bioRxiv preprint doi: https://doi.org/10.1101/235267; this version posted December 15, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Title: 2 Germ layer specific regulation of cell polarity and adhesion: insight into the evolution of 3 mesoderm. 4 5 6 Authors: 7 Miguel Salinas-Saavedra1*, Amber Q. Rock1, and Mark Q Martindale1† 8 9 10 Affiliation: 11 1 The Whitney Laboratory for Marine Bioscience, and the Department of Biology, University of 12 Florida, 9505 N, Ocean Shore Blvd, St. Augustine, FL 32080-8610, USA 13 14 15 16 *Corresponding author 17 Email: [email protected] (MS-S) 18 †Lead contact: 19 Email: [email protected] (MQM) 20 21 22 1 bioRxiv preprint doi: https://doi.org/10.1101/235267; this version posted December 15, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 23 24 25 Summary 26 In triploblastic animals, Par proteins regulate cell-polarity and adherens junctions of both 27 ectodermal and endodermal epithelia. But, in embryos of the diploblastic cnidarian Nematostella 28 vectensis, Par proteins are eliminated altogether in the bifunctional endomesodermal epithelia. 29 Using immunohistochemistry, CRISPR/Cas9, and overexpression of specific mRNAs, we describe 30 the functional association between Par proteins, ß-catenin, and snail genes in N. 31 vectensis embryos. We demonstrate that the aPKC/Par complex regulates the localization of ß- 32 catenin in the ectoderm by directing its role in cell adhesion, and that endomesodermal epithelia 33 are organized by a different cell adhesion system. We also show that snail genes, which are 34 mesodermal markers in bilaterians, are sufficient to downregulate Par proteins and translocate ß- 35 catenin from the junctions to the cytoplasm in N. vectensis. These data provide insight into the 36 evolution of epithelial structure and the evolution of mesoderm in metazoan embryos. 37 2 bioRxiv preprint doi: https://doi.org/10.1101/235267; this version posted December 15, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 38 Introduction 39 Bilaterian animals comprise more than the 95% of the extant animals on earth and exhibit 40 enormous body plan diversity (Martindale and Lee, 2013). One of the most important 41 morphological features in bilaterian evolution is the emergence of the mesoderm, an embryological 42 tissue that gives rise important cell types such as muscle, blood, cartilage, bone, and kidneys. The 43 emergence of mesoderm clearly contributed to the explosion of biological diversity throughout 44 evolution (Martindale, 2005; Martindale and Lee, 2013). Cnidarians (e.g., sea anemones, corals, 45 hydroids, and “jellyfish”) are the sister group to bilaterians, and despite their surprisingly complex 46 genomes (Putnam et al., 2007), do not possess a distinct mesoderm. Instead, the gastrodermal 47 lining to their gut cavity consists of a bifunctional endomesoderm with molecular characteristics of 48 both bilaterian endodermal and myoepithelial mesodermal cells (Jahnel et al., 2014; Martindale 49 and Lee, 2013; Martindale et al., 2004; Technau and Scholz, 2003). For example, brachyury and 50 snail, among other genes, contribute to the specification of the endomesodermal fates in both 51 bilaterian and cnidarian embryos (Magie et al., 2007; Martindale et al., 2004; Servetnick et al., 52 2017; Technau and Scholz, 2003; Yasuoka et al., 2016). Yet in bilaterians, mesodermal cells 53 segregate from a endomesodermal precursor (Davidson et al., 2002; Maduro and Rothman, 2002; 54 Martindale et al., 2004; Rodaway and Patient, 2001; Solnica-Krezel and Sepich, 2012) to form both 55 endoderm and a third germ layer not present in the embryos of diploblastic cnidarians. How 56 mesodermal cells originally segregated from the endomesodermal epithelium during animal 57 evolution is still unclear (Martindale, 2005; Martindale and Lee, 2013; Technau and Scholz, 2003). 58 During the last decade, several studies have described molecular and cellular characteristics 59 related to the segregation of mesoderm during bilaterian development (Darras et al., 2011; Keller 60 et al., 2003; Schäfer et al., 2014; Solnica-Krezel and Sepich, 2012). Here we investigate the 61 cellular basis of morphogenesis during embryogenesis of the “diploblastic” sea anemone, 62 Nematostella vectensis. 63 64 In most bilaterian embryos described to date, after a series of synchronous and stereotyped 65 cleavage patterns, maternal determinants redistribute and induce the localization of nuclear ß- 66 catenin to blastomeres of the vegetal pole (Martindale and Lee, 2013). Hence, gastrulation and the 3 bioRxiv preprint doi: https://doi.org/10.1101/235267; this version posted December 15, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 67 specification of endomesodermal fates is restricted to the vegetal pole. In these species, brachyury 68 is expressed at the border of the blastopore and snail is expressed in the prospective mesodermal 69 tissues (Technau and Scholz, 2003). The formation of mesoderm involves a variety of cellular 70 processes including the downregulation of E-cadherin, loss of apicobasal cell polarity, and in some 71 cases, the induction of epithelial-to-mesenchymal transition (EMT) (Acloque et al., 2009; Lim and 72 Thiery, 2012; Schäfer et al., 2014; Solnica-Krezel and Sepich, 2012). 73 74 Embryos of the cnidarian starlet sea anemone N. vectensis develop under non-synchronous and 75 random cleavage pattern with non-comparable cleavage stages from one embryo to another 76 (Fritzenwanker et al., 2007; Salinas-Saavedra et al., 2015). During blastula formation, embryonic 77 cells of N. vectensis begin to organize to form an embryo composed of a single hollow epithelial 78 layer. Epithelial cells of the animal pole, characterized by the nuclear localization of Nvß-catenin 79 prior to gastrulation (Lee et al., 2007; Wikramanayake et al., 2003), invaginate by apical 80 constriction to form the endomesodermal epithelium (Magie et al., 2007; Tamulonis et al., 2011). 81 The expression of Nvbrachyury around the presumptive border of the blastopore and Nvsnail 82 genes in the presumptive endomesodermal gastrodermis of N. vectensis embryos (Röttinger et al., 83 2012; Scholz and Technau, 2003) occurs even before the morphological process of gastrulation 84 begins. 85 86 Interestingly, the components of the Par system (NvaPKC, NvPar-6, NvPar-3, NvPar-1, and 87 NvLgl), which show a highly polarized “bilaterian-like” sub cellular distribution throughout all 88 epithelial cells at the blastula stage in N. vectensis, are degraded or down-regulated from the 89 endomesoderm during the gastrulation process (Figure 1A)(Salinas-Saavedra et al., 2015). We 90 have previously suggested that the expression of bilaterian “mesodermal genes” (e.g. Nvsnail) 91 might induce the loss of apicobasal cell-polarity indicated by the absence of the components of the 92 Par system in the endomesoderm of N. vectensis embryos (Salinas-Saavedra et al., 2015). 93 Compelling studies in N. vectensis and bilaterians have given information that supports this 94 hypothesis. For example, recent studies have shown that snail is necessary and sufficient to 95 downregulate Par3 in Drosophila mesoderm, inducing the disassembly of junctional complexes in 4 bioRxiv preprint doi: https://doi.org/10.1101/235267; this version posted December 15, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 96 these tissues (Weng and Wieschaus, 2016, 2017). In addition, we have shown that Nvbrachyury 97 regulates epithelial apicobasal polarity of N. vectensis embryos, suggesting conserved 98 mechanisms with bilaterian systems (Servetnick et al., 2017). Together, this evidence suggests a 99 plausible cellular and molecular mechanism for the segregation of a distinct cell layer in bilaterian 100 evolution from an ancestral endomesodermal tissue. Thus, in this study we describe the functional 101 association between the components of the Par system, apical junctions, epithelial integrity, and 102 the nuclearization of Nvß-catenin in a cnidarian embryo. In addition, we demonstrate that the 103 endomesoderm of N. vectensis embryos is organized by different junctional complexes that 104 confers different functional properties than the overlying ectoderm. And finally, we investigate the 105 putative interactions between the components of the Par system, Wnt canonical pathway, and snail 106 gene expression, giving insights on the evolution of the mesoderm and EMT. 107 108 Results and Discussion 109 110 Ectodermal and endomesodermal epithelium are joined by different cell-cell adhesion 111 complexes. 112 Components of the Par system are not present in the cells of endomesodermal epithelium of N. 113 vectensis during gastrulation, even though the very same cells express these proteins during the 114 blastula stage (Salinas-Saavedra et al., 2015) (Figure 1). This absence is consistent with the 115 absence of apical Adherens Junctions (AJs) in the endomesoderm of N. vectensis (Figure S1) and