Experimental workflow and gating scheme used for sorting lcr:GFP⁺ erythroid cells. (A) Individual zebrafish are homogenized into a single-cell suspension in PBS containing 1% bovine serum albumin. Single cells were collected by filtration through a 70-μM cell strainer. Cells/debris caught in the filter were rinsed and collected for genotyping analysis by polymerase chain reaction (PCR) and Sanger sequencing of the PCR product. The filtrate (single-cell suspension) was processed for FACS and analysis. When indicated, cells were sorted onto microscope slides and processed for histological analysis. (B) Gating scheme to sort lcr:GFP⁺ and lcr:GFP⁻ cells. Gates were set up using WT AB zebrafish embryos as a negative control. The single-cell population is gated by FSC and SSC (left panels; black outline). The GFP⁻ population is defined by the FITC signal on cells from the negative controls that do not express GFP (teal outline); any FITC signal above that is defined as GFP⁺ (green outline). (C) lcr:GFP marks erythroid cells during development. At 2 dpf, primitive erythroid cells emerge from the ICM; at 4 dpf, definitive erythroid cells emerge from the CHT. By 6 dpf, the KM becomes the primary site of erythropoiesis. From 2 dpf onward, circulating erythroid cells (green) are also found in blood vessels. (D) Giemsa, Benzidine, and Hoechst staining of sorted lcr:GFP⁺ erythroid cells from pooled zebrafish embryos. Erythroid cells are hemoglobinized by 2 dpf but acquire a more elliptical shape at ∼4 dpf. (E) Erythroid cells grow in area through 4 dpf, largely due to increase in (F) cytoplasmic area. (G) Nuclear area is mostly constant from 2 to 6 dpf. (H) Cytoplasmic/nuclear area markedly increased ∼4 dpf, attributable to the increase in cytoplasmic area around this time. Magnification ×63. ∗∗∗P < .001. CHT, caudal hematopoietic tissue; FSC-A, forward scatter; ICM, intermediate cell mass; KM, kidney marrow; SSC-A, side scatter.