Tcf as Wnt effector
In 1991, we reported the cloning of a T cell specific transcription factor that we termed TCF1 (1). Related genes exist in genomes throughout the animal kingdom.We have shown in frogs (4), flies (7) and worms (11) that the TCF proteins constitute the effectors of the canonical Wnt pathway. Upon Wnt signaling, ß-catenin binds and activates nuclear TCFs by providing a trans-activation domain. For these studies, we designed the widely used pTOPFLASH Wnt reporters. In the absence of Wnt signaling, we found that Tcf factors associate with proteins of the Groucho family of transcriptional repressors to repress target gene transcription (9).
Wnt signaling in cancer
The tumor suppressor protein APC forms the core of a cytoplasmic complex which binds ß-catenin and targets it for degradation in the proteasome. In APC-deficient colon carcinoma cells, we demonstrated that ß-catenin accumulates and is constitutively complexed with the TCF family member TCF4, providing a molecular explanation for the initiation of colon cancer (5).
Wnt signaling in adult stem cells
In mammals, physiological Wnt signaling is intimately involved with the biology of adult stem cells and self-renewing tissues (18,19). We were the first to link Wnt signaling with adult stem cell biology, when we showed that TCF4 gene disruption leads to the abolition of crypts of the small intestine (8), and that TCF1 gene knockout severely disables the stem cell compartment of the thymus (2). The Tcf4-driven target gene program in colorectal cancer cells is the malignant counterpart of a physiological gene program in selfrenewing crypts (13, 14, 21).
Lgr5 as adult stem cell marker
Amongst the Wnt target genes, we found the Lgr5 gene to be unique in that it marks small cycling cells at crypt bottoms. These cells represent the epithelial stem cells of the small intestine and colon (23), the hair follicle (24), the stomach (28) and many other tissue stem cell types.They also represent the cells-of-origin of adenomas in the gut (25) and within adenomas Lgr5 stem cells act as adenoma stem cells (36). Lgr6 marks multipotent skin stem cells (29).
Lgr5 stem cell biology
Lgr5 crypt stem cells behave in unanticipated ways: Against common belief, they divide constantly and in a symmetric fashion. Stem cells numbers remain fixed because stem cells compete 'neutrally' for niche space. Thus, they do not divide asymmetrically (31), a phenomenon that was confirmed by in vivo imaging (43). Daughters of the small intestinal stem cells, the Paneth cells, serve as crypt niche cells by providing Wnt, Notch and EGF signals (30).
The Wnt target gene encoding the transcription factor Achaete scute-like 2 controls the fate of the intestinal stem cell (26).
Lgr5 is the R-spondin receptor
Lgr5 resides in Wnt receptor complexes and mediates signaling of the R-spondin Wnt agonists (32), explaining the unique dependence of Lgr5 stem cells on R-spondins in vivo and in vitro. Two other Wnt target genes, RNF43 and ZNRF3, encode stem cell-specific E3 ligases that downregulate Wnt receptors. They serve in a negative feedback loop to control the size of the stem cell zone (34). Independent work by the Feng Cong lab has first shown that R-spondin, when bound to Lgr5, captures and inactivates RNF43/ZNRF3.
Long-term clonal culturing of organoids from Lgr5 stem cells
Wnt signaling intimately interacts with the BMP and Notch cascades to drive proliferation and inhibit differentiation in intestinal crypts and adenomas (17, 20). Based on these combined insights, we have established Lgr5/R-spondin-based culture systems that allow the outgrowth of single mouse or human Lgr5 stem cells into ever-expanding mini-guts (27), mini-stomachs (28), liver organoids (38, 45), prostate organoids (44) and organoids representing other adult tissues. These epithelial organoid cultures are genetically and phenotypically extremely stable, allowing transplantation of the cultured offspring of a single stem cell, as well as disease modeling by growing organoids directly from diseased patient tissues (45).
As proof-of-concept, the CFTR locus was repaired in single gut stem cells from two Cystic Fibrosis patients, using CRISPR/Cas9 technology in conjunction with homologous recombination. Repaired stem cells were clonally expanded into mini-guts and shown to contain a functional CFTR channel (42).
About the group leader
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