Fluorescence stereomicroscopy for live imaging of zebrafish embryos and larvae has revolutionized our ability to gain insight into human developmental and disease processes. It allows visualization in real time of cell lineages that build the complex tissues and organs that define vertebrate animals. Examining how these processes are disrupted by injury or disease leads to novel discoveries of the cellular mechanisms that can be manipulated to improve human health. The vast amount of single cell expression data generated from distinct developmental stages and tissues has provided a wealth of new information about key transcription factors that specify cell lineages. Until now, methods to create transcription factor-specific cell lineage tracing reporters, such as Cre recombinase, have been inefficient and limited by the ability to clone gene specific promoters. With our GeneWeld CRISPR precision targeted integration strategy, we can generate a Cre driver in any gene of interest, unlocking the potential to examine cell lineages that were previously inaccessible, and to transform single cell gene expression data into functional tools for cell lineage specific analysis. With our R24 resource grant, we have recovered Cre drivers in transcription factor genes that define mesodermal and immune cell lineages and are expanding this to early mesendoderm, vascular, and hematopoietic cells. Fluorescence microscopy is at the heart of the transgenic resource we are building and functional fluorescence stereomicroscope equipment is critical to efficiently generate and characterize new lines. To meet the imaging needs of the R24, we propose 2 aims that will significantly expand our fluorescence stereomicroscope capabilities. In Aim 1 we propose funds to support our imaging capabilities through the purchase of 3 Leica M165 FC imaging systems for routine daily screening and documentation of new transgenic lines. In Aim 2 we propose funds to elevate our capacity for real time live imaging of cell lineage labeling through the purchase of 1 Leica M205 FCA Thunder imaging system. Together, these systems will allow us to successfully grow the zebrafish Cre driver community resource and thoroughly characterize the spatial and temporal cell lineage labeling activity of each Cre line.
|Effective start/end date||6/1/16 → 3/28/23|
- NIH Office of the Director: $294,484.00
- NIH Office of the Director: $62,320.00
- NIH Office of the Director: $779,247.00
- NIH Office of the Director: $31,160.00
- NIH Office of the Director: $828,987.00
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