The media formulations necessary for deriving and sustaining organoids from epithelial tissues such as prostate, colon, gastric, liver, pancreas, and others have been established. Critical components of organoid media are a set of growth factors that include R-spondins and BMP signalling antagonists such as Noggin or Gremlin 1. Currently, the practical limitations for formulating organoid media of reproducible potency and larger-scale media production that have hampered further technological applications of organoid technology include: the cost of growth factors such as R-spondins and Gremlin 1/Noggin and their production as defined specific activities free of contaminants that may affect organoid growth. Here we report the production of highly pure recombinant Gremlin 1 and R-spondin 1 from bacterial expression for use in organoid media. We detail the workflow for Gremlin 1 and R-spondin 1 expression, purification, quantification of cellular activity, quality control and use in media formulated for culturing organoids derived from a number of tissues. The development of precisely formulated, cost-effective media of defined specific activity will engender the development of novel applications for organoid technology.
The adenomatous polyposis coli (Apc) protein regulates diverse effector pathways essential for tissue homeostasis. Truncating oncogenic mutations in Apc removing its Wnt pathway and microtubule regulatory domains drives intestinal epithelia tumorigenesis. Exuberant cell proliferation is one well-established consequence of oncogenic Wnt pathway activity; however, the contribution of other deregulated molecular circuits to tumorigenesis has not been fully examined. Using in vivo and organoid models of intestinal epithelial tumorigenesis we found that Wnt pathway activity controls intestinal epithelial villi and crypt structure, morphological features lost upon Apc inactivation. Although the Wnt pathway target gene c-Myc (also known as Myc) has critical roles in regulating cell proliferation and tumorigenesis, Apc specification of intestinal epithelial morphology is independent of the Wnt-responsive Myc-335 (also known as Rr21) regulatory element. We further demonstrate that Apc inactivation disrupts the microtubule cytoskeleton and consequently localisation of organelles without affecting the distribution of the actin cytoskeleton and associated components. Our data indicates the direct control over microtubule dynamics by Apc through an independent molecular circuit. Our study stratifies three independent Apc effector pathways in the intestinal epithelial controlling: (1) proliferation, (2) microtubule dynamics and (3) epithelial morphology.This article has an associated First Person interview with the first author of the paper.
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statement 25 We have determined that APC control of intestinal epithelia form and function can be divided 26 by three independent effector pathways controlling: (i) cell proliferation; (ii) epithelial 27 morphology and (iii) intracellular organisation. 28 29 Abstract 30 The tumour suppressor adenomatous polyposis coli (Apc) regulates diverse effector pathways 31 essential for cellular homeostasis. Truncating mutations in Apc, leading to the loss of its Wnt 32 pathway and microtubule regulatory domains, are oncogenic in human and murine intestinal 33 epithelia and drive malignant transformation. Whereas uncontrolled proliferation via Wnt 34 pathway deregulation is an unequivocal consequence of oncogenic Apc mutations, it is not 35 known whether loss of its other control systems contribute to tumorigenesis. Here we employ 36 in vitro models of tumorigenesis to unmask the molecular barriers erected by Apc that 37 maintain normal epithelial homeostasis in the murine intestinal epithelia. We determine that 38 (i) enterocyte proliferation, (ii) microtubule dynamics and (iii) epithelial morphology are 39 controlled by three independent molecular pathways, each corrupted by oncogenic Apc 40 mutations. The key result of the study is to establish that Apc regulates three individual 41 biological fates in the intestinal epithelia, through three distinct effector pathways, a 42 significant advance to our understanding of normal tissue homeostasis, the molecular 43 architecture of epithelial tissue and the aetiology of intestinal cancer. 44 45 67 heterozygous for a germline mutation inactivating one allele of APC (Su et al., 1992) exhibit 68 spontaneous loss of heterozygosity that leads to hundreds of tumours, all of which are 69 restricted to the intestinal epithelium. The murine model of FAP, Apc Min/+ (multiple intestinal 70 neoplasia; Min), follows a similar pattern of tumour development-despite mono-allelic 71
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