Mechanisms of epithelial stratification
All stratified epithelia initially begin as simple epithelia, but this single layer of cells rapidly expands into many specialized layers during only a few days of murine development. The deepest basal layer is where progenitors reside, and is overlaid by multiple layers of “suprabasal” cells. Epidermal cells, or keratinocytes, thus become progressively more differentiated as they move outward toward the body surface, being replaced by new cells generated within the basal layer. But how basal cells generate their suprabasal progeny has been a matter of debate. Historically, it was believed that detachment of basal cells from their underlying basement membrane, termed “delamination,” was the driving force for differentiation. While a post-doc in Elaine Fuchs’ lab, we provided the first functional evidence that oriented cell divisions are a major driving force for epidermal stratification during development (Williams et al, Nature 2011). Basal cells can divide both symmetrically, within the plane of the epithelium, to give rise to two basal progenitors. Alternatively, by rotating their mitotic spindle, they can divide perpendicularly to divide asymmetrically, yielding one basal and one suparabasal daughter.
Asymmetric divisions promote differentiation
Asymmetric cell divisions require the conserved spindle orientation adapter protein LGN (known as Pins in Drosophila), which localizes asymmetrically to the apical cell cortex of basal cells. LGN binds both apical polarity cues (Inscuteable and the heterotrimeric G-protein Gαi3) as well as the microtubule-binding protein NuMA, which facilitates its interaction with the motor complex dynein-dynactin, exerting pulling forces on the astral microtubules that reorient the mitotic spindle. Using a powerful genetic tool we term LUGGIGE (Lentiviral Ultrasound-Guided Gene Inactivation and Gene Expression) [embed LUGGIGE figure], we can utilize in utero delivery of a vast toolbox of lentiviral vectors to transduce stratified epithelia with high efficiency during embryonic development (Beronja, Livshits, Williams and Fuchs, Nat Med 2010). This tool has enabled us to identify the pathways necessary for perpendicular asymmetric divisions to occur, and demonstrate that without them, epidermal stratification is dramatically impaired (Williams et al, Nat Cell Biol 2014; Williams et al, Nature 2011). More recently, we’ve also used this technique, in combination with genetic lineage tracing, to show that delamination also occurs in vivo (Williams et al, Nat Cell Biol 2014). This has led us to build a model whereby delamination drives early stages of stratification while perpendicular “asymmetric” divisions drive later stages.
Current research interests
While we now have considerable understanding of the intrinsic cues that orient mitotic spindles to undergo asymmetric divisions, several questions remain. Our current research in this area to addresses two broad areas which remain poorly understood.
How are planar divisions regulated? Is this an active or “default” mechanism?
Our studies in the oral epithelia demonstrate that in some tissues LGN can localize laterally rather than apically, thus promoting divisions within the epithelial plane (Byrd et al, Development 2016). Thus, different stratified epithelia utilize LGN in distinct ways, by localizing the protein to distinct subcellular domains. However, during epidermal development, we have evidence that planar divisions do not require LGN, Gαi3 or Insc, and may be actively regulated by other active mechanisms.
What are the extrinsic cues that influence the choice that basal cells make to undergo either a symmetric or asymmetric division?
We know from genetic lineage tracing that any given basal cell can undergo both symmetric and asymmetric divisions during development, but we understand little about how these choices are made. We also know from studies in C. elegans (Goldstein lab @ UNC) and other model systems that adhesions with neighboring cells can influence division orientation. Our studies focus on components of the adherens junction complex (Peifer lab @ UNC), which plays a critical role in mediating cell-cell adhesions within the epidermis. We have uncovered spindle orientation defects in key mediators of the adherens junction complex, including afadin, alpha-catenin, vinculin, and classical cadherins.
We continue to seek to understand the interplay between cell polarity, adhesion, and spindle orientation pathways, and how they influence tissue architecture and differentiation.