YAP Activity Is Necessary and Sufficient for Basal Progenitor Abundance and Proliferation in the Developing Neocortex

摘要

class="head no_bottom_margin" id="sec1title">IntroductionThe neocortex, the seat of higher cognitive functions, undergoes substantial expansion during the evolution of certain mammalian brains such as human. A major factor in neocortical expansion, notably regarding the increase in the number of cortical neurons, is thought to be an increased proliferative capacity of cortical neural progenitor cells (cNPCs) (, , , , , , ).Two principal classes of cNPCs exist in the developing neocortex, referred to as apical progenitors (APs) and basal progenitors (BPs) (, , ). The defining feature of APs is that they undergo mitosis at the ventricular (apical) surface of the ventricular zone (VZ), the primary germinal zone where the AP cell bodies reside (, ). At the onset of neurogenesis, apical (or ventricular) radial glia (aRG) are the major AP cell type (, , , href="#bib39" rid="bib39" class=" bibr popnode">Namba and Huttner, 2017). The defining feature of BPs is that they undergo mitosis away from the apical surface, typically in a secondary germinal zone called the subventricular zone (SVZ) where the BP cell bodies reside (href="#bib21" rid="bib21" class=" bibr popnode">Haubensak et al., 2004, href="#bib36" rid="bib36" class=" bibr popnode">Miyata et al., 2004, href="#bib42" rid="bib42" class=" bibr popnode">Noctor et al., 2004). BPs originate from APs, delaminate from the apical surface, migrate beyond the VZ, and thus form the SVZ. There are two main types of BPs, basal intermediate progenitors (bIPs) and basal (or outer) radial glia (bRG). In contrast to aRG, which are epithelial cells exhibiting apical-basal polarity with contact to the ventricle and (in the canonical form) to the basal lamina, bIPs are non-epithelial cells that no longer exhibit apical-basal polarity and have lost contact with both the ventricle and the basal lamina (href="#bib21" rid="bib21" class=" bibr popnode">Haubensak et al., 2004, href="#bib36" rid="bib36" class=" bibr popnode">Miyata et al., 2004, href="#bib42" rid="bib42" class=" bibr popnode">Noctor et al., 2004). bRG, however, though lacking an apical process that reaches the ventricle, retain epithelial features in that they (in the canonical form) possess a basal process that contacts the basal lamina (href="#bib64" rid="bib64" class=" bibr popnode">Betizeau et al., 2013, href="#bib14" rid="bib14" class=" bibr popnode">Fietz et al., 2010, href="#bib19" rid="bib19" class=" bibr popnode">Hansen et al., 2010, href="#bib45" rid="bib45" class=" bibr popnode">Reillo et al., 2011).BP composition and proliferative capacity may differ greatly between a developing lissencephalic neocortex (e.g., mouse) and a developing gyrencephalic neocortex (e.g., ferret and human). In the developing mouse neocortex, BPs mostly comprise bIPs that typically undergo neurogenic consumptive divisions giving rise to two neurons; compared to the aRG they derive from, these neurogenic bIPs characteristically upregulate the transcription factor Tbr2 and downregulate the transcription factor Sox2 (href="#bib21" rid="bib21" class=" bibr popnode">Haubensak et al., 2004, href="#bib36" rid="bib36" class=" bibr popnode">Miyata et al., 2004, href="#bib42" rid="bib42" class=" bibr popnode">Noctor et al., 2004). Only a minor portion of mouse BPs are bRG, and their proliferative potential is limited (href="#bib50" rid="bib50" class=" bibr popnode">Shitamukai et al., 2011, href="#bib55" rid="bib55" class=" bibr popnode">Wang et al., 2011). In contrast, in the developing ferret and human neocortex, the majority of BPs are proliferative bRG (highly proliferative in human) that do not express Tbr2 but rather maintain expression of Sox2 (href="#bib14" rid="bib14" class=" bibr popnode">Fietz et al., 2010, href="#bib19" rid="bib19" class=" bibr popnode">Hansen et al., 2010, href="#bib45" rid="bib45" class=" bibr popnode">Reillo et al., 2011). Moreover, as first shown in a seminal contribution for the developing monkey neocortex (href="#bib51" rid="bib51" class=" bibr popnode">Smart et al., 2002), the SVZ in a gyrencephalic neocortex is characteristically split into two morphologically distinct zones, an inner SVZ (iSVZ) and an outer SVZ (oSVZ). Of note, the evolutionary expansion of the neocortex has been linked to an increase in the proliferative capacity and abundance of BPs in the oSVZ, especially of bRG (href="#bib9" rid="bib9" class=" bibr popnode">Dehay et al., 2015, href="#bib13" rid="bib13" class=" bibr popnode">Fietz and Huttner, 2011, href="#bib33" rid="bib33" class=" bibr popnode">Lui et al., 2011, href="#bib39" rid="bib39" class=" bibr popnode">Namba and Huttner, 2017). However, the molecular players that differentially promote the proliferative capacity of BPs across the various mammalian species remain largely unknown.To gain insight into this issue, we examined a major molecular mechanism known to regulate organ size, the Hippo-YAP signaling pathway (href="#bib2" rid="bib2" class=" bibr popnode">Barry and Camargo, 2013, href="#bib5" rid="bib5" class=" bibr popnode">Camargo et al., 2007, href="#bib30" rid="bib30" class=" bibr popnode">Lian et al., 2010, href="#bib59" rid="bib59" class=" bibr popnode">Yu et al., 2015). The core of Hippo-YAP signaling is the YAP protein, whose ability to activate transcription is regulated by phosphorylation. Phosphorylated YAP (phospho-YAP) is largely retained in the cytoplasm, whereas dephosphorylated YAP (dephospho-YAP) can translocate to the nucleus and activate the expression of genes linked to cell proliferation (href="#bib60" rid="bib60" class=" bibr popnode">Zanconato et al., 2015, href="#bib61" rid="bib61" class=" bibr popnode">Zhao et al., 2007, href="#bib62" rid="bib62" class=" bibr popnode">Zhao et al., 2008, href="#bib63" rid="bib63" class=" bibr popnode">Zhao et al., 2010). Recent studies dissecting the roles of the cadherin family members Dchs1 and Fat4 (href="#bib6" rid="bib6" class=" bibr popnode">Cappello et al., 2013) and the tumor suppressor neurofibromatosis 2 (href="#bib28" rid="bib28" class=" bibr popnode">Lavado et al., 2013, href="#bib29" rid="bib29" class=" bibr popnode">Lavado et al., 2014) and investigating heterotopia formation (href="#bib46" rid="bib46" class=" bibr popnode">Saito et al., 2018) in mouse brain development have reported that YAP promotes the proliferation of mouse APs. These studies, however, have not focused on a potential role of YAP in regulating the proliferation of BPs, nor have they addressed whether differences in YAP activity may underlie the differences in the proliferative capacity of BPs across various mammalian species in the context of the evolutionary expansion of the neocortex.In the present study, we have identified differences in YAP expression and YAP activity between the developing lissencephalic mouse and gyrencephalic ferret and human neocortex that match the differences in the proliferative capacity of BPs across these species. Enhancing YAP activity in mouse BPs induced their proliferation and therefore shifted their fate from neurogenic to proliferative. In contrast, inhibition of endogenous YAP activity by verteporfin, administration of a dominant-negative YAP, or CRISPR-Cas9-mediated disruption of YAP expression reduced BP proliferation in developing ferret and human neocortex. Taken together, these findings suggest that an upregulation of YAP levels and presumably activity contributed to the increased proliferative capacity of BPs in the context of the evolutionary expansion of the neocortex.