Defining the metabolic determinants of intestinal stem cell homeostasis

The intestine is the fastest renewing organ. This ability is enabled by the presence of fast proliferating intestinal stem cells (ISCs) that reside in the crypts of Lieberkühn at the base of the intestinal villi. The mechanisms that drive ISC self-renewal are incompletely understood. The non-essential amino acid proline is synthesized from its precursor pyrroline-5-carboxylate (P5C) through the activity of the pyrroline-5-carboxilate reductase (PYCR) enzymes. There are 3 PYCRs: mitochondrial PYCR1 and 2 are highly homologous (84%), whereas cytosolic PYCR3 is the least conserved (45%). Our data show that PYCR1 enzyme is expressed at the bottom of the mouse intestinal crypts. This finding was confirmed by analysis of transcriptomic data showing high expression of PYCR1 in the proliferative area of the intestinal crypts, where ISCs are located. Moreover, we observed a dramatic increase in PYCR1 expression in a genetically modified mouse model of stem-cell driven colorectal cancer and genetic depletion of PYCR1 blocks proliferation of human colorectal cancer cells in vitro. However, whether proline biosynthesis is necessary for the survival and functions of the ISCs is currently unknown. Here, we intend to approach this possibility using different experimental models:

Mouse: Expression of Pycr1 in the ISCs will be confirmed using confocal microscopy and double staining with the ISC marker Olfm4. Moreover, we will use a mouse model that expresses the green fluorescent protein (GFP) selectively in the ISCs. GFP-positive and negative cells will be sorted at the cytofluorimeter and the expression of proline metabolism enzymes will be investigated in the two cell populations using real-time PCR. In addition, we will isolate intestinal crypts from mice and grow them in-vitro using a cutting-edge, 3-dimensional (3D) culture protocol. The growth of these organoid cultures is fuelled by ISCs. We will then deplete expression of Pycr enzymes using retroviruses to deliver short hairpin (sh)RNA to the isolated crypts. The impact of these genetic manipulations on 3D cultures will be assessed investigating markers of proliferation and stemness.

Drosophila: The molecular pathways that control ISCs homeostasis in mammals are highly conserved in the fruit fly Drosophila melanogaster. This model also enables a fast and versatile in vivo approach to investigate the impact of disrupting the expression of the fly PYCR orthologues (P5cr, P5cr-2) in the intestine by genetic manipulation. We will obtain transgenic Drosophila lines from the Vienna Drosophila Resource Center, which bear RNA-interference knockdown constructs of the reductase genes and we will use confocal microscopy to investigate intestinal homeostasis, including expression of stem cell markers and ISC proliferation in the intestine of these flies.

Metabolomics and transcriptomics. Intestinal crypts and differentiated villi will be isolated and subject to metabolomics analysis by mass spectrometry. The data will then be cross-analysed with publicly available data of single-cell mRNA sequencing of intestinal cells to identify the landscape of selective metabolic adaptations in the different intestinal cell types, with a specific focus on ISCs.

Techniques that will be undertaken during the project
• Drosophila genetics and characterization of transgenic fly lines
• In vivo work with genetically modified mouse models
• Genetic manipulation of gene expression
• 3D intestinal organoid cultures
• Confocal microscopy
• Real-time PCR
• Mass-spectrometry enabled-metabolomics
• Bioinformatics mining of transcriptomic datasets to identify metabolic networks active in the ISCs

Available to UK/EU applicants only
Application information
https://www2.le.ac.uk/research-degrees/doctoral-training-partnerships/bbsrc