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Supracellular contractility dynamics and cell communication during collective chemotaxis.

Periodic Reporting for period 1 - SUPRACELL_COMMUN_CCT (Supracellular contractility dynamics and cell communication during collective chemotaxis.)

Reporting period: 2016-10-01 to 2018-09-30

Collective chemotaxis, whereby groups of cells move in response to a chemoattractant, is a highly coordinated process underlying development and tumour migration. The Neural Crest (NC) is an invasive mesenchymal cell population that, responding to chemoattractant signals such as Sdf1, migrates throughout the embryo to differentiate into a variety of cell types, in a way that is reminiscent of metastatic migration in some cancer types. NC cells perform chemotaxis much more effectively as a cluster than as single cells, indicating that cell-cell communication improves coordination and directionality. In this project, we addressed the cluster behaviour as a ‘supracellular’ entity, where cell-cell intercellular chemical communication is maintained in order to collectively polarize and migrate.
Defective migration of the NC during embryogenesis leads to many human syndromes and birth defects, collectively known as neurocristopathies, and this is why the project is highly relevant for addressing development and diseases.
From the results we conclude that ATP, secreted through Connexin hemichannels and sensed via P2Y8 and P2Y11 receptors, controls NC dispersion by regulating cellular levels of the adhesion molecule E-cadherin. Our data support the hypothesis that purinergic and calcium signaling play a main role in regulating the collective behaviour of migrating NC, by coordinating their supracellular organisation. Further investigation is needed to extend these observation to our recently developed Zebrafish model for in-vivo calcium imaging during NC migration, and to assess which other molecular players take part to the pathway regulating E-cadherin expression levels.
The results of this project surely will contribute to increasing competitiveness and economical impact of European research worldwide.
Work performed:
Analysis of the biomechanics of the cell cluster and cell-cell communication. Further experiments on the relationship between the actomyosin ring and connexin/gap junction signalling were not as convincing as initially thought by the analysis of preliminary data. However, we found, even more interestingly, that ATP, a molecule never involved in neural crest migration before, has effect on synchronising cell cluster movements. This work package was therefore reduced in size and we started to focus on how the ATP controls coordination of the migrating cluster, without further characterisation of actomyosin ring.
Analysis of connexins/gap junctions and purinergic signalling. The work included expression analysis of ATP receptors and knockdown experiments in vitro and in vivo to study the function of these receptors and their involvement in response to ATP stimulation, dispersion effect and migration (including all appropriate controls), as well as expression analysis and knockdown experiments for connexins in vitro and in vivo (Milestone III). Furthermore, the effect of these knockouts on E-Cadherin expression levels was addressed.
Analysis of diffusible messenger and development of a zebrafish transgenic line to study calcium signals in the neural crest during migration in-vivo. The work involved analysis and characterisation of ATP-induced intracellular calcium signals and some of the inhibitors and analogues experiments (Milestone V). Characterisation of the effect of ATP stimulation (including apyrase experiments and all appropriate controls) on collective migration and coordination of the neural crests cluster, as well as effect on chemotaxis, motility and speed of neural crest cells both at the single-cell and collective level (Milestone V). Furthermore, the effect of ATP stimulation on E-Cadherin expression levels was addressed. Additionally, a zebrafish transgenic line was established to express the GCaMP5 calcium sensor specifically in the neural crest. This line was used to observe calcium transient patterns in vivo, using confocal imaging, in migrating neural crest cells in developing zebrafish embryos (Milestone IV). All experiments were carried out by the Researcher.

Result Dissemination:
Due to the early termination of the Fellowship, publication of the result in international scientific journals has not been achieved yet, however, the results obtained in the Fellowship months are of the highest relevance in the collective cell migration field, and future publication is still possible if other researchers in the lab will carry out the final needed experiments.
Besides participating in outreach activities, such as New Scientist Live 2016 London in collaboration with The Royal Society of Biology, I also set up a Twitter account of the Host Lab for the first time. This account had been well received by the scientific community and beyond, and it has quickly collected hundreds of followers in just a few months of being set up ( The page is being regularly updated by all members of the Host Lab to share interesting new research in the fields of cell and developmental biology, exciting new results and interesting discussion and presentations from the top scientists in the field during international conferences. Given the wide audience of this social media, this information is likely to reach not only scientists and collaborators, but also the general public. Evidence of this outreach potential is in several comments received from non-scientists to some posts on the page.
This project contributed to shed light on some of the basic principles underlying collective chemotaxis. This question has implications in developmental craniofacial conditions and cancer metastasis, therefore it’s of the highest scientific importance. Besides the established in vivo and in vitro techniques in the Host lab, my contribution with this project helped establishing a new calcium imaging method in cultured Xenopus neural crest cells as well as in vivo in zebrafish, resulting in experimental advantages as very little is known about the relevance of calcium signalling for collective migration. Specifically, I established a new transgenic zebrafish line that will be used further in the lab by other researchers after my departure to address different aspects of neural crest collective migration and calcium signalling. This interdisciplinary project also constituted a platform for development and sharing of new methods and approaches with the scientific community.
The novelty of this project’s results and discoveries, as well as the experimental approach adopted, is going to have high impact in the field of collective cell migration, generating a new platform of techniques potentially useful also for studying collective chemotaxis in other models, like cancer cells. The work carried out during the Fellowship strongly helped to strengthen existing collaborations of the Host Lab.
The early termination of the project determined a deviation from the original proposal, however, several objectives of the proposal have been achieved (see above). In fact, this project provided important insights into the biology of collective migration and the cellular pathways involving connexins, calcium and ATP, leading to coordination of a collectively migrating NC cell cluster. However, further work will be required to achieve a publishable level for these observations.