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. 2018 Aug;103(8):e336-e340.
doi: 10.3324/haematol.2018.191338. Epub 2018 Mar 22.

GATA1 s exerts developmental stage-specific effects in human hematopoiesis

Sofia Gialesaki  1 , Anna Katharina Mahnken  1 , Lena Schmid  1 , Maurice Labuhn  1 , Raj Bhayadia  2 , Dirk Heckl  3 , Jan-Henning Klusmann  4
Affiliations

GATA1 s exerts developmental stage-specific effects in human hematopoiesis

Sofia Gialesaki et al. Haematologica. 2018 Aug.
No abstract available

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Figures

Figure 1.
Figure 1.
GATA1s mutation is essential for the development of transient abnormal myelopoiesis (TAM). (A) Schematic representation of the GATA1 locus, the translation of wild-type (WT) GATA1 product in normal fetal hematopoiesis (top), and the translation of GATA1s after the introduction of mutations as seen in TAM (middle) or as generated using CRISPR-Cas9 genome editing (bottom). (B) The efficacies of 4 different sgRNAs targeting exon 2 of GATA1 as assessed using a fluorescence-based reporter assay. The sgRNAs selected for later studies are marked red and blue. (C-F) K562 cells lentivirally transduced with GATA1-targeting (GATA1.1 and GATA1.2) or control (Luc.1) sgRNAs. (C) Western blot of GATA1 and GATA1s proteins in WT K562 cells and in two representative GATA1s-K562 clones (#1 and #2; used in subsequent experiments). (D) Number of WT and GATA1s-K562 cells (clone #1 and #2) grown in liquid culture. (E) Flow cytometric analysis of WT and GATA1s-K562 cells (clone #1 and #2) for expression of the indicated cell surface markers. (F) Expression of CD235a represented as mean fluorescence intensity (MFI), in WT K562 cells and GATA1s-K562 clone #1 and #2.
Figure 2.
Figure 2.
GATA1s mutations in fetal hematopoietic stem and progenitor cells (HSPCs) promote the hyperproliferation of megakaryocytic progenitors. Fetal, neonatal and adult HSPCs were transduced with GATA1-targeting (GATA1.1 and GATA1.2) or control (Luc.1) sgRNAs. (A) Relative number of transduced HSPCs (normalized to the number of Luc.1 cells) grown in liquid cultures supporting megakaryocytic differentiation. Data from 1 of 4 independent experiments performed in replicates are shown as mean±Standard Deviation (SD). *PANOVA<0.05, **PANOVA<0.01. (B) (Left) Representative flow cytometry plots (from 3 independent experiments), showing expression of the common megakaryocytic markers CD41/CD61 and CD42b in fetal HSPCs (pre-gated on live cells). Percentages of each population are indicated. (Right) Percentage of fetal CD41/CD61+ cells on day 10 of megakaryocytic differentiation. Data from 2 independent experiments performed in replicates are shown as mean±SD. *PANOVA<0.05, **PANOVA<0.01. (C) (Left) Photographs (scale bar 5 mm) of CFU-MK assays using lentivirally transduced fetal HSPCs. (Right) Number of CFU-MKs. **PANOVA<0.01. (D) Microscopic images of CFU-MK assays (400× original magnification; scale bar 100 μm).
Figure 3.
Figure 3.
GATA1s perturbs megakaryocytic/erythroid differentiation of neonatal hematopoietic stem and progenitor cells (HSPCs), resulting in accumulation of erythroid progenitors. Fetal, neonatal and adult HSPCs were transduced with GATA1-targeting (GATA1.1 and GATA1.2) or control (Luc.1) sgRNAs. (A) Relative number of transduced HSPCs (normalized to the number of Luc.1 cells) grown in liquid cultures supporting combined megakaryocytic/erythroid differentiation. Data from 1 of 4 independent experiments performed in replicates are shown as mean±Standard Deviation (SD). *PANOVA<0.05, **PANOVA<0.01. (B) Microscopic images (MGG staining; 1000x original magnification; scale bar 20 μm) of sorted neonatal CD34+ HSPCs on day 10 of combined megakaryocytic/erythroid differentiation. A representative mature erythroid cell is indicated by an arrow (left photograph), while characteristic immature erythroblast-like cells are indicated by an asterisk (middle and right photographs). (C) Representative flow cytometry plots (from 3 independent experiments), showing CD71+CD117−, CD71+CD117med and CD71+CD117+ neonatal HSPC-derived cells on day 10 of combined megakaryocytic/erythroid differentiation (pre-gated on live cells). Percentages of each population are indicated. (D) Percentage of neonatal CD71+CD117med cells on day 10 of combined megakaryocytic/erythroid differentiation. Data from 3 independent experiments are shown as mean±SD. **PANOVA<0.01. (E) Representative flow cytometry plots (from 2 independent experiments), depicting the CD41−CD235a− and CD41−CD235a+ cells within the neonatal CD71+CD117med population. Percentages of each population are indicated. (F) Percentage of the CD41−CD235a+ population. Data from replicates of 2 independent experiments are shown as mean±SD. *PANOVA<0.05.
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References

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