The external development and transparent property of zebrafish embryos enable us to directly visualize and trace fluorescence-labeled cells in vivo during early embryonic development. In this study, we proposed to identify and characterize HSCs using a label-retaining strategy, a method which is well characterized in defining niche and functional analysis of epithelial stem cells. This method is based on slow cell cycling and relative quiescent state of stem cells, resulting in retaining a specific transient labeling for a longer time, compared to other progenitor cells. Inducible CreER/lox recombination system is introduced into this cell-labeling and tracing study, achieving to provide a transient genetic labeling manner. First, two transgenic zebrafish lines are generated, one is PAC...[ Read more ]

The external development and transparent property of zebrafish embryos enable us to directly visualize and trace fluorescence-labeled cells in vivo during early embryonic development. In this study, we proposed to identify and characterize HSCs using a label-retaining strategy, a method which is well characterized in defining niche and functional analysis of epithelial stem cells. This method is based on slow cell cycling and relative quiescent state of stem cells, resulting in retaining a specific transient labeling for a longer time, compared to other progenitor cells. Inducible CreER/lox recombination system is introduced into this cell-labeling and tracing study, achieving to provide a transient genetic labeling manner. First, two transgenic zebrafish lines are generated, one is PAC-scl-CreER in which CreER expression is controlled by scl promoter with all its regulatory elements harbored in this PAC construct, and the other is a transgenic reporter fish, termed as ef1α-loxP-dsRed-SV40-loxP-eGFP- SV40. Before Cre/lox recombination, the transgenic reporter fish will only express dsRed and cells are only labeled by dsRed, and further it will transit to express eGFP upon Cre treatment. By mating reporter fish with PAC-scl-CreER transgenic fish, Cre/lox recombination can be specifically induced in SCL-positive cells upon 4-OH-tamoxifen treatment at different time points. Therefore, dsRed gene is deleted in SCL-positive cells and they will change to express eGFP, attaining a transient labeling method by existed dsRed. Conformed to principle of label-retaining study, we can achieve to divide cells into three different pools according to presence of eGFP and dsRed: SCL-negative cells with only dsRed expression; SCL-positive progenitor and mature hematopoietic cells with newly expressed eGFP in which dsRed is rapidly diluted and degraded owing to replication and differentiation, and the last SCL-positive label-retaining hematopoietic stem cells (LR-HSCs) with retained dsRed owing to slow cell cycling and relative quiescent state which will collectively represent as both eGFP and dsRed positive under fluorescence microscope. Through this scheme, we can attain to resolve HSCs from other hematopoietic progenitor cells, which can further facilitate to evaluate and monitor the behavior of HSCs in vivo using time-lapsed live imaging technique.