Function of mechanical forces in the construction of neuronal circuits in vivo – NEUROMECHANICS

We used a multidisciplinary approach that combines imaging of neuronal behavior (cellular movements, axon growth), physical approaches to measure and disturb forces in vivo and more traditional molecular/genetic tools.
To establish a map of the mechanical forces in our system, we mainly used the laser ablation technique. It involves destroying a structure within a tissue (such as a contact between two cells, or a small group of cells) with a laser and analyzing the immediate reaction of the tissue. If the structure was under tension, it opens upon ablation. The more the structure was initially tense, the faster the opening following the ablation. We can therefore use this speed as a proxy for mechanical tension within the biological structure. We performed these laser ablations at the tissue, cellular (Monnot et al., 2022) and subcellular (Baraban et al., 2023) scales. We have also started to develop, in collaboration with the physicist Laetitia Pontani, a new approach for measuring forces in vivo, which consists of injecting small oil droplets into biological tissues, then monitoring their deformations by microscopy, and to deduce the forces which are applied to these droplets by the surrounding cells/tissues.
To perturb the mechanical forces and analyze their role, we used:
- a pharmacological approach: we treated zebrafish embryos with drugs that target molecules that generate forces in cells and tissues (actomyosin);
- genetic approaches, such as for example a mutant line in which the eyes, whose morphogenesis is a source of mechanical forces, do not develop (Monnot et al., 2022), or lines allowing to genetically perturb actomyosin in certain tissues only (Baraban et al., 2023).

Our previous results suggested that extrinsic mechanical forces control the movement of olfactory neurons and the extension of their axons by causing the cell body to move passively away from the axonal extremity which remains fixed (Breau et al., 2017). Thanks to the ANRJC funding, the team has identified the origin of these extrinsic forces, which come from the morphogenesis of a neighboring tissue, the developing eye, and are transmitted by the extracellular matrix located at the interface between the two tissues. We also used the ANRJC funding to initiate a new line of research in the team and characterize the mechanical interaction between olfactory neurons and the skin epithelium, which leads to the opening of the zebrafish nostril, a developmental step that is essential for the olfactory function.

The objectives have been largely achieved with regard to the in vivo mapping of the forces and the perturbation of the forces in the developing olfactory circuit. The ECM has been identified as a central player in force transmission between the eye and the OP, but the mechanisms underlying this force propagation remains to be characterised in detail. In this sense, oil droplet-based sensors for forces and material properties in the ECM are currently being developed in collaboration with the physicist Laetitia Pontani (Laboratoire Jean Perrin, pending ANR PRC application, collaborator: M. Breau, partner: L. Pontani).

We anticipate that this interdisciplinary research will bring new knowledge on the development of functional neuronal circuits. Our work challenges the long accepted view that chemical cues are solely responsible for neuronal movements and axon pathfinding during development and regeneration of the nervous system. By unraveling important roles for mechanical inputs, we are making a step forward in the nascent field of in vivo neuromechanics and reach a more integrated view of neuronal development. We develop novel tools to probe and dissect the role of mechanical forces and material properties during neuronal morphogenesis in 3D environments, which can be used in many other systems, in vivo as well as in ex vivo or organoid/organ on chip systems.
Beyond its fundamental impact in basic neuroscience, this project has biomedical implications in the physiopathology of neuronal disorders and bioengineering of neural tissues. Here we focus on the contribution of forces in neuronal movements and axon formation, two key events of neuronal circuit construction. If one of these two processes fails to occur properly, the function of the nervous system is affected, as exemplified by severe neurodevelopmental disorders resulting from neuronal migration and axon formation defects, such as lissencephalies, mental retardation, epilepsies and Kallmann syndrome. The contribution of mechanical inputs in the etiology of these diseases is far from being understood and requires more attention. In addition, deciphering the basic rules of neuron response to mechanical cues will provide valuable information for the design of neuronal culture systems and associated 3D scaffolds dedicated to brain and spinal cord repair. Our work will thus potentially feed future developments in biomedical engineering to help treating brain and spinal cord neuronal injuries in the long-term.

Scientific output (research articles)
• Monnot P, Gangatharan G, Baraban M, Pottin K, Cabrera M, Bonnet I, Breau MA. Intertissue mechanical interactions shape the olfactory circuit in zebrafish (2022) EMBO Rep 23: e52963.
In this article we have identified the source of the extrinsic forces involved in the construction of the olfactory circuit. We have shown that the mechanical forces come from the morphogenesis of the eye, transmitted to the olfactory neurons by the extracellular matrix.

• Baraban M, Gordillo Pi C, Bonnet I, Gilles JF, Lejeune C, Cabrera M, Tep F, Breau MA. Actomyosin contractility in olfactory placode neurons opens the skin epithelium to form the zebrafish nostril (2023) Dev Cell S1534-5807(23)00043-6.
The olfactory neurons of zebrafish larvae are exposed to odors through an opening in the skin that prefigures the future nostril. We analyzed the mechanical interactions between olfactory neurons and skin during nostril opening, and showed that olfactory neurons pull on skin cells to trigger the opening of the skin and thus nostril formation.

The aim of this project is to investigate the role of mechanical forces in the sculpting of neuronal circuits, the functional building blocks of the nervous system. Motion is highly involved in neuronal circuit development: neurons migrate to their final location, axons and dendrites emerge and grow before connecting through synapses. So far, most studies have focused on attractive and repulsive chemical cues guiding neuronal migration and axon navigation. Yet the movement of neurons and their protrusions is likely to be influenced by extrinsic mechanical forces, whose functions have started to be studied in vitro, but remain largely unexplored in vivo. Thus, dissecting out the role of mechanical cues in complex 3D neural tissues represents a major challenge for modern neurosciences.

I will address this challenge with a pluridisciplinary strategy combining multiscale live imaging, in vivo force measurement and force perturbation approaches and molecular functional studies.
I will follow three main lines of research:

(1) Obtain a spatiotemporal map of forces deployed in a developing neuronal circuit in vivo
(2) Identify the origin and contribution of these forces in the assembly of the circuit
(3) Decipher the molecular bases of force propagation and sensing in the forming circuit

I will take advantage of the olfactory sensory circuit in zebrafish as model system, as its superficial location underneath the skin makes it amenable to live imaging, mechanical manipulation and drug screening. I already characterised neuronal movements and axon extension in this context, analysed the role of two cytoskeleton components that produce forces within tissues -microtubules and actomyosin-, and started to map tension in the forming circuit. My findings highlight a novel mechanism of neuronal circuit construction, where extrinsic mechanical forces mediate the retrograde extension of axons by driving the displacement of cell bodies away from their static axon tips. This system thus represents a unique opportunity to dissect the deployment and function of mechanical forces in a developing neuronal circuit in vivo.

Building on these exciting results, this project will bring new mechanistic knowledge on the development of the nervous system by unravelling significant roles for mechanical inputs. This step forward in the nascent field of in vivo neuromechanics will contribute in adding a new paradigm in the understanding of neuronal development. Beyond its importance for basic neuroscience, this novel knowledge will potentially bring valuable insights on the etiology of neurodisorders including lissencephalies, mental retardation and Kallmann syndrome, and fuel neural tissue engineering for brain and spinal cord repair.