We also find that PRKCZ fails to accumulate normally

We also find that PRKCZ fails to accumulate normally at the apical membrane in Gata3 mutant embryos, even though its expression and activation levels are maintained. PRKCZ is part of the apical Par complex comprising PARD3 and PARD6B and acts as a key regulator of mitotic spindle orientation in other systems (Durgan et al., 2011; Guilgur et al., 2012; Niessen et al., 2013). Of interest, the subcellular redistribution of PRKCZ is found in the presence of normal PARD3 and PARD6B apical expression, suggesting a decoupling of PRKCZ from the Par complex. Very few examples of such a decoupling phenotype have been documented so far. Only loss of Patj (Inadl) in MDCK angiotensin ii receptor blockers or Dlg5 during mouse lung development have been associated with a mislocalization of PRKCZ, possibly through PARD3 and PARD6B protein misexpression, which is not observed upon Gata3 loss (Adachi et al., 2009; Nechiporuk et al., 2013; Shin et al., 2005). The detailed mechanisms by which loss of Gata3 leads to PRKCZ mislocalization will require further investigation. Using ATM, a small molecule that affects the interaction between PRKCZ and the Par complex (Erdogan et al., 2006), we showed that PRKCZ decoupling from PARD6B is sufficient to cause mitotic spindle orientation and epithelial defects. This is in line with the known role of PRKCZ as a kinase for spindle pole proteins (Hao et al., 2010). Phosphorylation by apical PRKCZ has been proposed to prevent spindle machinery from interacting with the apical domain, thereby preventing asymmetric cell division (Chatterjee and McCaffrey, 2014; Hao et al., 2010). Together, our results suggest a model by which the mislocalization of PRKCZ by loss of Gata3 leads to spindle randomization by ectopic inhibition of the interaction between astral microtubules and the cell cortex (Figure 7). The epithelial hyperplasia resulting from spindle randomization is likely to be an important underlying cause of the branching morphogenesis defects observed in Gata3-deficient prostates. Hyperplastic tissue remained epithelial in this system as evidenced by the maintenance of the epithelial markers E-cadherin, ZO-1, and Par complex components, suggesting that the role of Gata3 in prostate progenitor cells is different from the regulation of epithelial-mesenchymal transition reported in metastatic prostate cancer cells (Jiang et al., 2016; Wang et al., 2015). An interesting consequence of these findings is that they provide a mechanism by which tissue hyperplasia occurs in the absence of cellular transformation. It is likely that this intermediate population generated by aberrant spindle orientation defects constitutes an epithelial cell population prone to oncogenic growth and dissemination upon transformation. Spindle orientation defects may therefore contribute to the hyperplastic and tumor progression phenotypes observed in the adult prostates and in other systems such as the skin and mammary gland in the absence of Gata3 (Kaufman et al., 2003; Kouros-Mehr et al., 2006, 2008; Nguyen et al., 2013). Together, this work highlights the critical importance of regulating mitotic spindle orientation in progenitor cells to control the stepwise cellular differentiation process and maintain tissue architecture and homeostasis.
Experimental Procedures
Author Contributions
Acknowledgments We are grateful to members of the Bouchard laboratory for critical reading of the manuscript. We would like to thank the McGill Advanced BioImaging Facility (ABIF) for their technical support with spectral unmixing and confocal microscopy, as well as the Flow Cytometry platform of the McGill University Life Sciences Complex for cell sorting. This work was supported by grants from the Canadian Institutes of Health Research (CIHR; MOP-130460) and the Cancer Research Society (CRS, Canada) to M.B. M.B. holds a Senior Research Scholar Award from the Fonds de la Recherche du Québec-Santé (FRQS). M.E.R.S. was supported by a Graduate Studentship from Prostate Cancer Canada, and a Lloyd-Carr Harris Graduate Studentship from McGill University. A.H.T.N. was supported by a studentship from the CIHR and the McGill Integrated Cancer Research Training Program (MICRTP). M.T. was supported by a Dr. Gerald B. Price Fellowship (Cancer Research Society), and MICRTP and FRSQ postdoctoral fellowships.