Multicolor Cell Barcoding Technology for Long-Term Surveillance of Epithelial Regeneration in Zebrafish

Abstract

SUMMARY Current fate mapping and imaging platforms are limited in their ability to capture dynamic behaviors of epithelial cells. To deconstruct regenerating adult epithelial tissue at single-cell resolution, we created a multicolor system, skinbow, that bar-codes the superficial epithelial cell (SEC) population of zebrafish skin with dozens of distinguishable tags. With image analysis to directly segment and simultaneously track hundreds of SECs in vivo over entire surface lifetimes, we readily quantified the orchestration of cell emergence, growth, repositioning, and loss under homeostatic conditions and after exfoliation or appendage amputation. We employed skinbow-based imaging in conjunction with a live reporter of epithelial stem cell cycle activity, and as an instrument to evaluate the effects of reactive oxygen species on SEC behavior during epithelial regeneration. Our findings introduce a platform for large-scale, quantitative in vivo imaging of regenerating skin and reveal unanticipated collective dynamism in epithelial cell size, mobility, and interactions.

Regeneration Genetics

“…In vivo imaging platforms enable the direct visualization of intricate cell activities in live regenerating tissues (Figure 2) (4; 16; 23; 125). This has been effectively employed to visualize the behaviors of labeled cells.…”

“…Instead of tracking one cell and its progeny, the ability to assign many color tags enables monitoring of many cells in a population and their collective behaviors. With Brainbow-based genetic technology, Chen et al recently developed a transgenic line, referred to as skinbow , to simultaneously monitor individual superficial epithelial cells (SECs) in a large population during tissue homeostasis and regeneration (16) (Figures 1 and 2c). Live imaging of skinbow surface tissue allows tracking of hundreds of cells over long periods of time, permitting acquisition of epithelial cell size, mobility, and cell-cell interactions during cell turnover and injury-induced regeneration.…”

“…Moreover, pre-existing SECs and de novo emerging SECs could be identified. For example, major injuries like fin amputation induced massive new cell creation, whereas minor injuries such as exfoliation recovered through accelerated differentiation (16). In an analogous study, transgenic “Limbow” axolotls were employed to define cell behaviors in the connective tissue of amputated digit tips (23) (Figure 2d).…”

“…In zebrafish fins, epithelial cell sheets migrate at different velocities corresponding to ray and interray tissue, covering the fin stump within 30–60 minutes (16). Mechanical properties of epithelial cells are likely to mobilize epithelial cell sheets (16), but the molecular basis of their differential motility and its significance are unclear. Studies of wound healing responses in zebrafish larvae and Drosophila embryos indicate clues to early molecular responses (83; 95).…”

Quantitative Analysis of Synthetic Cell Lineage Tracing Using Nuclease Barcoding

“…Existing approaches are limited in the resolution provided, the ability to combine lineage information with other system readouts, and the need for real-time observation. 34 We were motivated by the great potential for data storage and manipulation within DNA and thus developed our dynamic, sequence-based barcoding technology to address the aforementioned shortcomings. Previous work has exploited the natural occurrence of mutations in microsatellite loci 18–22 or mitochondrial DNA 35 to trace lineages.…”