Su1659: DEMONSTRATING WHOLE-ORGAN LINEAGE TRACING OF FLUORESCENT MARKERS IN INTESTINAL STEM CELLS USING WIDEFIELD FLUORESCENCE IMAGING IN A ZFP148CREERT2 MOUSE MODEL

Daigle, Noelle; Song, Heyu; Sontz, Ricky; Merchant, Juanita L.; Sawyer, Travis W.

doi:10.1016/s0016-5085(22)61555-5

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“…Thus, tissue and organ-level expression of fluorescent reporter proteins cannot be evaluated directly. Further knowledge of biological mechanisms could be elucidated by measuring the expression of labeled cell populations across entire organs to determine cell behavior and protein expression on a macroscopic scale 19 . One possible solution is to leverage a technique similar to whole slide imaging to obtain both a microscopic and macroscopic view of fluorescent expression.…”

Section: Introductionmentioning

confidence: 99%

“…Further knowledge of biological mechanisms could be elucidated by measuring the expression of labeled cell populations across entire organs to determine cell behavior and protein expression on a macroscopic scale. 19 One possible solution is to leverage a technique similar to whole slide imaging to obtain both a microscopic and macroscopic view of fluorescent expression. In this case, the specimen would be imaged using an epifluorescent microscope on an XY displacement stage to acquire tiles over the entire spatial area with some degree of overlap.…”

Section: Introductionmentioning

confidence: 99%

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Wide field-of-view fluorescence imaging for organ-level lineage tracing of rare intestinal stem cell populations

Daigle,

Duan,

Song

et al. 2023

J. Biomed. Opt.

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Lineage tracing using fluorescent reporters is a common tool for monitoring the expression of genes and transcription factors in stem cell populations and their progeny. The zinc-binding protein 89 (ZBP-89/Zfp148 mouse gene) is a transcription factor that plays a role in gastrointestinal (GI) stem cell maintenance and cellular differentiation and has been linked to the progression of colon cancer. While lineage tracing is a useful tool, it is commonly performed with high-magnification microscopy on a small field of view within tissue sections, thereby limiting the ability to resolve reporter expression at the organ level. Furthermore, this technique requires extensive tissue processing, which is time consuming and requires euthanizing the animal. Further knowledge could be elucidated by measuring the expression of fluorescent reporters across entire organs with minimal tissue processing.Aim: We present the application of wide-field fluorescence imaging for whole-organ lineage tracing of an inducible Zfp148-tdTomato-expressing transgenic mouse line to assess the expression of ZBP-89/Zfp148 in the GI tract.Approach: We measured tdTomato fluorescence in ex vivo organs at time points between 24 h and 6 months post-induction. Fluctuations in tdTomato expression were validated by fluorescence microscopy of tissue sections.Results: Quantification of the wide field-of-view images showed a statistically significant increase in fluorescent signal across the GI tract between transgenic mice and littermate controls. The results also showed a gradient of decreasing reporter expression from proximal to distal intestine, suggesting a higher abundance of ZBP-89 expressing stem cells, or higher expression of ZBP-89 within the stem cells, in the proximal intestine. Conclusions:We demonstrate that wide-field fluorescence imaging is a valuable tool for monitoring whole-organ expression of fluorescent reporters. This technique could potentially be applied in vivo for longitudinal assessment of a single animal, further enhancing our ability to resolve rare stem cell lineages spatially and temporally.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%