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Analysis of the neural transcriptome of the sea anemone Nematostella vectensis

Final Report Summary - ANTSAN (Analysis of the neural transcriptome of the sea anemone Nematostella vectensis)

Summary

The objective of ANTSAN was to investigate nervous system development in the sea anemone Nematostella vectensis and thereby address the current paucity of data on the molecular control of cnidarian neurogenesis. This was to be achieved by documenting the cell-type specific gene expression profile of N. vectensis neurons, and then using this dataset to identify candidate genes to analyse at a functional level.

Due to technical difficulties in achieving the objective of isolating and analysing the RNA content of a population of N. vectensis neurons, the methodology of ANTSAN was adjusted such that the project instead focused around the study of the opposing activities of two molecular regulators of N. vectensis neurogenesis - Notch signalling which restricts neurogenesis, and a SoxB gene, NvSoxB(2), which promotes neurogenesis. We used functional and descriptive techniques, including morpholino knockdown, transgenic technologies and drug treatments, to examine the activity and interplay of these two components. From this work, we have achieved significant progress in understanding the cellular and molecular events of neurogenesis in N. vectensis, and a manuscript describing these results is currently in preparation for submission to the journal Neuron.

Results

The goal of ANTSAN was to describe the neural transcriptome of N.vectensis larvae expressing a fluorescent reporter protein in their nervous systems by dissociating them into single cells and then isolating the neurons via fluorescence-activated cell sorting (FACS). The RNA content of the neural and non-neural cell populations would then be compared using microarrays. Towards these goals, we developed a protocol for creating viable single-cell suspensions of N. vectensis, and we were successful in separating the neurons from the rest of the cells via FACS. The project then stalled as we had great difficulty in attaining a sufficient quantity and quality of RNA from the isolated cells.

As such we refocused ANTSAN on the investigation of an established transgenic line of N.vectensis - pSoxB(2)::mOrange - in which the promoter regions of a neural gene of interest, NvSoxB(2), drives the expression of the reporter gene mOrange. We analysed the line using a range of descriptive methodologies and investigated the function of NvSoxB(2) using morpholino knockdown. We discovered that NvSoxB(2) plays a key role in early neurogenesis, being expressed in neural progenitor cells that go on to form the cells of both the ectodermal and endodermal nervous systems, and being required for the promotion of neurogenesis. We continued our investigations by considering the interplay between NvSoxB(2) and other neural regulators, focusing on the Notch signalling pathway and proneural basic helix-loop-helix (bHLH) genes. We found that NvSoxB(2) and Notch have opposing roles in the development of the N. vectensis nervous system, and that NvSoxB(2) interacts with Notch signalling at the level of the proneural gene activity.

The insights into N. vectensis neurogenesis achieved by ANTSAN will be built upon in several ongoing projects within the Rentzsch research team. Primarily, a PhD student will make use of the described opposing functionalities of NvSoxB(2) and Notch to perform a microarray analysis in which the gene expression of N.vectensis larvae with depleted nervous systems (NvSoxB(2) knockdown) will be compared against those with expanded nervous systems (Notch knockdown). This will provide a list of candidate genes specific to nervous system development, and the PhD student will select both novel and conserved genes of interest from this list for detailed functional analysis. Additionally, commencing in September 2013, a Masters student will examine the role of Notch signalling in apical organ formation.

Conclusions / impact

The regulatory basis of early neurogenesis has been studied in only few bilaterian animal groups, leaving the evolutionary origin and the variability of this process largely unexplored. In ANTSAN, we have investigated neurogenesis in the sea anemone N. vectensis and in doing so have identified conserved and variable roles of Notch signaling, Sox B and bHLH genes in the development of neural cell types. By use of a transgenic reporter line, we discovered that NvSoxB(2) is transiently expressed in neural progenitor cells that will give rise to sensory cells, ganglion cells and nematocytes of both the ectodermal and endodermal nervous systems, throughout the course of development. By use of morpholino knockdown and drug treatment assays, we found that NvSoxB(2) is a positive regulator of neurogenesis and that conversely, the Notch signalling pathways negatively regulates the development of neurons and nematocytes. Double inhibition experiments revealed that the increase in neurogenesis caused by blocking Notch signalling is dependent on functional NvSoxB(2). Our data show that the broad neurogenic potential of N.vectensis is based on a gene regulatory network with significant similarity to that of bilaterians.

The value of these data is two-fold. Firstly, functional analyses of neurogenic genes in N. vectensis represent a move beyond the gene expression studies that had previously dominated the field, and have allowed us to unequivocally determine the consequence of gene expression in N. vectensis neural cells. Secondly, these data contribute to our understanding of the relationship between cnidarian and bilaterian nervous systems, and provide new insight into longstanding evolutionary questions about the emergence of simple nervous systems in the animal kingdom.

In a biomedical context, the worth of investigating neurogenesis in N. vectensis lies in the possibility of attaining significant insights into the molecular mechanisms behind the impressive neurogenic potential of cnidarians. These animals make neurons throughout both the internal and external layers of their bodies, across their entire lifespan, and possess an immense regenerative capacity. We anticipate that an understanding of cnidarian neurogenesis could potentially be applied to encourage axon regrowth and regeneration after injury or disease in humans. Currently, biomedical applications for ANTSAN data are distant projections, but they are nonetheless credible prospects that may provide a significant service to human health research.

Contact details

All work was carried out in the lab of Fabian Rentzsch at the Sars International Centre for Marine Molecular Biology, Thormøhlensgate 55, Bergen N-5008, Norway.