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Project titleThe role of embryonic glial cells in the development of peripheral nervous systems and neurodegenerative pathologies

AffiliationNational Scientific Center of Marine Biology of the Far Eastern Branch of the Russian Academy of Sciences,

KeywordsNeural crest cells, nervous systems, development, glial cells, genetic tracing, mouse

Annotation
The project is aimed at solving the fundamental problem of developmental biology - elucidating the mechanisms of specialization of stem cells and their use as a regenerative cellular material in the pathological conditions of the body. The mechanisms of directed specialization of embryonic cells remain not yet understood, and molecular scenarios obtained in in vitro systems are often not repeated in vivo. One source of a wide variety of cellular types in early development is neural crest cells, capable of producing many differentiated cell types in the early stages of embryo development (Le Douarin, 1986, Prendergasr, Raible, 2014, Le Douarin et al., 2004, Le Douarin, Kalcheim, 1999). It is generally accepted that the cells of the neural crest after migration and reaching the final location under the influence of a specific set of factors give one or another cellular type, thus providing a variety of cellular variations (Le Douarin et al., 2004). For a long time it was believed that the neural crest cells directly (or through blast forms) differentiate into terminally differentiated states, contributing to the overall development of the embryo (Rohrer, 2011). Our data changed the idea of the hierarchy of cells of nervous rowing and forced to reconsider the mechanisms of their specialization. Thus, we found that some cell types specialize not directly from neural crest cells, but through the "glial" stage (Adameyko et al., 2009; Dyachuk et al., 2014; Kaukua et al., 2014; Furlan et al., 2017). The cells of the neural crest after migration are able to specialize in cells with a glial phenotype, they are tightly associated with peripheral nerves, which in turn serve as specific rails for the migration of these cells over long distances (Dyachuk et al., 2014). After reaching the final location in the embryo, the glial (Schwann) cells disconnect from the nerve and differentiate. Such a mechanism for delivery of multiplet cells with subsequent specialization has been shown for some cell types: melanocytes (Adameyko et al., 2009), parasympathetic neurons (Dyachuk et al., 2014), mesenchymal stem cells, odontoblast (Kaukua et al., 2014). However, after describing the biological phenomenon of the glial origin of some cellular types and clarifying the hierarchy of neural crest cells, the molecular mechanisms of glial cell specialization, the role of peripheral nerve signals in maintaining the glial stem niche and their role in tissue morphogenesis remain unexplored. Nerve-associated cells are of particular interest in regeneration processes, pathological processes whose mechanisms are largely unknown (Johnston et al., 2013). Undoubtedly, such stem niches have great potential, which we still have to study and understand not only in early development, but also in adult animals. This project suggests studying the mechanisms of specialization of parasympathetic, sympathetic and enteric neurons, the source of which is embryonic glia. The project is relevant, as it raises complex and still unresolved issues of developmental biology with direct access to biomedical research of a fundamental and applied nature. The set of genetically modified animal lines used in the project is unique and is used for the first time to solve similar problems. To solve the main goal of the project, modern methods of embryonic genetic tracing, transcriptomal analysis, genetic ablation of glia and peripheral nerves will be used. Such a modern and multifactorial range of approaches will allow unambiguously to obtain data not inferior to the world level.

Expected results
This project is aimed at studying the possibilities of stem cells to differentiate into different terminal cell types during development, as well as studying the role of glial cells in pathological processes in genetically modified model objects. The idea of the project is new and unique, since the proposed approach to solving problems of treatment with stem glial cells of neurodegenerative diseases is proposed for the first time. The proposed project is a logical continuation of previous studies with immersion in the molecular mechanisms of differentiation, elucidation of the signaling pathways and the search for new possibilities for controlling the specializations of glial cells in embryonic development. As a result of the project, new data on the biology of stem cells will be obtained. The main results planned to be obtained during the project implementation: 1. To study new potentialities of differentiation of glial stem cells in early development; 2. To identify molecular mechanisms and signaling pathways for the specialization of glial cells in neurons of peripheral nervous systems. 3. To determine the role of glial stem niches in tissue embryonic morphogenesis; 4. To study the role of glial cells and their descendants in neuropathological processes. Successful implementation of this project will open new opportunities for the use and management of stem niches in embryonic development. The results of the project will have a wide range of applications: from the possibilities of correcting the dysfunctions of autonomic nervous systems to the prevention of malignant tumors in which the peripheral nervous systems play an important role. The main consumer of the data obtained will be biological and biomedical research groups engaged in the development of therapy and treatment, primarily neurodegenerative diseases, in the medicine of catastrophes, the restoration of the innervation of extremities after trauma, and a wide range of nerve-associated pathologies of embryonic development. The project for the Russian Federation is new and relevant, as there are no scientific groups involved in peripheral innervation and nerve-associated stem cells in the country.

Annotation of the results obtained in 2020
During the project frame (2020-2021), all the planned experiments were carried out and completed, new data on the nature and behavior of the peripheral heterogeneous population of Schwann cells in the development and adult lines of rodents and zebrafish were obtained and published. For the first time, it was shown that in the embryos of transgenic mice activated on 10 day of development and analyzed on 11 day of development, glial cells sitting on the cranial nerves have a high proliferative status. At the same time, Schwann's tracer cells almost do not divide from the stage of 15 days. All proliferating glial cells do not express myelin sheath markers. We conclude that in the late stages of development and postnatally, non-myelinated Schwann cells are subject to rare division, and myelinated cells do not divide normally. The most valuable data were obtained using the transplantation of “fluorescent” glioblastoma cell lines, as well as the patient's material into blastula and zebrafish Hb9-GFP larvae. Monitoring the behavior of the grafts using time-lapse microscopy, as well as real-time light microscopy, showed that the transplanted glioblastoma cells quickly migrate to the developing nervous system, where they create an orthotopic intracranial tumor within 5 days after transplantation. The problem of the potentials of the peripheral glia of adult animals has been solved. Long-term genetic tracing of embryonic (12-18hpf) and larval (2dpf-5dpf) Sox10+ glia (up to 45 days) for the Sox10-CreERT2 line; Ubi:Zebrabow-S showed that the Schwann cells of the larvae produce enteric (enteral) neurons of adult zebrafish. Surprisingly, the fact that glia in the enteric nervous system is generally absent (or immunonegative for classical markers) and, therefore, can not be source cells for neurons.

Publications

1. Maxim Shirokov, Valentin Milichko, and Vyacheslav Dyachuk Zebrafish as model system for cancer development AIP Conference Proceedings, - (year - 2020)

2. Milichko V., Dyachuk V. Novel glial cell functions: extensive potency, stem cell-like properties, and participation in regeneration and transdifferentiation Frontiers in Cell and Developmental Biology, - (year - 2020)

Annotation of the results obtained in 2018
We have been discovered a new mechanism of cell specialization in the early development of mice. A new type of pluripotent cells has been found, glial progenitor stem cells, which have a characteristic set of glial markers and are always associated with peripheral nerves, support migration processes of a given cell type and their survival in development. Using the method of genetic tracing and transgenic mice, it was shown that glial progenitor cells are able to specialize into neurons of the autonomic nervous systems, adrenal gland cells and Zuckerkandl organ, as well as in pigment cells, odontoblasts, as previously shown. These data are strongly changing our understanding of the hierarchy of stem cells and the mechanisms of cell specialization in early development. Moreover, the transcriptional data showed that the choice that the neural crest cells make to become sensory, glial, autonomous neurons or mesenchymal cells can be signed as a sequence of binary solutions. As a result of this type of solution, a group of cells with a similar set of gene expression, markers of differentiated cells, branches off. And the determination of the specific fate of the cell is achieved by increasing the synchronization of certain programs and the simultaneous suppression of competing programs that decide the fate of the cell. The data obtained show how complex cell hierarchy is, which is realized in the development of vertebrate animals. The data are not only of fundamental importance but also applied, as the impairment in the development of the neural crest, migrating cells, as well as glial precursor cells and the expression of their specific gene assemblies are realized in developmental anomalies up to their mortality.

Publications

1. Maria E. Kastriti, Polina Kameneva, Dmitry Kamenev, Viacheslav Dyachuk, Alessandro Furlan, Marek Hampl, Fatima Memic, Ulrika Marklund, Francois Lallemend, Saida Hadjab, Laura Calvo-Enrique, Kaj Fried, Patrik Ernfors, Igor Adameyko Schwann cell precursors generate the majority of chromaffin cells in Zuckerkandl organ and some sympathetic neurons in paraganglia Frontiers, - (year - 2019)

2. Ruslan Soldatov, Marketa Kaucka, Maria Eleni Kastriti, Julian Petersen, Tatiana Chontorotzea, Lukas Englmaier, Yunshi Yang, Viacheslav Dyachuk, Jean-Francois Brunet, Gian Giacomo Consalez, Patrik Ernfors, Kaj Fried, Peter V. Kharchenko, Igor Adameyko Spatio-temporal structure of cell fate decisions in murine neural crest Science, - (year - 2019)

3. - Cпособ иммуноокрашивания и химического обесцвечивания крупных биологических объектов для микроскопии -, - (year - )

4. - Вручение премий Президента в области науки и инноваций для молодых учёных Официальные сетевые ресурсы Президента России, Опубликован в разделах: Новости, Выступления и стенограммы Дата публикации: 7 февраля 2019 года, 14:00 (year - )

Annotation of the results obtained in 2019
Sympathetic neurons and neuroendocrine chromaffin cells control basic functions of the body, being main depots of catecholamines outside CNS. Schwann cell precursors (SCPs) are multipotent nerve-navigating cells found throughout the body. To investigate the multipotency of SCPs in Danio rerio, we utlized lineage tracing and demonstrated that SCPs traverse peripheral nerves and give rise to the majority of sympathetic neurons and chromaffin cells in an ERBB3-dependent way. Consistently, the ablation of SCP-carrying ventral motoneurons through CAS9-mediated knockout of Olig2 resulted in a reduction of sympathetic neurons and chromaffin cells. Based on CAS9-mediated knockout of Chat, our results indicate an important role of cholinergic signaling from peripheral nerves in morphogenesis of sympathetic ganglia. Altogether, we revealed that peripheral innervation and nerve-associated SCPs contribute to the development of the sympathoadrenal system in a teleost fish. This provides strong support to the notion that the multipotency of nerve-associated SCPs is an evolutionary preserved trait. We hypothesize that multipotent SCPs might be present in adult tissues and retain capacities to differentiate into autonomic neurons and chromaffin cells. This has obvious implications for regenerative medicine.

Publications

1. Kamenev D., Kotsyuba E., Kalachev A., Milichko V., Dyachuk V. Glia-neuronal transitions in development: significance in medical research and treatment of pathologies Journal of Physics: Conference Series, - (year - 2020) https://doi.org/10.1088/1742-6596/1461/1/012101

2. Xie M, Kamenev D, Kaucka M, Kastriti ME, Zhou B, Artemov AV, Storer M, Fried K, Adameyko I, Dyachuk V, Chagin AS. Schwann cell precursors contribute to skeletal formation during embryonic development in mice and zebrafish Proceedings of the National Academy of Sciences, - (year - 2019) https://doi.org/10.1073/pnas.1900038116

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