Primary cilium remodelling mediates a cell signalling switch in differentiating neurons Gabriela Toro-Tapia1 and Raman M. Das1. 1Division of Molecular and Cellular Function, Faculty of Biology Medicine and Health, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK. INTRODUCTION • Neurons are amongst the most highly polarised cell types [1,2]. • Cells undergoing neuronal differentiation shed their apical membrane via the regulated process of apical abscission [3], leading to acute loss of cell polarity and delamination from the neuroepithelium (A,B). • Apical abscission also results in disassembly of the apical primary cilium, leading to curtailment of Shh signalling and onset of cell cycle exit (C). • Newborn neurons repolarise in response to extracellular signals, leading to extension of a nascent axon in the correct orientation. Figure 1: A) Cartoon represents a two-day-old chick embryo. The transverse section of the spinal cord shows the apicobasal polarity of neural progenitors and the sequence of events leading to neuronal differentiation. B) The primary cilium and centrosome are located at the apical pole of cells in the early stages of neuronal differentiation. C) Apical abscission leads to acute loss of apical polarity and primary cilium dis-assembly. B C RESULTS CONCLUSIONS References: [1] Takano, T., et al. (2015). "Neuronal polarization." Development 142(12): 2088-2093;[2] Barnes, A. P. and F. Polleux (2009). "Establishment of axon-dendrite polarity in developing neurons." Annu Rev Neurosci 32: 347-381;[3] Das, R. M. and K. G. Storey (2014). "Apical abscission alters cell polarity and dismantles the primary cilium during neurogenesis." Science 343(6167): 200-204. A I) The primary cilium is dynamically disassembled and remodelled during neuronal differentiation II) Intraflagellar trafficking is inactive in the Arl13b+ particle and reactivated during apical process retraction • Cells delaminating from the neuroepithelium undergo primary cilium dis-assembly and inherit a small Arl13b+ particle, leading to acute cessation of canonical Shh signalling. • Arl13b+ particle elongates and progressively initiates intraflagellar trafficking (IFT) as it transits towards the cell body, indicating primary cilium remodelling. • Ciliary accumulation of the Shh transducer Smoothened (Smo) is gradually restored during cilium remodelling in the absence of Gli transcriptional activity. • The remodelled primary cilium directs neuron polarisation and subsequent axonogenesis via a non-canonical Shh signalling. • This work indicates that cilium remodelling provides differentiating neurons the opportunity to configure a new signalling centre to facilitate a qualitatively different response to the common signalling environment. Apical process { Figure 2:. A) Time-lapse sequence showing Arl13b+ particle retention following apical abscission. White arrow indicates apical abscission, white arrowhead the retained Arl13b+ particle and yellow arrowhead the abscised primary cilium. Dashed magenta lines indicate ventricular surface. Scale bar: 10µm. Boxed regions are enlarged in right-hand panels. Primary cilium is highlighted by dashed white lines. Scale bar: 0.7µm. B) Time-lapse sequence of a cell undergoing primary cilium reassembly during apical process retraction. Dashed cyan lines demarcate cell body of differentiating neuron and white arrowhead the reassembling primary cilium. Boxed regions are enlarged in right-hand panels. Scale bar: 5µm and 0.7µm (enlarged region). C) Cell undergoing axonogenesis transfected with Arl13b-GFP to label the ciliary membrane (green), mKate2-GPI to label cell membrane (red), and Ngn2-GFP (green), Scale bar: 5µm. D) Representation of differentiating neurons at different stages of apical process retraction and early stages of axonogenesis. E) Immunostaining to detect Tuj1+ cells (green) and endogenous Arl13b (red) at different stages of apical process retraction. Scale bar: 5µm an