SIGNALLING TISSUE RENEWAL AND CRYPT SURVIVAL IN THE HUMAN COLONIC EPITHELIUM AND BARRETT’S OESOPHAGUS

Abstract Stem cell driven tissue renewal in the intestinal epithelium is a tightly regulated and controlled process. The colonic epithelium is organised into millions of invaginations called crypts, each of which represents the self-renewing unit of the tissue. In the mouse, renewal of the intestinal epithelium is regulated by signalling cross-talk between the Wnt, Notch, EGF and TGFβ/BMP pathways. The molecular mechanisms that regulate the processes of tissue renewal in the human are of great interest because they are disrupted in colorectal cancer and inflammatory diseases. Barrett’s oesophagus is an intestinal metaplasia arising in response to inflammation and ulceration provoked by gastroesophageal reflux. Detailed knowledge of the processes and signalling pathways involved in tissue renewal in Barrett’s oesophagus is still lacking and is required to understand more fully the risk and pathogenesis of this metaplasia and oesophageal adenocarcinoma. Intact human colonic crypts were isolated and placed into 3D tissue culture conditions optimised for steady-state tissue renewal. The role of Wnt and TGFβ/BMP signalling pathways in tissue renewal was investigated. Native human colonic crypts exhibited distinct activation profiles for canonical Wnt, TGFβ and BMP pathways. A population of intestinal Lgr5/OLFM4+ stem cells were found to be interspersed between goblet cells at the base of the crypt. Exogenous Wnt signals maintained Lgr5/OLFM4+ stem cells, whilst BMP and TGFβ inhibited and caused complete loss of stem cells. Wnt signals also rescued the inhibitory effects of Dkk1, IWP2 and dnTCF4 on Wnt target gene expression, cell proliferation and crypt length. BMP and TGFβ inhibited Wnt target gene expression, cell proliferation and crypt length. A near-native human Barrett’s oesophagus ex vivo culture model was developed similar to the colonic model which was amenable to real-time time-lapse microscopy and imaging techniques. The Wnt and NFκB signalling pathways exhibited distinct activation profiles. A population of OLFM4+ stem cells were found to reside in the lower third of the Barrett’s crypt. Steady-state tissue renewal in the human colonic epithelium is dependant on Wnt signals combined with suppressed TGFβ/BMP pathways. The human colonic crypt model and the Barrett’s oesophagus crypt model will permit functional interrogation of the mechanisms underlying tissue renewal and risk of inflammatory diseases and adenocarcinoma. *[N.B.: A DVD was attached to this thesis at the time of its submission. Please refer to the author for further details

Canonical Wnt signals combined with suppressed TGFβ/BMP pathways promote renewal of the native human colonic epithelium

Background: A defining characteristic of the human intestinal epithelium is that it is the most rapidly renewing tissue in the body. However, the processes underlying tissue renewal and the mechanisms that govern their coordination have proved difficult to study in the human gut. Objective: To investigate the regulation of stem cell-driven tissue renewal by canonical Wnt and TGFβ/bone morphogenetic protein (BMP) pathways in the native human colonic epithelium. Design: Intact human colonic crypts were isolated from mucosal tissue samples and placed into 3D culture conditions optimised for steady-state tissue renewal. High affinity mRNA in situ hybridisation and immunohistochemistry were complemented by functional genomic and bioimaging techniques. The effects of signalling pathway modulators on the status of intestinal stem cell biology, crypt cell proliferation, migration, differentiation and shedding were determined. Results: Native human colonic crypts exhibited distinct activation profiles for canonical Wnt, TGFβ and BMP pathways. A population of intestinal LGR5/OLFM4-positive stem/progenitor cells were interspersed between goblet-like cells within the crypt-base. Exogenous and crypt cell-autonomous canonical Wnt signals supported homeostatic intestinal stem/progenitor cell proliferation and were antagonised by TGFβ or BMP pathway activation. Reduced Wnt stimulation impeded crypt cell proliferation, but crypt cell migration and shedding from the crypt surface were unaffected and resulted in diminished crypts. Conclusions: Steady-state tissue renewal in the native human colonic epithelium is dependent on canonical Wnt signals combined with suppressed TGFβ/BMP pathways. Stem/progenitor cell proliferation is uncoupled from crypt cell migration and shedding, and is required to constantly replenish the crypt cell population

Drosophila Araucan and Caupolican Integrate Intrinsic and Signalling Inputs for the Acquisition by Muscle Progenitors of the Lateral Transverse Fate

A central issue of myogenesis is the acquisition of identity by individual muscles. In Drosophila, at the time muscle progenitors are singled out, they already express unique combinations of muscle identity genes. This muscle code results from the integration of positional and temporal signalling inputs. Here we identify, by means of loss-of-function and ectopic expression approaches, the Iroquois Complex homeobox genes araucan and caupolican as novel muscle identity genes that confer lateral transverse muscle identity. The acquisition of this fate requires that Araucan/Caupolican repress other muscle identity genes such as slouch and vestigial. In addition, we show that Caupolican-dependent slouch expression depends on the activation state of the Ras/Mitogen Activated Protein Kinase cascade. This provides a comprehensive insight into the way Iroquois genes integrate in muscle progenitors, signalling inputs that modulate gene expression and protein activity