Project 1 - Understanding the identity and characteristics of adult gastric stem cells
Recently, we identified Troy+ chief cells as a novel stem cell population in the corpus epithelium of the stomach. Troy+ chief cells proliferate slowly, indicating a rather quiescent nature compared to other known gastro-intestinal tract stem cells. Interestingly, these stem cells actively divide upon the depletion of proliferating progenitors by the chemotherapy agent 5-fluorouracil (5-FU). This suggests a distinct status of these stem cells under homeostasis and injury. As Troy+ stomach stem cells (StSCs) exhibit interchangeable characteristics, i.e. quiescent and proliferative, they represent a unique model of adult stem cells. This allows us to study:
1) The dynamics of stem cell propagation in homeostasis and regeneration, as well as the underlying mechanism of this switch. Based on the changes in the mRNA expression and epigenetic profiles of Troy+ stem cells in homeostasis versus injury repair, we will generate a list of gene candidates that potentially play a role in cell fate decision. We will subsequently investigate the role of these candidates by using in vitro 3D organoid culture and in vivo genetic mouse models.
2) The StSC programme in homeostasis and regeneration using in vitro and in vivo functional genetics. For this project, my team uses lineage tracing approaches with ubiquitous and cell type-specific CreERT2 transgenic mice, combined with diphtheria toxin receptor-mediated cell-type specific ablation models, tissue clarification, thick tissue imaging, and expression analysis at tissue, cell type-specific and single cell levels.
Project 2 - Tracing the effect of oncogenic signals on adult stem cell behaviour
Cancer develops through a multi-stage evolutionary process, proceeding from early meta-/neoplastic transformation to invasive carcinoma and metastasis. Current methods struggle to define mutation-containing early pre-tumorigenic lesions due to their phenotypic similarity with the surrounding normal tissue. We aim at defining the earliest steps of pre-neoplastic transformation, and identifying which tumorigenic mutations are the most threatening and require early medical intervention. To advance this field of research, we developed novel variants of the Rosa-Confetti 4-colour reporter allele (Red2cDNA series). These variants harbour different oncogenes and site-specific recombinase (e.g. FLP, for tumour suppressor knockout) coupled to a red fluorescent protein (RFP). Drawing upon the development of mathematical modelling methods, as well as advanced imaging and image-analysis, our team will define the clonal dynamics of tumorigenic mutation-containing clones. We will also evaluate the response of surrounding normal clones in homoeostasis and different tissue contexts, such as regeneration and inflammation. In this way, we can compare and contrast clonal dynamics in transformed mutant (red) cells with neighbouring normal clones (in green, yellow and cyan). This will allow us to:
1) Investigate the clonal behaviour of mutant clones in cell-to-cell competition in the tumour niche
2) Probe the remodelling of the tumour niche and the involvement of non-mutated surrounding tissue in this process
3) Analyse competition of large clones in the wide field of tissue.
Project 3 - Analysing the role of E3 ubiquitin ligases in the maintenance of adult stem cells
Homeostatic turnover in adult tissues is governed by the interplay of a multitude of signalling pathways. De-regulation of these pathways can result in pathogenic conditions, such as uncontrolled proliferation or stem cell depletion. Activation of these pathways is often triggered by niche cells, which provide diverse ligands to support the stem cells. Therefore, ligand-receptor interaction-initiated signalling cascades must be thoroughly and tightly controlled in order to sustain the functionality of the stem cells. An important class of such modulators are E3 ubiquitin ligases that specifically conjugate ubiquitin tags to their target proteins for ubiquitin-mediated degradation by proteasomes. Particularly, ubiquitination of membrane proteins directs trafficking decisions related to both biosynthetic delivery of proteins to the plasma membrane (PM) via the secretory pathway, and removal of proteins from the PM via the endocytic pathway. Previously, I showed the crucial role of Mib1 (an E3 for Notch ligands) in Notch ligand activation in niche cells that, in turn, promoted Notch signalling in stem cells. We found that RNF43 and ZNRF3 attenuate Wnt activation in intestinal stem cells by functioning as E3s for Wnt receptors. We aim at identifying other E3 ligases that play important roles in adult stem cell biology. For this project, three advanced technologies are combined to characterise the function of diverse E3 ligases playing an important role in tissue homeostasis.
First, we utilise intestinal and gastric organoid cultures to test the effect of E3 ligase overexpression and knockout.
Second, for selected E3 candidates, we perform extensive mass-spec analyses (e.g. surface proteome analysis, IP-Mass, and BIO-ID) to identify the substrate(s).
Lastly, we have developed diverse genetic tools for organoid culture, enabling loss- and gain-of-function approaches in organoids.