Imaging cell behaviour during neurogenesis

To understand better the impact of different signalling pathways on the behaviour of neuroepithelial cells we have recently developed (in collaboration with Jason Swedlow, University of Dundee) an assay which allows us to monitor live cells in the neural tube for up to 36h using the DeltaVision wide-field imaging system (Wilcock et al.2007). These cells exhibit the extraordinary process known as interkinetic nuclear migration – each cell retains attachments across the radius of the neural tube, but its nucleus moves between the two surfaces as the cell cycle progresses.

So, cells enter mitosis at the apical (luminal) surface of the neural tube and then progress through G1 as they move towards the basal surface where they either re-enter S-phase undergoing a further round of DNA synthesis or exit the cell cycle and differentiate as neurons. This cell behaviour can be seen in our movies, which also strikingly reveal that as neurons are born they release and then withdraw their apical process and re-orientate along the basal surface before putting out an axon (Wilcock et al. 2007).

The ability to monitor individual cells as they divide and their daughter cells adopt definitive fates (either dividing again or differentiating into neurons) allows us to examine the relationship between division orientation and cell fate choice. A number of studies in the developing mammalian cortex suggest that if cells divide with a cleavage plane perpendicular to the apical surface both cells will inherit a region of apical membrane and will remain progenitor cells. However, if a cell divides with a parallel cleavage plane one cell will inherit apical membrane and consequently becomes a neuron, while its sibling does not. Our movies reveal that in the early spinal cord a change in cleavage plane orientation does not correlate with the switch from progenitor to neuron generating divisions. However, it does distinguish stem cell and terminal modes of neuron production (Wilcock et al. 2007).

Progenitor cells in stem cell mode divisions (that generate a neuron and a progenitor) also have longer cell cycle times than cells that generate only further progenitors. This observation has also been made in the developing cortex and indicates that changes in cell cycle regulation are linked to acquisition of neuron generating ability.

Our recent work builds on our description of normal cell behaviour in the neural tube. In collaboration with Domingos Henrique (IMM, University of Lisboa) we have developed a fluorescent reporter for Notch signalling activity (Vilas-Boas et al 2011) and used this to investigate the relationship between signalling dynamics and cell fate choice during neurogenesis (Das & Storey 2012). Our imaging assay also lends itself to analysis of gene function, as it is amenable to delivery of small molecules and mis-expression of gain and loss of gene function constructs (Das et al 2012).

Most recently we have uncovered a new cell behaviour underlying neuronal differentiation, which involves abscission of the apical membrane of a new-born neuron. This results in loss of apical cell polarity and dis-assembly of key signalling organelle, the primary cilium, which help to re-configure the cell for neuronal differentiation (Das & Storey 2014). Current work addresses the molecular basis of apical abscission.