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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - TentaclesOfVenom (Unravelling the enigmatic origin of venom secreting cells in sea anemone)

Teaser

Summary

Evolution of venom, one of natureâ€™s most complex cocktails, has underpinned the predatory success of venomous animals. Until today, venom research has mostly focused on how genes coding venom proteins evolve, and our knowledge regarding the evolutionary origin of cells that produce venom and the mechanisms of injecting venom have been very limited. Understanding how venom and venom-secreting cells (VSCs) evolve in Cnidaria â€“ which are amongst the first venomous animals, is not only fascinating from an evolutionary perspective but could also prove to be instrumental in the development of efficient nematocyte (or stinging cell) based drug-delivery tools. Identifying variation in venom composition and sites of expression across various developmental stages and between the sexes (gender-specific expression) has also been a much neglected area of research. The former is particularly intriguing in sea anemones, as their larval stages do not feed, and yet, synthesize toxins.

Therefore, the major aims of this project were to 1) understand the origin and evolution of venom producing cells in Cnidaria; 2) characterize their expression profiles; and 3) identify variability in venoms across developmental stages, and between the sexes.

To address aims 1 and 2, we generated transgenic lines of the sea anemone Nematostella vectensis by modifying their genomes in such a way that VSCs produce a fluorescent protein. We then developed proteolytic cocktails that dissociate tissues and liberate individual cells. Following dissociation, the fluorescent VSCs were separated from all other types of cells using a Fluorescence Assisted Cell Sorter (FACS). We then sequenced the transcriptomes of these separated cell populations and employed bioinformatic analyses to identify genes that are differentially expressed (over- and underexpressed) within nematocytes, in comparison to the other types of cells. Our innovative strategy provided fascinating insights into the evolution of VSCs in Cnidaria. We have generated for the first time, the transcriptomic profiles of nematocytes in these animals. We have demonstrated that these evolutionarily unique cells develop through a number of transitionary stages, during which, they express different sets of toxins and physiological genes. We have made several other fascinating discoveries that will soon be submitted for publication in high-profile journals.

Citation: Sunagar K*, Columbus-Shenkar Y*, Fridrich A, Gutkovich N, Aharoni N, and Moran Y. Cell type-specific expression profiling sheds light on the development of a peculiar neuron, housing a complex organelle (in preparation).
* Equal contribution

For understanding the variability in venoms across different developmental stages, and between the sexes of N. vectensis (aim 2), we employed NanoString nCounter gene expression assay. Here, transcripts encoded by a large number of selected genes in various developmental stages and sexes were counted in a high-throughput manner. Our analyses revealed that toxin proteins are expressed throughout the development of this animal and that at least one class of toxin is maternally deposited in the eggs. Our results unravel a much more complex and dynamic venom landscape than initially appreciated, and highlight the importance of studying ontogenetic variability in venoms.

Citation: Columbus-Shenkar Y*, Sachkova M*, Fridrich A, Modepalli V, Sunagar K, and Moran Y. Dynamics of venom composition across a complex life cycle (under review).
* Equal contribution

Other research highlights
1. Widespread convergence in toxin resistance by predictable molecular evolution: â€œConvergence has a strong bearing on the fundamental debate about whether evolution is stochastic and unpredictable or subject to constraints. Here we show that, in certain circumstances, evolution can be highly predictable. We demonstrate that several lineages of insects, amphibians, reptiles, and mammals have utilized the same molecular solution, via

Work performed

During the course of this fellowship, we generated transgenic lines of sea anemone to trace the evolutionary origin of in the starlet anemone, Nematostella vectensis. We modified the genomes of these transgenic lines using CRISPR-cas9 system â€“ a tool for genomic DNA modification, so a wide population of VSCs produce a fluorescent (mOrange) protein. To accomplish this, we targeted a gene encoding a structural protein that is integral to building the capsules of nematocytes. We then developed proteolytic cocktails that dissociate tissues and liberate individual cells. Following dissociation, the fluorescent VSCs, which glow in orange when excited by green light, were separated from other types of (non-glowing) cells using a Fluorescence Assisted Cell Sorter (FACS). Next, we sequenced the transcriptomes of these separated cell populations and employed bioinformatic analyses to identify genes that are differentially expressed within nematocytes, in comparison to the other types of cells.

To identify the variability in venoms across different developmental stages, and the between the sexes of N. vectensis, we employed the state-of-the-art NanoString gene expression measurements.

We have also investigated how venoms evolve across large evolutionary time scales in the animal kingdom, and how animals can acquire resistance to various poisons because of similar and strong selective pressures. The results of these research projects have been published in influential journals in the fields of genetics and molecular biology and evolution, and many are either under review or in preparation for publication.

Final results

Research carried out during the tenure of this fellowship has advanced our understanding of the evolution of venom and venom producing systems across a very wide range of animal groups. Particularly, we have provided fascinating insights into the evolution of venom and venom producing cells in Cnidaria. These results have direct implications for the design of efficient nematocyte based drug delivery systems with tremendous therapeutic and cosmetic relevance.