The evolution of muscle cells: Development and structure of muscles in Cnidarians

Muscles are a major derivative of the mesoderm, the third germ layer found in all Bilateria and the evidence strongly suggests that bilaterian smooth and striated muscles both share a common origin in the Urbilateria. However, smooth and striated muscles can even develop in diploblastic animals (i.e. lacking the mesoderm), such as cnidarians, can develop, either from the ectoderm or the endoderm. In a previous FWF-funded project, we showed that despite their very similar appearance, even striated muscles, considered to be a complex cell type, evolved independently in cnidarians and bilaterians - on the basis of ancestral set of core proteins. Cnidarians are the sister group to the Bilateria and are therefore very informative for the evolution of key bilaterian traits. Here, we intend to use two cnidarian model species, the sea anemone Nematostella vectensis and the scyphozoan jellyfish Aurelia aurita to investigate the evolution of muscles in early branching animal lineages. In an unbiased transcriptome screen, we will try to identify muscle-specific genes involved in the developmental differentiation (postdoctoral project) or the structure (graduate student project) of the smooth and striated muscles. In the case of Nematostella, we will isolate retractor smooth muscle cells from a muscle-specific transgenic line by FACS. In Aurelia, we have developed a dissection technique to enrich striated muscles from the subumbrella. These samples of muscle cells will be used to generate a muscle-specific transcriptome and proteome from two diploblastic animals. Following the bioinformatic annotation and comparison, we will validate the differentially expressed genes by in situ hybridisation, and generate antibodies against a selected set of proteins to unravel the subcellular localization. The role of the developmental regulators and of the structural proteins will be investigated by gene knockdowns through morpholino injection (Nematostella) or electroporation of hairpin constructs / siRNAs (Aurelia). This project combines state-of-the-art techniques and unique expertise to answer a fundamental question in biology. It will hopefully shed light into how novel or newly recruited genes are integrated into an existing ancestral protein network in order to generate and evolve a complex cell type.

Muscle cells are one of the key innovations of animal evolution and the emergence of muscles probably allowed animals to diversify body plans and physiology within millions of animal species. Yet, how muscles evolved and whether muscles of different phyla are homologous to each other is unclear. Muscles in animals including humans can be smooth (like the lining of the gut) or striated (like the heart or the skeletal muscle) to fulfill different special tasks. Yet, by which mechanisms cells like muscle cells led to the diversification of cell types in evolution is also an enigma. In vertebrates and insects, which are part of the Bilateria, muscles arise mainly from the third germ layer, the mesoderm. In Cnidaria (sea anemones, jellyfish corals), which is an evolutionary early branch and sister to the Bilateria, muscles can be formed from both ectoderm and endoderm, as they lack the mesoderm. In this project we investigated the muscles in different cnidarians, emphasizing on a sea anemone, Nematostella vectensis. As each cell type differs by its set of activated and transcribed genes, the transcriptome reflects the identity of the cell type like a fingerprint. Using single cell transcriptomics we could determine the transcriptional profile of thousands of individual cells from the sea anemone. We were able to identify four distinct muscle cell types. When comparing them to each other and to muscles of Bilateria, we found that they group in two classes, fast-contracting and slow contracting muscles. The slow contracting muscles showed remarkable similarities in the set of regulatory proteins to the heart of vertebrates and insects, while the fast contracting muscles employed very different regulatory proteins. Notably, fast and slow muscles expressed numerous structural proteins, like myosins that arose from extensive gene duplications and that subsequently became specialised to either fast or slow muscles. Thus, extensive gene duplications facilitated the diversification of cell types. We postulate that the heart muscle of bilaterians has its evolutionary origin in slow contracting muscles in the common ancestor of cnidarians and bilaterians, some 700 Mio years ago. By contrast, the fast contracting muscles, like the striated skeletal muscles likely arose independently in the different animal lineages.