Determining the role and mechanism of spatially restricted basement membrane remodelling in regulating mesenchymal-epithelial interactions in skin and

The corneal limbus and epithelial hair follicles have long been a region of intense interest due to their essential roles in tissue homeostasis and their value as powerful models of post-embryonic tissue remodelling. Increasingly attention has focused these two regions as the sites supporting epithelial and mesenchymal stem cell populations. Recent studies, including those of this supervisory team, have revealed many determinants of the stem cells and their microenvironment. Importantly, collectively we have established that epithelial-stromal cross-talk in these niches is essential for the maintenance and the function stem cell. Moreover, using serial block-face scanning electron microscopy (SBF SEM) we have shown direct physical contact between limbal epithelial and stromal stem cells. These findings were surprising as the central dogma was that basement membranes between the epithelial and stromal compartments act as a barrier to such an interaction. In this studentship, we will combine the basement membrane expertise of supervisor 1 with the stem cell expertise of supervisors 2 and 3 to determine how basement membrane composition and organisation is locally disrupted to facilitate these epithelial-stromal interactions.
Every basement membrane comprises an interconnect structural networks of two families of proteins; type IV collagens and laminins. There are at least 16 different laminin family members which differ in their ability to polymerise and their relative affinity for cell surface receptors. Immunofluorescence microscopy has revealed enrichment of specific laminins in areas with high stem cell density, that laminin expression profiles change during stem cell activation, and that different laminins can maintain the stemness of cells in culture or drive lineage-specific differentiation. Although these findings strongly implicate laminins in defining the stem-cell niche, there are no studies of sufficient resolution to define the sites of epithelial-mesenchymal interactions occur within the niche. This studentship will use a combination of immunogold transmission electron microscopy, SBF SEM and superresolution light microscopy to fine-map the laminin and laminin-binding protein distribution around contact points (aim 1).
The second part of the studentship will address the mechanism through which basement membranes are locally disrupted within the nice. Here, we have two hypotheses. First, following a model of how tumours invade, enrichment or localised activation of matrix degradative enzymes occurs in a controlled manner at sites of basement membrane breach. This process would be energy expensive, with comparatively slow dynamics. We, therefore, propose a second hypothesis where localised matrix disruption occurs through the action of laminin-network disrupting proteins. This idea, which has not been proposed in this context, does have parallels to the discovery of temporary disruption of laminin networks during immune cell extravasation. Consistent with this concept, supervisor 1 has identified a laminin-related protein, LaNt 31, with hallmark features that suggest it could play this role. Specifically, this protein carries appropriate structural motifs to interact with laminins, influences laminin organisation in vitro (4), displays differential distribution throughout the limbal region and is enriched in the hair follicle, and is upregulated during times of stem cell activation (5, 6).
Using our established in vitro models, human explant culture and a suite of live imaging and antibody-based tools, we will directly test these two hypotheses. This will involve a combination of observational approaches to study the localisation, distribution of LaNt 31 and structural homologues in static and dynamic conditions (aim 2), and manipulative strategies to determine the effect of increased/decreased expression of laminin-network disrupting proteins in epithelial-mesenchymal interactions (aim 3).
Together, this proj