Fig. 2

(A) UMAP dimensional reduction and unsupervised clustering of drl:mCherry⁺;gata1:GFP^? cells at 1 and 2 dpf showing their original collection time point.

(B) mCherry expression level driven by the drl promoter for the different cell types collected at 1 and 2 dpf.

(C) (i) Schematic illustrating drl-specific, 4-OHT-inducible lineage-tracing system. Tg(drl:creER^T2) homozygous transgenic fish were crossed with hemizygous ubi:loxP-GFP-stop-loxP-mCherry (ubi:Switch). (ii) Zebrafish embryos were exposed to 12 ?M 4-OH-tamoxifen (4-OHT) or ethanol (EtOH) vehicle control for 20 h starting at either 30 hpf (labeled as Prog+, which includes embryonic progenitors and HSCs) or 54 hpf (labeled as HSC). Those embryos exposed to 4-OHT induce Cre recombination of loxP sites, leading to a permanent ?switch? excising the GFP cassette and resulting in expression of mCherry fluorescence. The resulting mCherry⁺ cells are the descendants of Prog+? and HSC-labeled cells. (iii) Representative flow cytometry analysis: forward (FSC) and side scatter (SSC) parameters were used to define the major blood cell populations (erythroid, myeloid, lymphoid, and precursor) in whole kidney marrow (WKM) (>3 months). (iv) The frequency of mCherry⁺ cells within each cell lineage was then calculated. Example flow cytometry plots (right) of mCherry contribution to the myeloid population. Gates were set based on EtOH-treated ?non-switched? controls.

(D) Quantification of mCherry⁺ percentage from experimental groups Prog+ and HSCs within each blood cell lineage found in WKM at >3 months post-fertilization (zebrafish adulthood). Data points represent individual zebrafish WKM with mean ± SEM (n = 5?12).

(E) Quantification of mCherry⁺ percentage at 1?5 days post-labeling in Prog+ and HSC-labeled cohorts. n = 5?11 samples, 7?10 pooled larvae per sample. Two-way ANOVA with Sidak?s multiple comparison test was used for analysis (mean ± SEM). *p < 0.05; ****p ? 0.0001. See also Figure S2.