Intestinal cylindrical growth peaks in mice a few weeks after birth, simultaneously with crypt fission activity. It nearly stops after weaning and cannot be reactivated later. Transgenic mice expressing Cd97/Adgre5 in the intestinal epithelium develop a mega-intestine with normal microscopic morphology in adult mice. Here, we demonstrate premature intestinal differentiation in Cd97/Adgre5 transgenic mice at both the cellular and molecular levels until postnatal day 14. Subsequently, the growth of the intestinal epithelium becomes activated and its maturation suppressed. These changes are paralleled by postnatal regulation of growth factors and by an increased expression of secretory cell markers, suggesting growth activation of non-epithelial tissue layers as the origin of enforced tissue growth. To understand postnatal intestinal growth mechanistically, we study epithelial fate decisions during this period with the use of a 3D individual cell-based computer model. In the model, the expansion of the intestinal stem cell (SC) population, a prerequisite for crypt fission, is largely independent of the tissue growth rate and is therefore not spontaneously adaptive. Accordingly, the model suggests that, besides the growth activation of non-epithelial tissue layers, the formation of a mega-intestine requires a released growth control in the epithelium, enabling accelerated SC expansion. The similar intestinal morphology in Cd97/Adgre5 transgenic and wild type mice indicates a synchronization of tissue growth and SC expansion, likely by a crypt density-controlled contact inhibition of growth of intestinal SC proliferation. The formation of a mega-intestine with normal microscopic morphology turns out to originate in changes of autonomous and conditional specification of the intestinal cell fate induced by the activation of Cd97/Adgre5.

Conditions which promote reversible growth arrest, such as hypoxia and high cell density, lead to activation of a diverse network of proteins known as growth arrest specific (GAS) genes. Fatty acid binding protein 4 (FABP4), a lipid chaperone involved in the regulation of metabolic and inflammatory responses, has been shown to be part of the GAS program. While the induction of FABP4 in oxygen-deprived environments is well characterized, its functionality and regulation in such conditions remains unclear. In this study, we describe how mis-expression of FABP4 affects cell viability and survival within low oxygen conditions. Loss of FABP4 using shRNA was shown to be associated with a significant increase in oxidative stress and lipid peroxidation, a reduction in lipid droplet formation and a greater incidence of apoptosis. Hypoxia-mediated expression of FABP4 was also found to be positively correlated with cellular levels of C/EBP-beta, an essential activator of p20K in quiescence. FABP4 and p20K are both lipocalins that have been shown to share similar induction patterns and ability to assist in the maintenance of lipid trafficking in cellular stress circumstances. Unexpectedly, the depletion of FABP4 or p20K results in loss of the other in limited oxygen concentrations. This occurs independently of disruption to the broad GAS gene program, suggesting the two proteins may be co-regulated in a shared hypoxic-signalling pathway. C/EBP-beta appears to be the transcriptional activator shared by FABP4 and p20K in quiescence, and the three may be part of an intricate system to sense and respond to reactive oxygen species and lipid radicals. However, the forced expression of either FABP4 or p20K when the other is repressed only moderately restores cell survival through alleviating oxidative stress, indicating the two are both necessary for optimal response to hypoxia. In all, these studies suggest that analogous to the p20K lipocalin, FABP4 plays a critical role in lipid homeostasis and cell survival in conditions of limited oxygen concentrations, and its stimulation is dependent on C/EBP-beta activity. / Thesis / Master of Science (MSc) / A study investigating the role of FABP4 and p20K in conditions of reversible growth arrest with an emphasis on cell survival, lipid homeostasis and mitigating the effects of oxidative stress, and regulation of the two lipocalins by C/EBP-beta.