Mechanism of the directive axis patterning in a sea anemone

Axial pattern formation is one of the key processes in animal development. After body axes are specified, the embryo is subdivided into regions giving rise to different primordia, which then develop into various morphological structures. In Bilateria, the Hox genes expressed in staggered domains specify cell identities along the anterior-posterior axis, and their misexpression leads to homeotic transformations where parts of the body acquire false molecular identities and develop ectopic structures. Cnidarians (corals, sea anemones, jellyfish and hydroids), the members of the diploblastic sister group of the Bilateria, are normally described as radially symmetric animals. However, the members of the class Anthozoa (corals, sea anemones) are bilaterally symmetric animals with a second directive body axis orthogonal to the primary oral-aboral axis. Several Hox genes and an Antennapedia class homeobox gene NvGbx are expressed in staggered endodermal domains along the directive axis of the sea anemone Nematostella vectensis. Recently, we have shown that BMP signaling specifies and maintains the directive axis in N. vectensis and that the staggered Hox and NvGbx expression is dependent on the BMP signaling in this animal. The aim of the proposed project is to understand the way the directive axis is patterned in Nematostella and the role of the Hox genes and NvGbx in this process. We hypothesize that the gradient of BMP signaling establishes the NvGbx and Hox gene expression in Nematostella, and the boundaries of the Hox and NvGbx expression domains specify the positions of the endodermal mesenteries; this hypothesis will be tested in the course of our work. We will search for all the direct targets of the BMP signaling by chromatin precipitation with the use of the anti-pSMAD1/5 antibody followed by next generation sequencing. We will also detect indirect targets via perturbing BMP signaling by morpholino knockdown of the genes coding for two BMP ligands and one BMP antagonist followed by transcriptome sequencing. The resulting lists of genes will provide us with candidates for further functional analysis. In parallel, we will also directly assess the function of the NvGbx and Hox genes in patterning of the directive axis by performing loss of function and overexpression experiments. We will learn whether the mode of Hox genes action is similar to that known in Bilateria and find out if changes in the expression of the NvGbx and Hox genes lead to a morphological change, including homeotic transformations known in bilaterian model systems. This will significantly advance our understanding of the axial patterning processes in early branching metazoan phyla and also promote Nematostella vectensis further as an Evo/Devo model organism as new genetic tools allowing tissue-specific inducible gene expression will be developed in the course of this study.

BMP signaling is central in patterning the dorsal-ventral body axis in Bilateria the huge group uniting all conventional animals with the bilateral body symmetry, such as vertebrates, insects, worms and mollusks. Bilateria have two body axes: a head-to-tail one, a back-to-belly one, as well as the left and the right side of the body. The evolutionary sister group of Bilateria are the Cnidaria (jellyfish, corals and sea anemones), which were all initially described as radially symmetric. Later it became clear that only the jellyfish and their closest relatives were truly radial, while corals and sea anemones were bilaterally symmetric, and in addition to the oral-aboral body axis common to all cnidarians, they possessed a second directive body axis. Even more strikingly, the directive axis turned out to be regulated by BMP signaling, just like the back-belly axis of the Bilateria. In order to find out how the two body axes of cnidarians relate to the two body axes of Bilateria and to understand whether the last common evolutionary ancestor of Cnidaria and Bilateria was radially or bilaterally symmetric, we first need to understand how BMP-driven axial patterning works in bilaterally symmetric cnidarians, which was the topic of this FWF project. We discovered that, similar to the situation in Bilateria, BMP signaling intensity forms a gradient along the directive axis of the sea anemone Nematostella vectensis, and this gradient regulates the subdivision of the directive axis into morphological compartments. Combining experimental analysis of the function of individual genes and mathematical modeling, we deciphered the gene regulatory network maintaining the BMP signaling gradient and compared it to the regulatory networks patterning the back-to-belly axis in bilaterian animals. We found striking similarities in the logic of the cnidarian and bilaterian BMP networks, but big differences in their topologies. Using next generation DNA sequencing technology, we identified all the direct target genes of BMP signaling, among which were Hox genes and Gbx, which were shown recently by us and others to be crucial for the formation of the compartments along the directive axis and giving each of the compartments their identities. In addition to the known regulatory genes, working under BMP control, we also identified many new ones with yet unclear roles. One of them, ZSWIM, turned out to be a completely novel modulator of BMP signaling in Nematostella. Nematostella ZSWIM encodes a nuclear protein, which appears to have a role explicitly in BMP signaling mediated gene repression, but not in BMP-dependent gene activation. Although ZSWIM is a conserved gene also present in Bilateria, its function is a mystery. Association studies suggest ZSWIM6 involvement in the development of the facial skeleton in humans. We established collaborations to find out what ZSWIM does in vertebrates and sea urchins. Taken together, this project has provided us with the basic understanding of the BMP-dependent axial patterning in the sea anemone and allowed us to compare BMP-dependent axial patterning in bilaterally symmetric cnidarians and in Bilateria.