Stem cells and development : studies on SUFU and LGR5

Abstract: Embryonic development and cancer formation appear quite different from each other at first glance, yet they share common characteristics such as the reliance on certain signalling pathways. The Hedgehog (HH) and WNT signalling cascades play key roles in a variety of developmental processes where deregulation of these pathways result in a diversity of birth defects. In adult tissue, they are crucial for homeostasis and repair. Therefore, tight regulation of these pathways is of utmost importance, since aberrant activation can lead to numerous types of cancer. Suppressor of fused (SUFU) is an essential negative regulator of the HH pathway, and consequently pivotal for embryonic development such that Sufu-/- embryos die at around E9.5. The leucine-rich repeat containing Gprotein- coupled receptor 5 (LGR5), a WNT target gene, acts as a co-receptor in WNT signalling, and is up-regulated in basal cell carcinoma (BCC). Additionally, Lgr5 is a marker for adult stem cells in various tissues. In Paper I, we set out to explore the role of SUFU in lineage differentiation processes during early embryonic development, using mouse embryonic stem cells (mESC). Sufu-/- mESCs expressed typical pluripotency markers and showed characteristic morphology demonstrating that mESCs lacking Sufu can be kept in an undifferentiated state. Upon embryoid body formation in vitro and teratoma development in vivo, Sufu-/- mESCs exhibited limited differentiation capacity, with cartilage and bone completely missing in vivo. This result highlights the importance of HH signalling and in particular SUFU for regulating cell fate specification processes. In Paper II, we generated a hypomorphic Sufu allele and investigated the effects of significantly reduced SUFU levels on embryonic development in vivo. Sufu hypomorphic (Sufuhypo/hypo) embryos were viable up to E18.5 and showed a diverse range of developmental defects. Remarkably, despite the importance of HH signalling in skin development and BCC formation, Sufuhypo/hypo skin remained unaffected and did not show signs of hyperplasia or defects in epidermal differentiation. In stark contrast, bone development was severely impaired affecting numerous skeletal structures. Our findings further strengthen the role of SUFU in bone development, and demonstrate that tissues require different levels of SUFU for accurate development. In Paper III, we developed a transgenic mouse line in which expression of human (hu) LGR5 in basal cells of the skin could be reversibly controlled. We discovered that activation of huLGR5 during embryo development, with sustained expression after birth, affected skin development and homeostasis. Mice exhibited a sparse fur coat, hyperplasia of the interfollicular epidermis, and accelerated sebaceous gland maturation. However, no tumour formation was observed. Remarkably, the observed phenotype could be reversed when expression was stopped during early adulthood. Interestingly, induction of huLGR5 in juvenile mice did not result in any apparent phenotypic changes. In the preliminary study, we investigated the capacity of Sufu-/- hair follicle (HF) stem cells to initiate tumour formation. We unveiled that conditional inactivation of Sufu in Lgr5-expressing cells in juvenile or adult mice did not suffice to induce changes in the HF, or tumour formation. No changes were observed up to 80 weeks after Sufu deletion, and could furthermore not be provoked by wounding. This indicates that the level of HH signalling activity necessary for inducing skin changes is not achieved by deleting Sufu in the HF.