regeneration. Materials and Methods Phylogenomic analysis Putative integrin genes from the Nematostella vectensis Stephenson, 1935, genome (Putnam et al., 2007) were identified using the domain architecture analysis feature at Pfam (http://pfam.sanger.ac.uk/) to identify predicted proteins with domains unique to integrins (e.g., ␣ integrin  propeller domains and  integrin head domains). The identity of the domains and their boundaries in the predicted proteins were identified using Interpro (Hunter et al., 2011), and molecular weights were determined at ExPASy (Gasteiger et al., 2005). For phylogenetic analysis, sequences were aligned using MUSCLE (Edgar, 2004) and trees were constructed with PhyML 3.0 (maximum-likelihood) using default parameters at Phylogeny.fr (Dereeper et al., 2008). Raising and transecting Nematostella vectensis Nematostella vectensis polyps derived from the CH2 and CH6 clones of Hand and Uhlinger (1992) were maintained in microfiltered 1/3 seawater at room temperature and fed a mixture of egg yolk and Artemia nauplii. Adults used for regeneration studies were immobilized with 7.5% MgCl2 and cut with a razor blade through the center of the body column in the region where gonad-bearing mesenteries are found. Both halves were maintained in 1/3 seawater for 48 h prior to quantitative PCR studies (see below), or just the aboral half was maintained for 48 h prior to processing for in situ hybridization (see below). Molecular and histological methods RNA was isolated from either juvenile (⬃4-week-old) or adult polyps using an RNeasy Mini Kit (Qiagen) and treated with amplification grade DNase I (Invitrogen) prior to determining the concentration and quality of the RNA with a nanodrop spectrophotometer. Reverse transcription PCR
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was performed using a Super Script III One Step RT-PCR Kit (Invitrogen) as described previously (Tucker, 2010). Intron-spanning primer pairs (Eurofins MWG/Operon) used were (1) 5⬘-ccaagggcaatgaagtgtct-3⬘ and 5⬘-tccagcatgtctcctcacag-3⬘ for ␣238305 (499bp); (2) 5⬘-actctgccccgtcagaagta-3⬘ and 5⬘-gcgtaagtcccgttaccaga-3⬘ for 143492 (298bp); (3) 5⬘-tgactgccctacatgtccag-3⬘ and 5⬘-caccacacccagaatgacag-3⬘ for 80868 (301bp); and (4) 5⬘-ggatgtcgcgaagaaaacat-3⬘ and 5⬘-caaatgatccaaaccccaac-3⬘ for 91193 (304bp). All PCR products were subcloned into pCR2.1 or pCRII by TOPO TA cloning (Invitrogen) and sequenced by Davis Sequencing (Davis, CA). For in situ hybridization, adult polyps were immobilized with 7.5% MgCl2 and fixed in ice-cold 4% paraformaldehyde in 0.01 mol l⫺1 phosphate buffered saline (PBS; Sigma P-3813) overnight, then rinsed in PBS and dehydrated in a series of ethanol baths, cleared in xylene, and embedded in Paraplast. Sections 8-m thick were cut on a microtome and collected on Superfrost Plus (Fisher) presubbed microscope slides. In situ hybridization was carried out essentially as described in Tucker and Gong (2014) with the following modifications. Both sense (control) and antisense (experimental) digoxigenin-labeled riboprobes were made using pCRII clones mentioned above as templates, and Proteinase K treatment was carried out at room temperature for 3 min. After postfixation, rinsing, and dehydration, riboprobes at 1 g/ml were put on the slides that were pre-warmed to 60 °C, and hybridizations were carried out at 65 °C overnight. Quantitative reverse transcription PCR was performed using the primer pairs described above. In brief, three adult polyps, either whole or transected 48 h previously, were sheared and homogenized with a 26-gauge needle in TRIzol reagent (Life Technologies), and the total RNA was precipitated from a chloroform suspension with isopropanol. cDNAs were obtained by reverse transcription with AMV reverse transcriptase and oligo dT primers. Quantitative reverse transcription PCR experiments were done in triplicate using SYBR Green chemistry on an ABI StepOnePlus system (Life Technologies). Expression levels of each integrin were standardized to the level of 18S rRNA using primer pair 5⬘-gactcaacacggggaaactc-3⬘ and 5⬘-gcaccaccacccatagaat-3’(Reitzel and Tarrant, 2009). The amplification protocol involved a 10-min denaturation step followed by 40 cycles of 95 °C and 60 °C (30 s each). To verify the quality of each primer pair, melting curves were obtained from the first step starting from 50 °C to 95 °C. The cycle threshold (Ct) and the standard deviation were calculated for each replicate. The average Ct of the Gene of Interest (GOI) (avg. CtGOI) was normalized to the average Ct of the reference gene (avg. Ctref) for the same sample to calculate the normalized Ct for the GOI (⌬Ct ⫽ avg. CtGOI ⫺ avg. Ctref). The standard deviation of the ⌬Ct was calculated by (stdevGOI2 ⫹ stdevref2)(1/2). The calibrated value (⌬⌬Ct) for each sample was determined by ⌬⌬Ct ⫽ ⌬CtR ⫺ ⌬CtU. The
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stdev ⌬⌬Ct is the same as stdev ⌬Ct. The fold-induction for each sample relative to the calibrator ⫽ 2(⫺⌬⌬Ct). Standard deviations were accounted for within the calibrator by incorporating them into the equation as such: 2(⫺⌬⌬Ct ⫾ Stdev). Results The two ␣ and four  integrin subunits of Nematostella vectensis Domain analysis reveals two ␣ integrin subunits encoded in the genome of Nematostella vectensis. The first was given the locus tag NEMVEDRAFT_v1g196726; here this predicted protein is called ␣196726. The second ␣ integrin subunit was assigned the locus tag NEMVEDRAFT_ v1g238305 and is referred to here as ␣238305. ␣196726 (NW_001834411) is composed of 1025 amino acids (aa) and has a predicted molecular weight of 112640 Da. It is composed of six or seven (depending on the domain analysis program used)  propeller domains (aa 25– 459), an integrin ␣-2 domain (aa 443– 894), a transmembrane helix (aa 971–993) and a short cytoplasmic domain (Fig. 1B). ␣238305 (NW_001834409) is composed of 1283 aa and has a predicted molecular weight of 141245 Da. It has a head composed of a series of  propeller domains (aa 1–502), a tail composed of two integrin ␣-2 domains (aa 483–722 and aa 812–1143), a transmembrane helix (aa 1124 –1247), and a short cytoplasmic domain (Fig. 1B). Unlike ␣196726, ␣238305 does not have a predicted signal peptide, indicating that the N-terminus may be incomplete. Neither ␣ integrin subunit has an ␣A-domain, a domain that is characteristic of the laminin-collagen receptors (Campbell and Humphries, 2011). Domain analysis reveals four  integrin subunits that are similarly named here by using the numeric portion of their locus tag (Fig. 1B). 80868 (XP_001641468; EDO49405) is a 786 aa protein with a predicted molecular weight of 85470 Da. Following a signal peptide (aa 1–25), it is composed of an integrin  head domain (aa 33– 456), three EGF-like domains (aa 493– 624), an integrin  tail domain (aa 634 –715), a transmembrane helix (aa 717–739), and an integrin  cytoplasmic domain (aa 740 –786). 91193 (XP_001637894; EDO45831) lacks a signal peptide but has what appears to be a nearly complete  integrin head domain (aa 25– 437; that is, it has plexin-like folds at its N-terminus). It also has three EGF-like repeats (aa 490 – 611), an integrin  tail domain (aa 622–709), a transmembrane helix (aa 710 –732), and an integrin  cytoplasmic domain (aa 733–779). 123281 (XP_001627336; EDO35236) appears to be a complete  integrin subunit composed of 786 aa and a predicted molecular weight of 87020 Da. Following the signal peptide (aa 1–35) is a  integrin head domain (aa 34 – 455), four EGF-like repeats (aa 478 – 621), an integrin  tail domain (aa 631–715), a transmembrane helix (aa 717–739), and an integrin  cytoplasmic domain (aa 740 –786). Finally, 143492 (XP_001621822; EDO29722)
also appears to be a complete predicted protein composed of 795 aa with a predicted molecular weight of 87538 Da. An integrin  head domain (aa 34 – 451) follows a signal peptide (1–26). These are followed by three EGF-like repeats (aa 504 – 623), a  integrin tail domain (aa 634 –719), a transmembrane helix (aa 724 –746), and an integrin  cytoplasmic tail domain (aa 748 –794). Phylogenetic relationships of the Nematostella vectensis integrins Phylogenetic tree analysis of the Nematostella vectensis ␣ integrins shows that ␣196726 clusters with a clade that includes four ␣ integrins from the sponge Amphimedon queenslandica, the ␣1 integrin from the stony coral Acropora millepora, ␣PAT-2 from Caenorhabditis elegans, and the RGD-binding ␣ integrin subunits from Homo sapiens ␣2b, ␣V, and ␣8 (Fig. 2A). ␣238305 clusters with the LDV-binding ␣4 and ␣9 integrins from H. sapiens. 80868 and 123281 are related to each other and cluster with the 2 integrin of A. millepora. 91193 and 143492 are related to each other and cluster with Cn1 from A. millepora. These  subunits do not appear to be related to any of the major groups of chordate  integrins (Fig. 2B). Integrin expression in Nematostella vectensis The expression of one of the two predicted ␣ integrin subunits (␣238305) and three of the four predicted  integrin subunits (143492, 80868, and 91193) was confirmed by reverse transcriptase PCR using cDNA made from RNA isolated from juvenile polyps (Fig. 3). Multiple primer pairs as well as template made from RNA isolated from both juvenile and adult polyps were used to try to demonstrate the expression of ␣196726 and 123281, but without success. The pattern of expression of the ␣238305 subunit was determined using in situ hybridization on paraffin sections of adult polyps. At the oral end of the polyp there was scattered expression in the gastrodermis and epidermis, but there was a robust signal in large cells found in the complete mesenteries suspending the pharynx, as well as in the pharynx itself (Fig. 4A). Cross sections through the body column also revealed limited, scattered expression in the gastrodermis of the body wall, but there was a robust signal in cells adjacent to the parietal and retractor muscles, as well as in the gonadal region of the mesentery (Fig. 4B). A control section hybridized with a sense probe is shown in Figure 4C. The pattern of expression of ␣238305 is consistent with expression by gametes (it is unknown if this specimen is male or female) and in cells that make myonemes (the myofibril-like protrusions found in the pharynx, parietal muscle, and retractor muscle), but the cells expressing this integrin in the mesenteries near the pharynx are unknown. We previously identified cells with the morphology of neurons in the mesoglea of the pharynx and
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Figure 2. Phylogenetic tree analysis of Nematostella vectensis integrin subunits. (A) The ␣ integrin subunit ␣196726 clusters near the RGD-binding ␣ integrins from other species, including human ␣5, ␣8, ␣2b, and ␣V. The ␣238305 integrin clusters with the LDV-binding ␣4/␣9 integrin family. The N. vectensis integrins are highlighted with an asterisk. (B) The four  integrin subunits from N. vectensis (asterisks) are related to the 2 and Cn1 subunits of the hard coral Acropora millepora, but not to the human  integrins. Branch support (Approximate Likelihood-Ratio test) greater than 0.7 is indicated. Scale bars indicate substitutions/site.
retractor muscle; at least one cell in the mesoglea of the retractor muscle was positive for expression of ␣238305 (Fig. 4B). The expression of integrin subunits 143492, 80868, and 91193 was also studied by in situ hybridization on transverse paraffin sections through the pharynx and midbody column. All three probes revealed expression over background (Fig. 5). 143492 expression was strongest throughout the epidermis, gonad, and mesenteric filament (Fig. 5A, E). In contrast, 80868 hybridized in cells scattered in the epidermis of the oral region and in large cells found in the mesenteries of the body column (Fig. 5B, F). The 91193 probe gave an intense, uniform signal throughout the polyp (Fig. 5C, G). Changes in integrin expression during regeneration Changes in expression during regeneration (48 h after transection) of the ␣238305 subunit and two of the  integrin subunits, 143492 and 80868, were examined by quantitative PCR. The ␣238305 subunit expression increased 1.5-fold, while the expression of the 143492 and 80868 subunits increased 5-fold and 8-fold, respectively (Fig. 6A). Patterns of gene expression at this time point were also examined by in situ hybridization in longitudinal
sections through the aboral half of transected polyps (Fig. 6B). ␣238305 transcripts are found in the region where regeneration is taking place (Fig. 6C), but the pattern of expression (i.e., in scattered gastrodermal cells) is not different from the hybridization signal observed in the body wall of untransected polyps. The probe to 143492 transcripts also hybridizes in scattered gastrodermal cells in the region where regeneration is taking place, but this represents a novel site of expression (Fig. 6D). Consistent with the high level of expression observed after transection by quantitative PCR, the probe to the 80868 transcripts gives a strong hybridization signal at the site of regeneration, both in scattered gastrodermal cells and in patches within the epidermis (Fig. 6E). Discussion Integrin subunits from Nematostella vectensis have been described by others. The most detailed description comes from Knack et al. (2008), who cloned and sequenced ␣ and  integrin subunits from Acropora millepora and also identified three ␣ subunits and four  subunits from N. vectensis in silico. The four  subunits were named 1– 4, and when analyzed through phylogenetic tree construction they were found to cluster independently from the  integrins of
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Figure 3. Reverse transcription PCR using RNA isolated from juvenile Nematostella vectensis was used to demonstrate the expression of ␣238305, 1143492, 80868, and 91193. Base pair size standards are indicated to the left.
chordates. We identified the same four  subunits here, but chose to name them in such a way that they would not be confused with the 1– 4 subunits of chordates, to which they are not related (80868 corresponds to their 1, 123281 to 2, 91193 to 3, and 143492 to 4). Our phylogenetic analysis of these integrin subunits is in total agreement with theirs; our unique contributions here include the domain analysis, confirmation of expression, and patterns of expression in normal and transected adult polyps. Knack et al. (2008) found three ␣ subunits, while we found only two. During the process of searching the N. vectensis genome we originally found three candidate ␣ subunits genes, but the sequences of two were ultimately determined to be identical. Only one ␣ integrin from N. vectensis was used in phylogenetic tree construction by Knack et al. (2008). This integrin, which they named NvItg␣1, clustered with the ␣4/9 family of chordate integrins, as does our ␣238305. The integrins of N. vectensis are also mentioned in a comprehensive review of cell-cell interactions in cnidarians by Magie and Martindale (2008). They reported two ␣ integrin subunits and five  integrin subunits encoded in the N. vectensis genome, but did not include any identification numbers. Their phylogenetic analysis of the  integrin subunits, like that of Knack et al. (2008) and the results presented here, show significant divergence of the  integrin subunits after the appearance of chordates. The results of their analysis of the ␣ integrins, however, are somewhat different from ours and Knack et al. (2008). One of the ␣ integrins clusters with the RGD-binding integrins (as our analysis suggests for ␣196726), but the other clusters with the ␣A-domain containing laminin-binding integrins and not with the ␣4/9 family of LDV-binding integrins. Further
Figure 4. In situ localization of ␣238305 transcripts in sections through adult Nematostella vectensis. (A) In a section near the oral end of the polyp, a strong hybridization signal is observed in large cells (arrows) scattered throughout the gastrodermis of the mesenteries suspending the pharynx (p). There is also a strong hybridization signal in the pharynx itself (arrowheads). (B) In the gonad-bearing mesenteries ␣238305, transcripts are found in cells near the parietal muscle (pm), retractor muscle (rm), and the gonad (gc). The mesoglea between the gastrodermis (g) and epidermis (e) is indicated by an arrowhead. A single, positive cell in the mesoglea of a retractor muscle is indicated with an arrow. Mesenteric filament, mf. (C) A nearby section hybridized with a control sense probe. Scale bars ⫽ 500 m (A), 250 m (B, C).
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Figure 5. In situ localization of three  integrins in cross sections through an adult Nematostella vectensis. (A) 143492 is expressed primarily in the epidermis of the head region (arrow), but also weakly in the pharynx (p). (B) 80868 is widely expressed over background levels, but the hybridization signal is particularly intense in large, round cells found in the epidermis (arrows). (C) 91193 is expressed widely. (D) A control section showing background levels (91193 sense control probe). (E) In a cross section through the mid-column, 143492 is expressed primarily in the gonad (arrow) and the mesenteric filament (arrowhead). (F) 80868 is expressed in large cells scattered throughout the mesenteric filament (arrows). (G) There is widespread expression of 91193, including in the retractor muscle (arrowhead), mesenteric filament (arrow), and parietal muscle (double arrowhead). (H) Background levels in a section incubated with a control (91193 sense) probe. Scale bar ⫽ 250 m.
analysis may demonstrate the existence and expression of a fifth  integrin in N. vectensis, as is suggested by Magie and Martindale (2008). We were able to confirm the expression of only one ␣ integrin and three  integrins by reverse transcription PCR. This does not mean, however, that the other subunits are not expressed. It is possible that they are expressed during a narrow period of development (e.g., in the embryo or in the planula larva, neither of which were used as a source of RNA here), or that they are expressed by stressed or wounded animals but not in healthy polyps, or even that alternative primer pairs would prove successful in initiating the PCR. We did not determine the possible existence of alternative splicing of integrin subunits, which potentially adds more diversity to integrin-mediated signaling in N. vectensis. It is difficult to determine the precise cell types in the adult polyps where the integrins are expressed due to the paucity of suitable cell-type-specific markers and the tendency of single gastrodermal cells in sea anemones to play the roles of many different cell types in other organisms. However, the patterns of expression of ␣238305 are strongly suggestive of expression by cells that contribute to the formation of muscles. This would be consistent with the
known patterns of expression of other integrins in invertebrates, which are often associated with the anchoring of muscles to the extracellular matrix (e.g., Brabant and Brower, 1993). The distinctive pattern of expression of 80868 by large cells in the epidermis in the oral region and in large cells scattered throughout the mesenteries does not correspond to any particular class of neuron or cnidocyte, nor to any particular kind of mini-collagen, the intracellular matrix that reinforces the walls of the cnidocyst (Marlow et al., 2009; Zenkert et al., 2011). The upregulation of this integrin subunit at the site of regeneration in the aboral half suggests that this integrin may be expressed by the mitotically active stem cells that are abundant in this region during regeneration (Passamaneck and Martindale, 2012). Several of the integrin subunits are expressed in the gonads, including ␣238305 and 143492. It is interesting to note that 143492 is the apparent homolog of Cn1 from A. millepora, and that antibodies to Cn1 inhibit sperm-egg interactions in A. millepora (Iguchi et al., 2007). Moreover, ␣238305 is related to the ␣4/␣9 family of integrins, and ␣9 integrins mediate sperm-egg adhesion in mammals (Eto et al., 2002; Desiderio et al., 2010). In mammals, the spermbound ligands for the ␣9 integrin receptors found on the
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Figure 6. Changes in integrin expression 48 h after transection of Nematostella vectensis. (A) Quantitative PCR reveals increases in the expression of ␣238305, 143492, and 80868 during regeneration. Standard deviation is indicated by the bars. See text for details. (B) Diagrams indicating the location and plane of the transection and the general orientation of the sections shown in C–E. The oral and aboral ends of the polyps are indicated, and the area undergoing regeneration in the aboral half is indicated with an asterisk. Physa, ph; tentacles, t. (C) The ␣238305 probe hybridizes in cells scattered throughout the gastrodermis (arrowheads) in the region undergoing regeneration. The inset shows the whole aboral half of the polyp transected longitudinally and the regenerating region marked with an asterisk. Mesenteries, m. (D) Small cells scattered throughout the gastrodermis in the region undergoing regeneration are also positive for 143492 expression (arrowheads). (E) 80686 is expressed in cells found in both the epidermis (arrows) and gastrodermis (arrowhead) in the region undergoing regeneration. Scale bars ⫽ 1 mm (insets), 500 m (C–E).
surface of oocytes belong to the ADAM family of transmembrane peptidases, and homologs of ADAMs were recently identified in N. vectensis (Tucker and Adams, 2014). Thus, there is potential for ancient origins for this fundamental pair of sperm-egg binding partners. Note that while the polyp that was sectioned for studies of  integrin subunit expression was a female (oocytes were visible in some sections), the sex of the polyp sectioned for in situ hybridization with the probe to ␣238305 could not be determined.
Our phylogenetic tree analysis suggests that the two ␣ integrins of N. vectensis would recognize RGD and/or LDV-like motifs in their extracellular matrix ligands. The classic RGD- and LDV-dependent integrin ligand, fibronectin, is found only in craniates (Tucker and Chiquet-Ehrismann, 2009). A more likely ligand would be a member of the thrombospondin gene family, as these extracellular matrix molecules are expressed in N. vectensis and contain the same RGD motifs as their homologs in tetrapods (Tucker et
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al., 2013). Given the diversity of subtypes and patterns of integrin expression in N. vectensis, it seems likely that there are many more RGD or LDV-type integrin ligands present. Integrins are also receptors for laminins and collagens, both of which are found in N. vectensis (for review see Tucker and Adams, 2014). Future studies should be directed to understanding which integrin subunits dimerize as well as to identifying the actual ligands, both in the extracellular matrix and on the sperm surface, for these dimers. The observation that ␣238305 and 143492 have overlapping patterns of expression both in normal and in regenerating polyps leads us to predict that they may form a functional integrin dimer. However, the current level of our morphological analysis makes us reluctant to speculate further regarding potential functional dimers. This could be studied in the future using double-labeling techniques. Conclusions The starlet sea anemone Nematostella vectensis has at least two ␣ integrin subunits and four  integrin subunits available to dimerize and bind to the extracellular matrix or mediate sperm-egg interactions. The ␣ integrins are related to the RGD- and LDV-binding integrins of other animals. One, ␣238305, is expressed near the pharynx, parietal muscle, and retractor muscle, suggesting a role in the adhesion of myonemes to the extracellular matrix. This subunit is also expressed in the gonad and may play a role during fertilization. The  subunits have distinctive patterns of expression, which is suggestive of diverse roles for the integrins. One of the  subunits, 80686, is upregulated 8-fold during regeneration and is expressed by both gastrodermal and epidermal cells at the site of regeneration, which leads us to suggest that this subunit may play a role related to the significant cell proliferation taking place at this site. Acknowledgments We thank Carol Vines and Gary Cherr of the University of California, Davis, Bodega Marine Laboratory for their generous contribution to our project of Nematostella vectensis from Cadet Hand and Kevin Uhlinger’s CH2 and CH6 stocks, as well as for their advice and support. We also thank Jason Estep for his technical assistance with the in situ hybridization. Literature Cited Barczyk, M., S. Carracedo, and D. Gullberg. 2010. Integrins. Cell Tissue Res. 339: 269 –280. Bossert, P. E., M. P. Dunn, and G. H. Thomsen. 2013. A staging system for the regeneration of a polyp from the aboral physa of the anthozoan cnidarian Nematostella vectensis. Dev. Dyn. 242: 1320 – 1331. Brabant, M. C., and D. L. Brower. 1993. PS2 integrin requirement in Drosophila embryo and wing morphogenesis. Dev Biol. 147: 49 –59. Brower, D. L., S. M. Brower, D. C. Hayward, and E. E. Ball. 1997.
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