Hox gene transcription factors are important regulators of positional identity along the anterior–posterior axis in bilaterian animals. Cnidarians (e.g., sea anemones, corals, and hydroids) are the sister group to the Bilateria and possess genes related to both anterior and central/posterior class Hox genes. Here we report a previously unrecognized domain of Hox expression in the starlet sea anemone, Nematostella vectensis, beginning at early blastula stages. We explore the relationship of two opposing Hox genes (NvAx6/NvAx1) expressed on each side of the blastula during early development. Functional perturbation reveals that NvAx6 and NvAx1 not only regulate their respective expression domains, but also interact with Wnt genes to pattern the entire oral–aboral axis. These findings suggest an ancient link between Hox/Wnt patterning during axis formation and indicate that oral–aboral domains are likely established during blastula formation in anthozoan cnidarians.

In triploblastic animals, Par-proteins regulate cell-polarity and adherens junctions of both ectodermal and endodermal epithelia. But, in embryos of the diploblastic cnidarian Nematostella vectensis, Par-proteins are degraded in all cells in the bifunctional gastrodermal epithelium. Using immunohistochemistry, CRISPR/Cas9 mutagenesis, and mRNA overexpression, we describe the functional association between Par-proteins, ß-catenin, and snail transcription factor genes in N. vectensis embryos. We demonstrate that the aPKC/Par complex regulates the localization of ß-catenin in the ectoderm by stabilizing its role in cell-adhesion, and that endomesodermal epithelial cells are organized by a different cell-adhesion system than overlying ectoderm. We also show that ectopic expression of snail genes, which are expressed in mesodermal derivatives in bilaterians, is sufficient to downregulate Par-proteins and translocate ß-catenin from the junctions to the cytoplasm in ectodermal cells. These data provide molecular insight into the evolution of epithelial structure and distinct cell behaviors in metazoan embryos.

Summary 25In triploblastic animals, Par proteins regulate cell-polarity and adherens junctions of both 26 ectodermal and endodermal epithelia. But, in embryos of the diploblastic cnidarian Nematostella 27 vectensis, Par proteins are eliminated altogether in the bifunctional endomesodermal epithelia. 28Using immunohistochemistry, CRISPR/Cas9, and overexpression of specific mRNAs, we describe 29 the functional association between Par proteins, ß-catenin, and snail genes in N. 30vectensis embryos. We demonstrate that the aPKC/Par complex regulates the localization of ß-31 catenin in the ectoderm by directing its role in cell adhesion, and that endomesodermal epithelia 32 are organized by a different cell adhesion system. We also show that snail genes, which are 33 mesodermal markers in bilaterians, are sufficient to downregulate Par proteins and translocate ß- 34catenin from the junctions to the cytoplasm in N. vectensis. These data provide insight into the 35 evolution of epithelial structure and the evolution of mesoderm in metazoan embryos. 37All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx