Colorimetric Barcoding to Track, Isolate, and Analyze Hematopoietic Stem Cell Clones

Abstract

In zebrafish, hematopoietic stem cells (HSCs) are born in the developing aorta during embryogenesis. From the definitive wave of hematopoiesis onward, blood homeostasis relies on self-renewal and differentiation of progeny of existing HSCs, or clones, rather than de novo generation. Here, we describe an approach to quantify the number and size of HSC clones at various times throughout the lifespan of the animal using a fluorescent, multicolor labeling strategy. The system is based on combining the multicolor Zebrabow system with an inducible, early lateral plate mesoderm and hematopoietic lineage specific cre driver (draculin (drl)). The cre driver can be temporally controlled and activated in early hematopoiesis to introduce a color barcoding unique to each HSC and subsequently inherited by their daughter cells. Clonal diversity and dominance can be investigated in normal development and blood disease progression, such as blood cancers. This adoptable method allows researchers to obtain quantitative insight into clonality-defining events and their contribution to adult hematopoiesis.

Keywords: Colorimetric barcoding; Cre-loxP recombination; Hematopoietic stem cells (HSCs); In vivo multicolor imaging; Lineage analyses; Zebrabow system.

Fig. 1

Genotyping strategy for Tg(ubi:Zebrabow-M) zebrafish. (a) Schematic of the Zebrabow color cassette depicting the location of the ddPCR primer location within the tdTomato sequence. (b) Number of insertions are calculated from the event reads for each sample. Insertions vary and zebrafish with more than 20 insertions should be kept. The single insertion has one insertion detected and the wild-type control has no insertions, which indicated a successful genotyping procedure

Fig. 2

Experimental workflow for a Zebrabow analysis. Double transgenic Tg(ubi:Zebrabow-M);Tg(drl:creERT2) single-cell embryos are collected and a genetic manipulation could be performed. At 24 hpf, 4-OHT treatment is performed for 4 h to induce the color recombination in drl-CreERT2 positive cells. After growing the zebrafish to adulthood, the color barcodes can be analyzed via flow cytometry and clones of interest could be sorted for further analysis using RNA- or DNAseq techniques

Fig. 3

Representative flow cytometry gating strategy to prepare data for the Zebrabow Color Analysis Application. An example of flow cytometry data obtained from a single, dissociated kidney marrow of a Tg(ubi:Zebrabow-M);Tg(drl:creERT2) zebrafish treated with 4-OHT at 24 hpf. The gating strategy is depicted from left to right as indicated by the arrows. Single cells are identified via FSC-A and FSC-H and further by SSC-W and SSC-H, live cells are identified by the exclusion of DRAQ7 staining in the APC-Cy7 channel, and myelomonocytes are identified by their characteristic FSC-A and SSC-A properties. From the myelomonocyte gate, tdTomato, CFP, and YFP are used to identify all cells for analysis in the Zebrabow Color Analysis Application. Two gates are separated, either cells expressing CFP detected in the DAPI channel and/or YFP detected in the FITC and cells expressing tdTomato detected in the dsRED channel. From these gates a Boolean gate is generated, indicating all cells that will be analyzed by the Zebrabow Color Analysis Application. FSC-A Forward scatter area, FSC-H forward scatter height, SSC-A side scatter area, SSC-H side scatter height, SSC-W side scatter width, Comp compensation

Fig. 4

Example graphs obtained using the Zebrabow Color Analysis Application. (a) A three-dimensional rendering of myelomonocytes from the kidney marrow of a 3 mpf Tg(ubi:Zebrabow-M);Tg(drl:creERT2) zebrafish, 4-OHT treated at 24 hpf. (b) An individual cluster is highlighted in two-dimensional CFP/YFP and RFP/YFP plots. These plots can be used to inform gating strategies to sort specific clones using a flow cytometer. (c) The expression level of tdTomato, CFP, and YFP of each myelomonocyte is plotted in the ternary plot, respectively. (d/e) Two different methods of graphical representation are depicted, representing the percentage of analyzed cells belonging to each annotated color cluster