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ARHGEF12 in erythropoiesis of chemotherapy patients
throid differentiation of the cells, under hemin induction, decreased significantly compared to the non-targeted (NT) cells (Online Supplementary Figure S2C).
ARHGEEF12 or its orthologs is involved in erythroid differentiation in murine progenitor cells and in
a zebrafish model
To rule out potential effects by the neoplastic back- ground of K562 cells, we knocked down Arhgef12 expres- sion in mouse hematopoietic cell line 32D cells and in pri- mary mouse BM cells by lentiviral shRNA transduction. Erythropoietin-induced erythroid differentiation was sig- nificantly blocked by the interference of Arhgef12 expres- sion as by observed erythroid immunophenotyping by flow cytometry and in the burst forming units-erythroid and colony forming unit-erythroid colony forming assays (Online Supplementary Figure S3).
Zebrafish genome harbors two orthologs of ARHGEF12: arhgef12a on chromosome 15 and arhgef12b on chromo- some 5. By comparing their sequences (arhgef12a, ENS- DARG00000030532; arhgef12b, ENSDARG00000067634) with human ARHGEF12, we found that the similarities were 54% and 55%, respectively. Synteny analysis also showed the relatively conserved positions for both arhgef12a and arhgef12b (Online Supplementary Figure S4A). Arhgef12a is selectively enriched in early erythroid progen- itors (Online Supplementary Figure S4B) whereas arhgef12b is expressed in early erythroid progenitors (Online Supplementary Figure S4C).
To study the role of arhgef12 in erythropoiesis, we per- formed microinjections of arhgef12a and arhgef12b mor- pholino both in combination (arhgef12 MO) and separate- ly (arhgef12a MO and arhgef12b MO). Firstly, we per- formed WISH at 22 hours post fertilization (hpf) to ana- lyze the primitive wave24 of hematopoiesis. Expressions of the erythroid progenitor marker gata1, the mature ery- throcyte marker αe1-globin, the hematopoietic lineage marker scl, and the myeloid markers pu.1 and lysozyme C remained unchanged in arhgef12 MO-injected embryos (Online Supplementary Figure S5). At 36 hpf, the definitive hematopoiesis stage of zebrafish, the expression of αe1-
Table 3. Gene function related to erythropoiesis.
globin was dramatically decreased in arhgef12a- and arhgef12b-deficient embryos, whereas the hematopoietic stem cells (HSC) markers runx1 and c-myb and the vascular morphology and marker flk1 were unchanged (Figure 3A). At four days post-fertilization (dpf), the markers repre- senting mature RBC including αe1-globin, βe1-globin, βe2- globin, band3, and alas2 were severely reduced (Figure 3B). Of interest, gata1 was associated with an obvious increase in caudal hematopoietic tissue (CHT) (Figure 3B), indicat- ing that the erythroid defect may be caused by an impaired differentiation. Of interest, gata1 was associated with an obvious CHT (Figure 3B), indicating that the ery- throid defect may be caused by an impaired differentia- tion. Consistent with this possibility, o-Dianisidine stained hemoglobin showed that erythrocytes from arhgef12 MO-injected embryos were more immature than those from the control group at 36 hpf and 4 dpf (Figure 3C). Subsequent examinations of arhgef12a and arhgef12b double knockout mutants by using the CRISPR/Cas9 method followed by o-Dianisidine staining found that mature erythrocytes were significantly decreased at 4 dpf in the mutant CHT and heart (Figure 3D and E). These results indicate that arhgef12a and arhgef12b are required for erythroid differentiation and maturation in zebrafish.
ARHGEF12 regulates erythroid differentiation through a RhoA-p38 pathway
RhoA is a well-defined substrate of ARHGEF12, which activates the exchange of RhoA bound GDP in the inacti- vated form for GTP to yield the active RhoA-GTP.26 We hypothesized that RhoA is the key target of ARHGEF12 to mediate its function in erythrocyte maturation. Because zebrafish harbors 5 rhoa genes27 and they all have an amino acid sequence which is quite similar (identity >90%) to human RhoA, we inferred that human RhoA mutant mRNA dominant-negative (DN) mutant RhoA T19N28 and constitutively active mutant RhoA Q6329 would also function in zebrafish. In fact, injection of dom- inant-negative RhoA mRNA led to anemia, which mim- icked the arhgef12 deficiency phenotype (Figure 4A), but the anemia seemed to be less severe than with MO injec-
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