Abstract
Development of neural progenitors depends upon the coordination of appropriate intrinsic responses to extrinsic signalling pathways. Here we show the deubiquitylating enzyme, Usp9x regulates components of both intrinsic and extrinsic fate determinants. Nestin-cre mediated ablation of Usp9x from embryonic neural progenitors in vivo resulted in a transient disruption of cell adhesion and apical-basal polarity and, an increased number and ectopic localisation of intermediate neural progenitors. In contrast to other adhesion and polarity proteins, levels of β-catenin protein, especially S33/S37/T41 phospho-β-catenin, were markedly increased in Usp9x−/Y embryonic cortices. Loss of Usp9x altered composition of the β-catenin destruction complex possibly impeding degradation of S33/S37/T41 phospho-β-catenin. Pathway analysis of transcriptomic data identified Wnt signalling as significantly affected in Usp9x−/Y embryonic brains. Depletion of Usp9x in cultured human neural progenitors resulted in Wnt-reporter activation. Usp9x also regulated components of the Notch signalling pathway. Usp9x co-localized and associated with both Itch and Numb in embryonic neocortices. Loss of Usp9x led to decreased Itch and Numb levels, and a concomitant increase in levels of the Notch intracellular domain as well as, increased expression of the Notch target gene Hes5. Therefore Usp9x modulates and potentially coordinates multiple fate determinants in neural progenitors.
Highlights
Neural progenitors (NPs) are the founding cell population driving brain development
Usp9x is required for normal brain development[6] but it was not clear what role, if any, it played in NPs
Nestin-Cre expression resulted in the complete deletion of Usp9x protein from NPs by E12.5 and expression of both Adherens junctions (AJ) and polarity proteins was significantly decreased in Usp9x−/Y NPs at this stage (Fig. 1A–C)
Summary
Neural progenitors (NPs) are the founding cell population driving brain development. cell fate decisions taken by NPs must balance their differentiation into neuronal and glial lineages, with self-renewal thereby facilitating ongoing growth. Overexpression of Usp9x in mouse embryonic stem cell-derived NPs significantly increased their polarity and self-renewal without affecting their differentiation capacity[3]. These studies demonstrate the importance of Usp9x in NP fate specification, the underlying molecular mechanism(s) remains to be elucidated. To date more than 35 proteins have been reported as Usp9x substrates, many of which are components of intrinsic and extrinsic signalling pathways known to regulate NP function[5]. In the current study we sought to directly determine if Usp9x regulates NP function in vivo and if so, determine the molecular mechanism(s) involved
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