Fig. 2
Extramedullary erythropoiesis in Vhlh knockout mice. a Erythrocyte progenitors are increased by ~10-fold in livers of HOXB7-Cre; Vhlhfl/fl knockout mice. Liver cell suspensions were prepared by mincing livers, followed by collagenase digestion and treatment with red blood cell lysis buffer (see Methods). Flow cytometric quantification of erythrocyte progenitors (TER119 + CD71+) was performed. Shown are representative FACS-plots (left panels) and quantification (right panel). Gate was set on live CD45+ cells, and doublets were gated out. b Quantification of erythroid progenitors with colony forming unit assay. Liver cell suspensions from HOXB7-Cre; Vhlhfl/fl (Vhlh-/-), HOXB7-Cre; Vhlhfl/fl; Hif2αfl/+ (Vhlh-/-; Hif2α+/-), HOXB7-Cre; Vhlhfl/fl; Hif2αfl/fl (Vhlh-/-; Hif2α-/-), and Vhlhfl/fl (WT) mice were prepared and cultured as described in Methods. Left panel: Representative image of erythroid progenitor colonies (BFU-E) from a Vhlh knockout mouse. Right panel: Quantification of BFU-Es. While Vhlh knockout increased the number of BFU-E compared to that obtained from liver cell suspensions of Cre negative mice (WT), no difference was seen in the number of BFU-Es from WT and HOXB7-Cre Vhlh-Hif-2α double knockouts. One Vhlh-/- and Hif-2α single allele knockout animal of the genotype HOXB7-Cre; Vhlhfl/fl; Hif-2αfl/+ (Vhlh-/-; Hif2α+/-) was used as a reference, which showed increased BFU-E number as in the Vhlh knockout. Mice were >3 months old. p-values were calculated using Student’s t-test with Welch’s correction. *: statistically significant (p < 0.05); ns: not significant