Stem cells and development : studies on SUFU and LGR5

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