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BACKGROUND: Cadherins are calcium-dependent transmembrane cell-cell adhesion proteins that are essential for metazoan development. They consist of three subfamilies: classical cadherins, which bind catenin, protocadherins, which contain 6-7 calcium-binding repeat domains, and atypical cadherins. Their functions include forming adherens junctions, establishing planar cell polarity (PCP), and regulating cell shape, proliferation, and migration. Because they are basal deuterostomes, echinoderms provide important insights into bilaterian evolution, but their only well-characterized cadherin is G-cadherin, a classical cadherin that is expressed by many embryonic epithelia. We aimed to better characterize echinoderm cadherins by conducting phylogenetic analyses and examining the spatiotemporal expression patterns of cadherin-encoding genes during Strongylocentrotus purpuratus development.
RESULTS: Our phylogenetic analyses conducted on two echinoid, three asteroid, and one crinoid species identified ten echinoderm cadherins, including one deuterostome-specific ortholog, cadherin-23, and an echinoderm-specific atypical cadherin that possibly arose in an echinoid-asteroid ancestor. Catenin-binding domains in dachsous-2 orthologs were found to be a deuterostome-specific innovation that was selectively lost in mouse, while those in Fat4 orthologs appeared to be Ambulacraria-specific and were selectively lost in non-crinoid echinoderms. The identified suite of echinoderm cadherins lacks vertebrate-specific innovations but contains two proteins that are present in protostomes and absent from mouse. The spatiotemporal expression patterns of four embryonically expressed cadherins (fat atypical cadherins 1 and 4, dachsous-2, and protocadherin-9) were dynamic and mirrored the expression pattern of Frizzled 5/8, a non-canonical Wnt PCP pathway receptor protein essential for archenteron morphogenesis.
CONCLUSIONS: The echinoderm cadherin toolkit is more similar to that of an ancient bilaterian predating protostomes and deuterostomes than it is to the suite of cadherins found in extant vertebrates. However, it also appears that deuterostomes underwent several cadherin-related innovations. Based on their similar spatiotemporal expression patterns and orthologous relationships to PCP-related and tumor-suppressing proteins, we hypothesize that sea urchin cadherins may play a role in regulating the shape and growth of embryonic epithelia and organs. Future experiments will examine cadherin expression in non-echinoid echinoderms and explore the functions of cadherins during echinoderm development.
Fig. 1. Main structures and functions of three cadherin subfamilies in deuterostomes. Domain architectures for representative classical cadherins, protocadherins, and atypical cadherins are derived from the SMART database. The domain structures for all the cadherins depicted are derived from Mus musculus with the exception of the type III classical cadherin, which instead originates from Lytechinus variegatus. The magenta bar encompasses the transmembrane regions of each protein, and the functions of each of the three subfamilies are outlined in blue. More in-depth descriptions of research on these functions are available in the Background section
Fig. 2. Phylogenetic analysis of amino acid sequences for echinoderm cadherins utilizing maximum likelihood and neighbor-joining methods. Both analyses were conducted on various cadherin sequences from echinoderm, non-echinoderm deuterostome, and protostome species using MEGA11 software. Clades and their respective orthologous sequences are labeled and color-coded. Two EGFLAM sequences from Mus musculus and Crassostrea gigas are included as an outgroup for both analyses. Full species and protein names for each taxon and their respective GenBank accession number(s) and amino acid sequences are listed in Additional file 1. A. A maximum likelihood analysis was performed using the WAG + F + G substitution model with 500 bootstrap replicates utilizing amino acid sites with ≥ 95% coverage across all taxa. B. A neighbor-joining analysis was performed using the p-distance substitution model with 5000 bootstrap replicates with pairwise deletion of amino acid sites
Fig. 3. Developmental expression of Sp-fat1, Sp-fat4, Sp-pcdh9, and Sp-dchs2 analyzed by whole mount in situ hybridization. All four genes show strikingly similar patterns of expression. Prior to the mesenchyme blastula stage, faint signal is seen in all cells and is highest in the basal cytoplasm (arrowhead, A’’’). By the late mesenchyme blastula (MB) stage, signal is enriched in the apical plate (arrowhead, B) and vegetal plate (arrowhead, B’’) but is absent from migrating primary mesenchyme cells (arrowhead, B’). At the early gastrula (EG) and mid-gastrula (MG) stages, intense signal is seen at the margin of the blastopore (arrowhead, C’’) and in the apical plate, but ingressing non-skeletogenic mesenchyme cells at the tip of the archenteron are unlabeled (arrowhead, D’). At the early prism (EP) stage, expression is detected primarily in the apical plate, the prospective foregut, and at the margin of the blastopore (arrowheads, E). At the pluteus (PL) stage, expression is elevated in the apical plate (arrowhead, G’), the portion of the ciliary band overlying the postoral arms (arrowhead, G), and in the gut (arrowhead, F’). FP- focal plane. PHB- pre-hatching blastula (16 hpf). MB- mesenchyme blastula (24 hpf). EG- early gastrula (28 hpf). MG- mid-gastrula (30–32 hpf). EP- early prism (44 hpf). PL- pluteus (72 hpf). Each pluteus was imaged in two different focal planes (FP1 and FP2). Scale bar = 50 μm
Fig. 4. Fluorescent whole mount in situ hybridization analysis of a representative cadherin gene (Sp-pcdh9). Each image shows a z-projection of confocal slices (15–73 slices/stack). Red- Sp-pcdh9 mRNA. Blue- Hoechst staining (nuclei). At the late mesenchyme blastula (MB) stage, strong signal is seen in the vegetal plate (large arrowhead) but is absent from migrating primary mesenchyme cells (small arrowhead). At the early gastrula (EG) stage, signal is elevated at the margin of the blastopore (large arrowheads) but is absent from migrating secondary mesenchyme cells (small arrowhead). At the early prism (EP) stage, expression is highest at what appears to be the foregut/mid-gut boundary and in the circumblastoporal region (small arrowheads), as well as in the apical plate (large arrowhead). At the pluteus (PL) stage, expression is elevated in the ciliary band, especially overlying the postoral arms (large arrowhead), and in the gut (arrowhead). Asterisks indicate the archenteron. MB- mesenchyme blastula (24 hpf). EG- early gastrula (28 hpf). EP- early prism (44 hpf). PL- pluteus (72 hpf). Early gastrula stage embryos are shown in lateral (lat) and vegetal pole (vp) views. Scale bar = 50 μm
Fig. 5. Diagram depicting the proposed evolutionary history of cadherin subfamily members in echinoderms. All cadherin subfamily members, which contain cadherin repeat (CA) domains, are depicted using blue, purple, or red rectangles. Classical cadherins are defined as cadherins that contain predicted intracellular p120 and beta-catenin-binding motifs. Both atypical cadherins and protocadherins lack these motifs, but protocadherins are defined as proteins that contain 6–7 CA domains. The ancient bilaterian cadherin toolkit suggested by our phylogenetic analysis is depicted using a yellow box. Additions, losses, or modifications of the proteins within this toolkit are depicted using black tick marks on the tree branches. Protein additions are represented using plus signs ( +), while losses are represented using minus signs (−). The conversion of a protein from one cadherin subfamily to a different classification is depicted using an arrow. If branch-specific loss is not noted for a taxon on the diagram, it is assumed that organism contains all the proteins within the ancient bilaterian cadherin toolkit. CDH23 = cadherin-23, CDH88C = cadherin-88C, CSTN1 = calsyntenin-1, DCHS2 = dachsous-2, Fat1 = fat atypical cadherin 1, Fat4 = fat atypical cadherin 4, GCDH = G-cadherin, PCDH9 = protocadherin-9, PCDH15 = protocadherin-15, UECDH = uncharacterized echinoderm cadherin
