Cell-biological mechanisms directing primary cilium mediated control of neuron polarisation

A key feature of neurons is their ability to respond to cues from the surrounding tissues which allow them to distinguish their front from their back and thus achieve polarisation. Newborn neurons in the spinal cord of the developing embryo shed their tips to move to their final location. As a result of this shedding event, these newborn neurons lose the key proteins that define their polarity as well as their cellular antenna, called the primary cilium, which allows these cells to sense external cues from the surrounding tissues. Consequently, newborn neurons stop responding to Shh signalling and exit the cell cycle, which is a key step of neuronal differentiation. Following this, the neuron must now rapidly re-establish its polarity. This step is crucial, as it allows the neuron to extend a long cell-process, called an axon, which makes connections with its targets, such as muscles or other neurons. In the developing embryo, the neuron polarises in response to external cues from the surrounding tissue. These external cues determine the orientation of this polarisation and therefore determine the direction in which the axon will travel, or if it forms at all. This is thus a critical event that is essential for the formation of functional neuronal circuitry. We have recently discovered that newborn neurons quickly reassemble a new primary cilium as they prepare to extend an axon. This new primary cilium now allows the newborn neuron to switch its response to Shh signalling, which now acts to determine the direction in which the axon projects.

This proposal aims to investigate the cell biological mechanisms that direct this switch in the interpretation of Shh signalling by the reassembled primary cilium. Furthermore, we also aim to investigate how signalling through the reassembled primary cilium directs remodelling of the neuronal cytoskeleton to generate the characteristic neuronal morphology. To achieve this, we will use cutting-edge microscopy techniques to make movies of polarising neurons in developing embryos and combine these with super-resolution fluorescence imaging of fixed embryonic tissue. This will allow us to identify and modulate the mechanisms that mediate the switch in the cellular response to Shh signalling and how this influences cytoskeletal remodelling in differentiating neurons. This work may lead to the development of novel clinical interventions to promote this process in the case of neurodevelopmental disorders or following injury during adulthood.

Newborn neurons undergo extensive morphological changes as they polarise in response to the environment to extend axons. Cells differentiating into neurons in the developing spinal cord undergo the regulated process of apical abscission, which leads to an acute loss of apical polarity and shedding of the primary cilium, followed by delamination from the neuroepithelium. These cells must now rapidly repolarise in response to extracellular signalling to specify a new axon. Strikingly, delaminating neurons rapidly reassemble a new primary cilium through a process called primary cilium remodelling, which corresponds with a switch from Gli transcription-dependent to Gli transcription-independent Shh signalling. This mode of Shh transduction directly modulates neuronal cytoskeletal organisation to direct axon extension. However, the mechanisms through which primary cilium remodelling directs this switch in Shh signal interpretation, and how this determines the dynamic microtubule remodelling that drives neuron repolarisation and axon extension remain unknown. These key questions will be studied using cutting-edge methods to image spinal cord neurogenesis in real time at high spatial and temporal resolution in ex-vivo tissue slices. This assay will therefore facilitate observation and manipulation of neuron repolarisation within the developing tissue, and is likely to uncover novel cell biological behaviours that only take place within a tissue context. This proposal aims to address two key questions:
i) How does the remodelled primary cilium switch function to direct neuron polarisation?
ii) How does primary cilium remodelling direct dynamic regulation of microtubule rearrangements during neuron polarisation?