Although it remains unclear how aPKCi promotes intermediate

Although it remains unclear how aPKCi promotes intermediate neurogenesis in the context of our “MGE” differentiation system, there are several intriguing possibilities. First, the aPKC subgroup contains two isoforms, iota (ι or λ) and zeta (ζ), which have been shown to have numerous, distinct functions in the regulation of cell polarity, proliferation, and neural differentiation (Fatt et al., 2015; Vorhagen and Niessen, 2014; Wang et al., 2012). Loss of aPKCλ in mouse stem pitavastatin enhances self-renewal through the activation of Notch1 and its downstream effectors (Mah et al., 2015). Similarly, in dorsal neocortex, knockdown of aPKCλ delays neural differentiation and expands the pool of Tbr2+ intermediate progenitors, whereas knockdown of aPKCζ promotes radial glia self-renewal (Wang et al., 2012). These studies show that aPKCλ and aPKCζ promote stem cell differentiation through partially overlapping pathways. In our system we use transient, partial inhibition of both aPKC isoforms to enhance the production of Ccnd2+ intermediate progenitors. We favor the idea that partial inhibition of both isoforms promotes a balance between differentiation and self-renewal, resulting in the expansion of basal progenitors. This idea is supported by studies in Drosophila melanogaster, which show that aPKC is required to restrict the localization of cell-fate determinants into the differentiating daughter cell via its interactions with the par complex. Inhibition of aPKC disrupts the par complex and promotes daughter cell self-renewal (Goulas et al., 2012). Additional studies focusing on the selective loss of either isoform during interneuron genesis are needed to determine their individual roles. Such knowledge might have profound implications for generating interneuron subtypes from stem cells. In the field of cancer biology, aPKCs have generated considerable interest due to their roles in driving cellular proliferation. Interestingly, in basal cell carcinomas, aPKCλ forms a complex with missing-in metastasis (MIM) that potentiates Shh signaling (Atwood et al., 2013). Genetic or pharmacological loss of aPKCλ blocks Shh signaling and cancer cell proliferation. Previous in vitro and in vivo studies from our laboratory have shown that lower levels of Shh signaling preferentially bias MGE progenitors to Pv-expressing interneuron fates (Tyson et al., 2015; Xu et al., 2010). It is tempting to speculate that aPKCi may also bias progenitors to produce Pv-fated interneurons through manipulation of Shh signaling. In fact, loss of Shh signaling in embryonic mice initially reduces proliferation in the MGE ventricular zone while simultaneously upregulating it in the MGE SVZ (Xu et al., 2005). Taken together, our study provides evidence that aPKCs play a role in cortical interneuron fate determination and may be doing so through interactions with the Notch and Shh signaling pathways.
Experimental Procedures
Author Contributions
Introduction With the development of methods to generate induced pluripotent stem cells (iPSCs) from somatic cells (Takahashi and Yamanaka, 2006; Yu et al., 2007), human cells with the potential to generate all body cell types in vitro became available. This advance led to tremendous progress in the development of protocols for the differentiation of iPSCs into various human cell types. In addition, disease-specific human iPSCs and their derived cell types are now widely used for in vitro disease modeling. However, particularly with regard to neuronal diseases, it is of importance to consider that the human brain is an extremely complex, three-dimensional (3D) structure. Consequently, the investigation of its development and modeling of disease processes in traditional, two-dimensional cultures has strong limitations. It has been demonstrated that the presence of a 3D matrix promotes many biologically relevant functions, such as differentiation capability (Baharvand et al., 2006; Greiner et al., 2013; Tanaka et al., 2004; Tian et al., 2008), cellular signaling, and lineage specification (Engler et al., 2006; Greiner et al., 2013; McBeath et al., 2004). In addition, 3D culture systems are more physiological concerning cell-cell and cell-matrix interactions (Lee et al., 2007). These observations have, in recent years, prompted the use of human iPSCs for the generation of 3D in vitro models of complete organs, the so-called organoids.