Fig. 3
Inactivation of Hif-2α rescues extramedullary erythropoiesis and suppresses hemangiomas. a Erythropoietin is over-expressed in livers of HOXB7-Cre; Vhlhfl/fl knockout mice: analysis of Erythropoietin (Epo) mRNA expression by quantitative PCR. b Serum erythropoietin levels were determined by ELISA for indicated genotypes. Data were pooled from mice of >2 months of age. Erythropoietin was elevated by ~5 folds in the serum of HOXB7-Cre; Vhlhfl/fl (Vhlh-/-) mice compared to HOXB7-Cre; Vhlh+/+ or Cre negative (WT) littermates. Upon Hif-2α inactivation (HOXB7-Cre; Vhlhfl/fl; Hif-2αfl/fl double knockout) (Vhlh-/-; Hif2α-/-) serum erythropoietin levels returned to normal, compared with wild-type (WT). Hif-2α single allele knockout did not significantly affect the Vhlh knockout Epo level (HOXB7-Cre; Vhlhfl/fl; Hif-2αfl/+) (Vhlh-/-; Hif2α+/-). Data were analyzed by Student’s t-test with Welch’s correction for unequal variance, followed by Bonferoni post-test. c Erythroid progenitors (TER119 + CD71+) were isolated from hemangiomas of two (labeled 1, 2) knockout mice with FACS (purity ~80 %). Genomic DNA was prepared and used for PCR for the recombined, the deleted Vhlh allele (Vhlh del), and the floxed allele (Vhlh flox). No deleted allele was detected in these erythrocyte progenitors. As controls, PCR without template (no template) and PCR with genomic DNA from liver of knockout mice (containing both deleted and floxed alleles; positive control) was performed. d Hemangiomas were rescued in HOXB7-Cre; Vhlhfl/fl; Hif-2αfl/fl double knockouts. Hemangiomas were counted in H&E stained liver sections of 2-3 months old mice of indicated genotypes. While the number of hemangiomas per liver section was significantly increased in Vhlh knockouts compared to the wild-type, the number of hemangiomas was significantly decreased in HOXB7-Cre; Vhlhfl/fl; Hif-2afl/fl double knockout mice compared with HOXB7-Cre; Vhlhfl/fl single knockouts. Each data point represents one liver sample. Counts were averaged from at least 5 sections per liver. p-values were calculated using Student’s t-test with Welch’s correction. *: statistically significant (p < 0.05); ns: not significant