Assembly of a dynamic spatio-temporal map of gene expression regulation during intestinal stem cell differentiation to enterocytes
Biophysics
Cell Biology
Final Report Abstract
Cellular gene expression programs are rewired during embryonic development and adult tissue renewal. During these processes stem cells give rise to the differentiated cell types that tissues and organs are comprised of. As aberrant gene expression is a hallmark of developmental disorders and cancer, precise control mechanisms need to ensure the formation and maintenance of healthy tissues. Lineage-determining transcription factors (TFs), which bind to specific DNA motifs to induce or repress the expression of target genes, provide one such regulatory level. However, it remains poorly understood how cell type identity and state transitions are modulated by the expression level and biophysical properties of TFs based on the cellular position within a differentiating tissue. I chose to address these questions in the rapidly renewing gut epithelium, in which stem cells differentiate and mature along the crypt-villus axis, thereby underlying a spatial differentiation hierarchy. To this end, I engineered mouse small intestinal organoids, so called mini-guts representing central features of the intestine in vitro, to express TF-HaloTag transgenes, which enabled live-cell single-molecule tracking (SMT) of TFs. To increase the throughput of this SMT approach, I co-developed an automated imaging and customizable analysis pipeline allowing me to sample single-molecule trajectories from hundreds of cells within heterogenous 2D enteroid monolayer cultures derived from intestinal organoids, target specific cell types, spatially reference cellular positions to tissue landmarks, and correlate TF diffusion parameters with relative TF expression level, cell type, and cellular morphological features. Studying two absorptive lineage-determining TFs, I found an expression level-independent contrasting diffusive behavior: While Hes1, key determinant of absorptive lineage commitment, displays a large cell-to-cell variability and an average fraction of DNA-bound molecules of ~32%, Hnf4g, conferring enterocyte identity, exhibits more uniform dynamics and a bound fraction of ~56%. These results suggest that TF diffusive behavior could indicate the progression of differentiation and modulate early versus late differentiation within the intestinal absorptive lineage. In conclusion, the developed broadly applicable automated live-cell SMT framework developed in this project bridges scales from the tissue to the single-molecule level, thereby enabling the study of molecular mechanisms underlying differentiation and tissue-level phenotypes. Based on this technology, future research will address a potential broader applicability of the found concept of lineage TF dynamics supporting cell state transitions in the intestinal differentiation context and beyond, such as during tissue homeostasis and regeneration, malignant transformation and cancer, and embryonic development.
Publications
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Single-molecule tracking (SMT): a window into live-cell transcription biochemistry. Biochemical Society Transactions, 51(2), 557-569.
Dahal, Liza; Walther, Nike; Tjian, Robert; Darzacq, Xavier & Graham, Thomas G.W.
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Transgene Expression in Cultured Cells Using Unpurified Recombinant Adeno-Associated Viral Vectors. Journal of Visualized Experiments(200).
Benyamini, Brian; Esbin, Meagan N.; Whitney, Oscar; Walther, Nike & Maurer, Anna C.
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Automated live-cell single-molecule tracking in enteroid monolayers reveals transcription factor dynamics probing lineage-determining function.
Walther, Nike; Anantakrishnan, Sathvik; Dailey, Gina M.; Tjian, Robert & Darzacq, Xavier
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Code for automated single-molecule tracking (SMT) and SMT data analysis including the correlation of diffusion parameters with cellular features as well as protein diffusion cluster analysis
Nike Walther
