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A major subject of study in biological research is the dynamics of stem cells in squamous epithelia. Given that most common human cancers develop from epithelia, understanding the rules of cell fate decision in these systems is key to explaining not only healthy tissue growth and maintenance but also the processes of mutagenesis and cancer. The aim of my project was to investigate the dynamics in squamous epithelial tissues both in homeostasis and preneoplasia, using cellular automata (CA) models. Stem cell dynamics has been shown to be accurately described by a simple mathematical model, the single progenitor (SP) model. Reliable parameterisation of this model would give access to valuable quantitative information on epithelial tissue maintenance and enable investigating how mutations affect tissue dynamics. I initially identified the most appropriate method for accurately parameterising the homeostatic system. I then sought to account for the spatial patterning of cells by implementing the SP model in two-dimensional space. The spatial model was able to reproduce the key signatures of homeostatic dynamics, thus showing that restrictions imposed by tissue organization do not alter the neutral dynamics. Furthermore, I studied non-homeostatic dynamics in stratified squamous epithelial tissues by spatially modelling the growth and competition of non-neutral mutations as well as the effects of wounding in the tissue. The studied dynamics of Notch and p53 mutant clones in mouse epithelia has been found to be highly distinct, with the former fully colonizing the tissue whereas the latter only partially. I demonstrated that the two mutants’ tissue takeover dynamics can be recapitulated by two distinct spatial feedback rules, on the basis of response to crowding, providing a mechanistic explanation of the observed distinct growth modes. Finally, mutant competition was explored. A striking effect resulting from the spatial interaction of the two mutations in a wild-type background is that the p53 mutant cell population was always outcompeted by the Notch mutant population and appeared to shrink. Considering this consistent emergent behaviour in the competition simulations and given the paucity of Notch mutations in human cancer datasets, it is tempting to speculate that the aggressive fitness of Notch may offer a tumour-protective effect.

Cell competition is a form of cell-cell signalling that results in the elimination of less fit cells from a tissue by their fitter counterparts. I take advantage of an established in vitro model of cell competition using Madin-Darby canine kidney (MDCK) cells to shed insight into the molecular basis of cell competition in epithelial cells. In this system, silencing of the tumour suppressor scribble (scribKD) results in a 'loser' phenotype whereby scribKD cells are specifically eliminated from the monolayer by surrounding wild-type cells. More specifically, scribKD cells are compacted into tight clones through activation of a directed, collective migration in the wild-type population: scribKD are 'mechanical losers' and delaminate and die due to an intrinsic hypersensitivity to high cell density. Remarkably, p53 activation is both necessary and sufficient for this mechanical loser cell status. I first investigate the role of E-, N-, and P-cadherin in the directed migration between scribKD and wild-type cells and in scribKD cell loser status. I show that differential expression of E-cadherin between scribKD losers and wild-type winners is required but not sufficient for directed migration and has no impact on loser cell status. I also show that elevation of neither E-cadherin nor N-cadherin is sufficient to induce directed migration or loser status, but that P-cadherin may play a role in both. I next focus on translating findings about the molecular details of competition from the scribKD set-up into a system where p53 differences alone drive the formation and elimination of mechanical losers. I show that the ROCK - P-p38 - p53 pathway activated in response to mechanical compaction in scribKD cells is conserved in p53-driven losers. In the latter part of my thesis, I characterise the directed migration observed during MDCK competition by drawing parallels to canonical leader-follower migration. Canonical leader cells emerge when epithelial sheets are wounded and, by becoming migratory, drive collective cell migration of follower cells, which results in wound closure. It was not known what confers the leader cell fate. I show that p53 and its effector p21 (and potentially other cyclin-dependent kinase inhibitors) are the key drivers of leader cell migration. I demonstrate that p53-induced leaders use the same molecular pathways that have been shown to drive leader cell migration during wound healing and, in fact, p53 and p21 are also elevated in leaders generated by wounding. Importantly, I establish that p53 activity drives efficient wound closure. Lastly, I show that leader cells are often eliminated by cell competition in the final stages of wound closure, as their elevated p53 mediates their hypersensitivity to density. The model incorporating these data proposes that cellular damage during wounding generates cells with elevated p53, which become leaders and drive wound healing, but these are then cleared once the wound is closed because their high p53 levels cause them to become mechanical losers.

Cell competition leads to elimination of a viable cell population, by fitter cells. Despite over forty years of research, the molecular mechanisms of competition in mammals are poorly understood. During my PhD I have investigated the mechanisms of competition by exploring an established mammalian cell culture system, in which wild-type MDCK cells eliminate scribble-deficient cells, and I have also developed a novel cell culture system to model mammalian competition. My work contributed to the discovery that scribble-deficient cells are eliminated not by biochemical exchange among cells, but by mechanical compaction. We termed this phenomenon mechanical competition. I employed transcriptional profiling to determine the molecular signature of mechanical losers, and identified activation of p53 signalling as their hallmark. My colleagues and I then demonstrated that elevation of p53 is both necessary and sufficient to trigger mechanical competition. In further investigating the mechanisms of mechanical competition, I found that compaction activates ROCK in scribble-deficient cells, and that this is required for their elimination. Inhibition of Src signalling in mechanical losers also protected them form out-competition, and integrin signalling is another pathway likely involved in mechanical competition. While investigating p53 competition, we observed that p53-high and p53-low cells engage in directional migration, with p53-high cells always at the migrating front. As a side-project, I investigated the role of p53 in directional migration, by exploring an established model with a single leader cell and multiple followers. We established a method to generate multinucleated leaders on demand. By creating leaders from p53-deficient cells, I established that p53 signalling is required for some, but not all multinucleated cells to trigger collective migration, thus implicating p53 signalling in a type of migration involved in wound healing. Finally, I successfully modelled p53-driven mechanical competition in a differentiated primary tracheal epithelial cell culture, thereby establishing a novel system to study mammalian competition, and also proving that p53 competition is conserved between different mammalian epithelia. Considering the involvement of p53, mechanical competition may play a major role in cancer.

Examples of metazoan regeneration can be seen within all animal phyla; however, the ability to restore lost tissues upon injury varies greatly. Some species can only partially replace tissues and organs, while others can repeatedly regenerate entire limbs, severed spinal cords, and portions of their brain. There are also extraordinary cases of whole-body regeneration (WBR) where animals can produce entire bodies from small fragments of leftover tissue after injury. A few species of botryllids can even utilize circulating blood borne stem cells for WBR. These animals are in the subphylum tunicata, and evolutionarily classified alongside vertebrata in the phylum Chordata. Botryllids are marine invertebrates that grow by repeated rounds of asexual reproduction to form a colony of clonally derived individuals called zooids. Under normal conditions, zooids are regenerated de novo on a weekly basis, and a colony increases in size by expanding the number of individuals. Two distinct processes that differ by the source of new bodies are responsible for zooid regeneration in the botryllids. In the first, called blastogenesis, new zooids arise from a multipotent epithelium of a pre-existing zooid. In the second, termed vascular budding (VB) or WBR, mobile cells in the vasculature are the source of the new zooid. In some botryllid species, blastogenesis and VB occur concurrently. In others, blastogenesis is exclusively used for expansion while WBR only occurs following injury or exit from dormancy. We studied WBR in two related species: Botryllus schlosseri and Botrylloides diegensis. Both have an extracorporeal vasculature that develops outside the bodies to connect all individuals within a colony. We isolated blood vessels to induce WBR by removing all zooids, primary buds, and secondary buds. This surgery was performed under a dissecting microscope with scalpels and extra-fine tip forceps to prevent damage to the vasculature. Regeneration was then analyzed using time-lapse microscopy with the animal being maintained in a temperature-controlled seawater recirculation basin. We compared and characterized early stages of WBR utilizing these days-long videos. Our data suggest that unlike other botryllid species, new zooid growth following injury in B. schlosseri is not due to WBR, but instead through ectopic development of tissues leftover from the blastogenic process. Despite these differences, we did find a common theme in the two species: developmental competition results in only a single zooid reaching maturity. We utilized this phenomenon to control the number of buds and their spatial orientation in B. schlosseri and found that competition is reversible and mediated by circulating factors and/or cells. This represents a new model to study resource allocation and competition within an individual.

How do different animals grow to be different sizes? The answer is programmed in their genes, encoding for the myriad of protein-based signaling pathways that regulate growth of cells, tissues, and the entire body. Whereas many developmental signaling pathways have been found to control cell-fate, some pathways have been discovered that specifically regulate growth. How exactly these signaling pathways control growth is still not fully understood and there are likely still undiscovered components. In addition, knowing the normal mechanisms cells use to coordinate growth can help us understand how it becomes misregulated in human diseases such as cancer. The goal of this thesis was to identify novel genes controlling tissue growth and characterize their function, using the imaginal discs in Drosophila melanogaster as a model tissue.In collaboration with a colleague in the lab, I identified Crumbs as a regulator of cell competition, a phenomenon that affects the survival of cells in growing tissues. Crumbs is a transmembrane protein known to control apical cell polarity and binds homophilically on adjacent cells. Cells that express higher levels of Crumbs die when in contact with cells that express lower levels of Crumbs. Therefore, Crumbs binding on adjacent cells may be a general mechanism for how cells "sense" competitive ability. I contributed by sequencing alleles of crumbs and finding that mutant cells behave as supercompetitors.I identified the Fbxl7 gene as a novel regulator of Fat and Hippo signaling. The atypical cadherin Fat localizes in a planar polarized manner in cells of the wing imaginal disc, which prevents the membrane localization of an atypical myosin named Dachs. It was not previously known how Fat keeps Dachs off of the membrane. I showed that Fbxl7 associates with the Fat intracellular domain and prevents Dachs from localizing to membrane. Fbxl7 is conserved in humans and may be a novel tumor suppressor. I identified a role for autophagy members in controlling tissue growth of the eye imaginal disc. Autophagy allows the recycling of cytoplasmic components during starvation via double membrane vesicles called autophagosomes. Atg2 and Atg18 form a complex and are critical for autophagosome formation. Loss of these genes in the eye imaginal disc disrupts formation of autophagosomes in developing photoreceptors and increases growth in the adult eye. Surprisingly, mutations in other autophagy genes do not produce the same phenotype, implying Atg2 and Atg18 may regulate growth through autophagy-independent mechanisms.I developed a unique genetic system called "CoinFLP" to facilitate the identification additional growth genes, as well as characterize cell-cell interactions occurring with tissues. CoinFLP uses stochastic recombination in a developmentally controlled fashion to produce patches of mutant and wild-type tissue in imaginal discs at predictable ratios. I used CoinFLP to conduct a large-scale screen and identified geminin as a novel regulator of cell competition. In addition, I used CoinFLP to manipulate gene expression in two separate populations and directly visualize cell-cell contacts between them.In conclusion, I have described three novel components regulating growth in Drosophila imaginal discs, and established a new genetic tool to identify and characterize additional regulators of tissue growth.

The survival and growth of cells can be influenced by the properties of adjacent cells. This reflects the ability of cells to communicate with each other and can result in either cooperation or competition for limited resources. Cell competition is an example of a situation in which cells exert a non-autonomous influence over the survival of their neighbors. The phenomenon was first discovered over forty years ago during studies of genetically mosaic Drosophila melanogaster. Clones of slow-growing, though viable, "Minute" cells were found to be eliminated from tissues in which they were surrounded by wild-type cells. It was later discovered that signaling between the two cell types at their clone borders causes the apoptosis and elimination of the Minute cells. Conversely, faster growing cells known as "supercompetitors", such as cells overexpressing the transcription factor, dMyc, can induce the elimination of wild-type cells. Thus, cell competition is a short- range interaction between cells with different growth rates in the same tissue and leads to the elimination of the slower-growing cells. The nature of this interaction is still unknown. Specifically, there are two important, unanswered questions regarding the mechanism of cell competition: 1) How do cells at clonal boundaries compare their properties and designate a "winner" and "loser"? 2) What are the signals that then induce the death of the designated losers?Understanding the mechanism of cell competition is important from several standpoints. First, cell competition has been proposed to play a role in regulating organ size and tissue composition during normal development. Thus, understanding the mechanism of cell competition may advance our understanding of basic developmental biology. Second, many of the genes involved in cell competition are also misregulated in certain cancers. Thus, cell competition would be expected to occur at the borders between such tumors and their host tissue. A competitive advantage could make the tumor more malignant, while a competitive disadvantage might make the tumor more manageable. Therefore, a better understanding of the mechanisms involved in cell competition could also advance our understanding of tumor progression and improve our ability to treat tumors more effectively. Finally, a thorough understanding of cell competition and the ability to manipulate it could eventually be useful in therapies. For example, it may become possible in regenerative medicine to give grafts the ability to replace injured or dysfunctional tissue.The goal of my thesis research has been to identify and characterize novel regulators of cell competition in order to gain a better understanding of the underlying mechanism. Accordingly, I devised an assay that would allow me to readily identify mutations that make cells into supercompetitors. The "supercompetitor assay" described here takes advantage of established mosaic analysis techniques in Drosophila. Small, marked clones of cells that are heterozygous for a mutation are created in an eye primordium that is primarily composed of homozygous-mutant cells. If the mutant cells are supercompetitors, the marked heterozygous clones are eliminated and cannot be recovered in the adult eye. Using this method I was able to test candidates from a collection of mutations in tumor suppressor genes from several different pathway that regulate growth. While mutations in components of some growth-regulating pathways, notably the Hippo pathway, caused cells to become supercompetitors, mutations in other pathways did not. Thus, only a subset of the pathways that regulate growth appear to be involved in cell competition.The supercompetitor assay was also used to conduct an unbiased genetic screen for novel mutations that make cells supercompetitors. Among the mutations found in the screen were several alleles of crumbs (crb), a gene that regulates apicobasal polarity. This gene had no previously defined role in cell competition or growth. I found that while loss of Crb causes cells to become supercompetitors, overexpression of Crb causes cells to be eliminated from wild-type epithelia. Furthermore, as expected in instances of cell competition, high levels of apoptosis were observed preferentially at boundaries between wild-type and crb mutant cells, as well as at boundaries between Crb-overexpressing and wild-type cells. Thus, cells that express higher levels of Crb appear to be eliminated through a mechanism that resembles cell competition when they are near cells that express lower levels of Crb.It is still unclear how cells compare their Crb levels. Cells may compare the levels of a molecule that is downstream of Crb. One candidate is the Hippo pathway, which has been repeatedly linked to cell competition. I found that crb mutant cells upregulate some of the transcriptional targets of the Hippo pathway suggesting that Crb impinges upon the Hippo signaling pathway. Alternatively, cells may directly compare their levels of Crb, as it is a transmembrane protein with a large extracellular domain of unknown function. Interestingly, the extracellular domain of Crb appears to be required to elicit some of the heterotypic interactions that I observe. For example, cells that express the intracellular domain of Crb alone are not eliminated. Furthermore, I see evidence of interactions between Crb molecules on adjacent cells that could be the basis of a direct comparison mechanism. Future work aimed at testing such models may yield important insights into a mechanism of cell competition.