The emergence and diversification of cell types is a leading factor in animal evolution. So far, systematic characterization of the gene regulatory programs associated with cell type specificity was limited to few cell types and few species. Here, we perform whole-organism single-cell transcriptomics to map adult and larval cell types in the cnidarian Nematostella vectensis, a non-bilaterian animal with complex tissue-level body-plan organization. We uncover eight broad cell classes in Nematostella, including neurons, cnidocytes, and digestive cells. Each class comprises different subtypes defined by the expression of multiple specific markers. In particular, we characterize a surprisingly diverse repertoire of neurons, which comparative analysis suggests are the result of lineage-specific diversification. By integrating transcription factor expression, chromatin profiling, and sequence motif analysis, we identify the regulatory codes that underlie Nematostella cell-specific expression. Our study reveals cnidarian cell type complexity and provides insights into the evolution of animal cell-specific genomic regulation.

BackgroundPlanktonic ciliated larvae are characteristic for the life cycle of marine invertebrates. Their most prominent feature is the apical organ harboring sensory cells and neurons of largely undetermined function. An elucidation of the relationships between various forms of primary larvae and apical organs is key to understanding the evolution of animal life cycles. These relationships have remained enigmatic due to the scarcity of comparative molecular data.ResultsTo compare apical organs and larval body patterning, we have studied regionalization of the episphere, the upper hemisphere of the trochophore larva of the marine annelid Platynereis dumerilii. We examined the spatial distribution of transcription factors and of Wnt signaling components previously implicated in anterior neural development. Pharmacological activation of Wnt signaling with Gsk3β antagonists abolishes expression of apical markers, consistent with a repressive role of Wnt signaling in the specification of apical tissue. We refer to this Wnt-sensitive, six3- and foxq2-expressing part of the episphere as the ‘apical plate’. We also unraveled a molecular signature of the apical organ - devoid of six3 but expressing foxj, irx, nkx3 and hox - that is shared with other marine phyla including cnidarians. Finally, we characterized the cell types that form part of the apical organ by profiling by image registration, which allows parallel expression profiling of multiple cells. Besides the hox-expressing apical tuft cells, this revealed the presence of putative light- and mechanosensory as well as multiple peptidergic cell types that we compared to apical organ cell types of other animal phyla.ConclusionsThe similar formation of a six3+, foxq2+ apical plate, sensitive to Wnt activity and with an apical tuft in its six3-free center, is most parsimoniously explained by evolutionary conservation. We propose that a simple apical organ - comprising an apical tuft and a basal plexus innervated by sensory-neurosecretory apical plate cells - was present in the last common ancestors of cnidarians and bilaterians. One of its ancient functions would have been the control of metamorphosis. Various types of apical plate cells would then have subsequently been added to the apical organ in the divergent bilaterian lineages. Our findings support an ancient and common origin of primary ciliated larvae.

Nematostella vectensis, an anthozoan cnidarian, whose genome has been sequenced and is suitable for developmental and ecological studies, has a complex neural morphology that is modified during development from the larval to adult form. N. vectensis' nervous system is a diffuse nerve net with both ectodermal sensory and effector cells and endodermal multipolar ganglion cells. This nerve net consists of several distinct neural territories along the oral-aboral axis including the pharyngeal and oral nerve rings, and the larval apical tuft. These neuralized regions correspond to expression of conserved bilaterian neural developmental regulatory genes including homeodomain transcription factors and NCAMs. Early neurons and stem cell populations identified with NvMsi, NvELAV, and NvGCM, indicate that neural differentiation occurs throughout the animal and initiates prior to the conclusion of gastrulation. Neural specification in N. vectensis appears to occur through an independent mechanism from that in the classical cnidarian model Hydra. ' 2009 Wiley Periodicals, Inc. Develop Neurobiol 69: 235-254, 2009

The puzzle of how complex nervous systems emerged remains unsolved. Comparative studies of neurodevelopment in cnidarians and bilaterians suggest that this process began with distinct integration centres that evolved on opposite ends of an initial nerve net. The 'apical nervous system' controlled general body physiology, and the 'blastoporal nervous system' coordinated feeding movements and locomotion. We propose that expansion, integration and fusion of these centres gave rise to the bilaterian nerve cord and brain.