Mechanism of the oral-aboral axis patterning in a sea anemone

Body axes are systems of molecular coordinates allowing different structures to develop at correct places in the embryo. Both, in Bilateria (insects, mollusks, worms and vertebrates i.e. all animals with a head-tail and back-belly axes) and in their ancient cousins sea anemones and corals, these coordinate systems are established by gradients of Wnt/beta-catenin and BMP signaling running perpendicularly to one another. Thus, every cell in an animal is located in a position characterized by a certain unique combination of Wnt/beta-catenin and BMP signaling intensity. Such molecular address instructs the cells, which genes they have to be activating or deactivating and which structures they have to be making as the embryo develops. Generating a set of molecular addresses along the body axes of an animal is called axial patterning. We are interested in understanding how axial patterning mechanisms evolved at the base of animal life. In order to understand this, a comparison of axial patterning mechanisms of Bilateria and sea anemones two evolutionary lineages that split some 600-700 million years ago is necessary, however, the information about how sea anemones pattern their body axes is very limited. This project will be devoted to understanding the molecular mechanism of how the sea anemone Nematostella vectensis is patterning its main, oral-aboral body axis by Wnt/beta-catenin signaling. By using chemically treated as well as mutant Nematostella, in which beta-catenin signaling is either abnormally strong or abnormally weak, we will find out expression of which genes is regulated by beta- catenin signal and which of them are involved in Wnt/beta-catenin dependent patterning of the oral-aboral axis. We will also identify, which of the Wnt ligands are capable of activating beta-catenin signaling and test their preferences for particular receptors. Finally, we will functionally test the roles of each Wnt ligand and frizzled receptor involved in beta-catenin signaling in Nematostella. This analysis will provide us with the core of the gene regulatory network allowing activation of different genes at correct positions along the oral-aboral axis in a sea anemone embryo, and allow comparison with the way bilaterian embryos pattern their bodies from tail to head.

Bilateria are a gigantic taxonomic group encompassing all vertebrates as well as the vast majority of invertebrates. The ancestral way of patterning the main, posterior-anterior (PA) body axis of bilaterian embryos is by a gradient of the Wnt/-catenin signalling activity, which has its maximum at the posterior end and a minimum at the anterior end. The evolutionary sister group of Bilateria are Cnidaria (corals, sea anemones, jellyfish) whose main oral-aboral (OA) body axis is also patterned by Wnt, however, the correspondence of the cnidarian and bilaterian body axes is disputed. To clarify this, we analysed the molecular mechanism of the Wnt/-catenin-dependent patterning in a model sea anemone Nematostella vectensis. We showed that, similarly to the situation in Bilateria, LRP5/6 and all four Frizzled receptors are involved in transmitting Wnt signals required for OA patterning in the early embryo of Nematostella and identified the Wnt ligands playing the main roles in this process. Curiously, we found that, unlike in Bilateria, Wnt/Frizzled/LRP5/6-mediated signalling is not responsible for the specification of the endomesoderm of the embryo - a critical process preceding axial patterning both in Cnidaria and Bilateria. Then we uncovered the molecular principle of the dose-dependent response of genes to -catenin signalling in the Nematostella embryonic ectoderm, which results in the subdivision of the OA axis into three major domains: the oral, the midbody, and the aboral. The subdivision happens as follows: a number of transcription factor coding genes, whose expression is positively regulated by -catenin signalling, start to be expressed in the oral hemisphere of the Nematostella embryo. Their expression resolves into specific domains along the OA axis because some of these genes, which are expressed more orally, encode transcriptional repressors acting on the genes, which are expressed more aborally. This creates the two main molecular boundaries of the early Nematostella embryo - the oral/midbody boundary, and the midbody/aboral boundary. By performing an RNA-Seq-based search for transcription factors positively or negatively affected by the modulation of -catenin signalling, subsequent in situ hybridization screen and loss-of-function experiments on multiple candidate genes, we showed that the oral/midbody boundary is established by the module of four transcription factors: Brachyury, Lmx, FoxA, and FoxB, while the midbody/aboral boundary is created due to the activity of the transcription factor Sp6-9. The regulatory logic and the transcription factors involved in the -catenin dependent OA patterning in Nematostella were strikingly similar to that in Bilateria, which allowed us to suggest that the cnidarian OA and the bilaterian PA body axes share a common evolutionary origin.