Objective 1 - Ctenophore nerve net connectivity and molecular composition.
Using 3D electron microscopy, we found that nerve-net neurons are not separate entities, but rather are interconnected through continuous neurite plasma membranes without evidence of synapses (syncytial nervous system). Our findings suggest fundamental differences of nerve net architectures between ctenophores and cnidarians/bilaterians and offer an alternative perspective on neural network organization and neurotransmission. We are currently working on a complete connectome of a 5-day old larva. 2500 of ca. 6000 sections have been prepared and imaging of the first 300 sections has started as well. This work is done in collaboration with the EM facility at the University of Bergen.
Objective 2 - Development of ctenophore nerve net neurons.
We found that the ctenophore Mnemiopsis leidyi is capable of reversal from mature lobate to early cydippid when fed following a period of stress. Our findings illuminate central aspects of ctenophore development, ecology, and evolution and show the high potential of Mnemiopsis as a unique model system to study reverse development and rejuvenation. Moreover, we described an easy and affordable setup to maintain a stable culture of the ctenophore Mnemiopsis which allows for stable laboratory lines, a continuous supply of embryos for molecular biological studies, and independence from population responses to environmental fluctuations. We are currently working towards understanding the development of the ctenophore nervous system at a single cell level.
Objective 3 - Functional characterisation of ctenophore neurons.
We are now able to perform imaging of the nerve net with fluorophores in live animals, allowing visualization of neurites and cell bodies, over short (second) to medium (min-hours) time scales. Apart from being able to identify the cells in living tissue, this has identified the dynamic nature of structures within the neurites, which are likely functionally relevant.
We have generated codon harmonized version of GCaMP6 for Mnemiopsis and cloned into an in vitro transcription vector followed by generation and purification of GCaMP6 mRNA for injection. Moreover, we generated a Mnemiopsis expression plasmid for injection. Both, mRNA and the plasmid generation were injected into 1, 2, and 4 cell stage embryos. To better allow ctenophore monitoring we developed mounting and imaging methods for visualizing the SNN (with cellular resolution) in living animals. For perturbations of the nerve net – splice blocking morpholinos targeting a neuropeptide have been designed and ordered, as well as gRNA targeting a neuropeptide has been designed and ordered for injection.
Outcome:
Soto Angel JJ, Burkhardt P (2024) Reverse development in the ctenophore Mnemiopsis leidyi. Proceedings of the National Academy of Sciences 121 (45): e2411499121.
Soto Angel JJ, Nordmann EL, Sturm D, Sachkova M, Pang K, Burkhardt P (2024) Stable laboratory culture system for the ctenophore Mnemiopsis leidyi. Methods in Molecular Biology 2757: 123–145.
Burkhardt P, Colgren J, Medhus A, Digel L, Naumann B, Soto Angel JJ, Nordmann EL, Sachkova MY, Kittelmann M (2023) Syncytial nerve net in a ctenophore adds insights on the evolution of nervous systems. Science 376 (6642): 293-297.
