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F. Yang et al.
Erythroid differentiation in vitro
E14.5 fetal liver cells were labeled with APC-conjugated anti-
mouse Ter119 antibody for 30 min followed by staining with anti- APC microbeads. Ter119- cells were purified and cultured in ery- throid-differentiation medium containing Iscove modified Dulbecco medium, 15% FBS, 1% detoxified bovine serum albu- min, 200 μg/mL recombinant human transferrin, 10 μg/mL recom- binant human insulin, 10-4 M β-mercaptoethanol, and 2.5 U/mL recombinant human EPO. After culturing the cells for 24 h, the medium was replaced with IMDM, 20% FBS and 10-4 M β-mer- captoethanol for another 24 h. Q-VD-OPh hydrate (#SML0063, Sigma) was added to the erythroid-differentiation medium for every 24 h.
Knockdown of TMEM30A by lentivirus in human CD34+ cells
Human CD34-positive (+) cells were purified and harvested from cord blood. The CD34+ cells were differentiated into ery- throid cells and the differentiation was assessed by flow cytome- try using GPA, Band 3 and α4 integrin as surface markers. pLKO1 vectors which express shRNA against the targeted gene of TMEM30A were purchased from Sigma-Aldrich. The sequences were as follows: sh-TMEM30A-1: GACAACCTGGAAGAAC- GATTT. sh-TMEM30A-2: GAGATTCTAGTGCTTTGCTTA. Lentivirus was prepared and transfected into CD34+ cells on the culture day 2, as described previously. Knockdown efficiency was checked by real-time polymerase chain reaction (RT-PCR). The primers for TMEM30A were: forward primer-5’-GCGATGAAC- TATAACGCGAAGG-3’; reverse primer-5’-GCCAATGCC- GATGGGAATGA-3’.
Statistical analysis
FACS analysis was performed using FlowJo software (BD, ver- sion 10). Statistical analysis was performed using GraphPad prism software (version 7). Band signal intensities were analyzed with ImageJ. The data were shown as the Mean±Standard Error of Mean (SEM). Differences among two groups were calculated by Student unpaired t-test. P<0.05 was considered statistically signif- icant.
Details of the other methods used in this study are available in the Online Supplementary Materials.
Results
Tmem30a deficient mice are embryonic lethal with anemia at mid gestation
To investigate the function of Tmem30a in hematopoiesis, we crossed Tmem30aflox/flox mice with B6.Cg-Commd10Tg(Vav1-icre)A2Kio mice to generate hematopoietic-specific Tmem30a-deficient mice (Online Supplementary Figure S1A and B). The Tmem30aflox/flox, VavCreTg/+ (Tmem30a cKO) mice were not viable. The surviving Tmem30a cKO embryos at embryonic day 14.5 (E14.5) are pale (Figure 1A), suggesting a defect in fetal hematopoiesis. Genotyping of the progeny embryos showed that the living embryos roughly followed the expected Mendelian ratio with 25% Tmem30a cKO embryos up to E12.5, but that ratio dropped to 0% by E16.5 (Figure 1B). The Tmem30a cKO fetal liver was noticeably smaller (Figure 1D), and the number of fetal liver cells was significantly reduced in the cKO mice com- pared to that of control mice at E14.5 (Figure 1C). Hematoxylin & Eosin staining of Tmem30a fetal liver sec- tions showed a severe atrophic phenotype (Figure 1E). Red
blood cell (RBC) count, hematocrits and hemoglobin lev- els were significantly decreased in the peripheral blood of the E14.5 Tmem30a cKO embryos compared with controls (Figure 1F-H). Wright-Giemsa staining of peripheral blood smears showed a large fraction of nucleated erythrocytes (Figure 1I) in Tmem30a cKO blood compared to control. To explore the reasons of the impaired hematopoiesis in Tmem30a-deficient embryos, we first analyzed the main- tenance of HSC in fetal livers by flow cytometry (Online Supplementary Figure S1C).27 The total number of HSC (Lin–Mac–1lowSac-1+ CD48–CD150+ cells) in cKO embryos was comparable to controls at E14.5 (Online Supplementary Figure S1D), indicating the impaired fetal hematopoiesis was not due to a defect in the FL HSC in cKO embryos, although Tmem30a is expressed in FL HSC as well as in T cells, B cells and erythroid cells (Online Supplementary Figure S1E). Interestingly, colony forming assay showed that the numbers of BFU-E and CFU-E colonies were dras- tically reduced in cKO embryos (Figure 1J and K). In con- trast, no differences were seen in the colonies of CFU-G, CFU-M and CFU-GM between cKO and control (data not shown). These findings suggest that loss of Tmem30a in mice resulted in a severe mid gestation anemia, likely due to impaired fetal liver erythropoiesis.
Tmem30a is necessary for definitive erythropoiesis in the fetal liver
Next, we investigated fetal liver erythroid progenitor cells in the fetal liver. The number of EryP in cKO fetal liv- ers was comparable with control (Online Supplementary Figure S2A and B). We also analyzed erythro-myeloid pro- genitors at embryo day 9.5 in the yolk sac by flow cytom- etry (Online Supplementary Figure S2C). The absolute num- ber of progenitors in cKO was comparable with the con- trol group (Online Supplementary Figure S2D). Finally, we analyzed definitive erythropoiesis using CD71 and Ter119 as surface markers (Figure 2A). The results showed that R3 erythroblast population (CD71hi/Ter119hi) was predominantly affected in the fetal livers of cKO embryos, suggesting a severe blockage in terminal erythroid differentiation from Ter119low to Ter119hi cells (Figure 2B). To further characterize terminal erythroid differentiation, we used FSC to separate Ter119hi cells into three populations: S1 (large), S2 (medi- um), and S3 (small) (Figure 2C). The number of erythrob- lasts in S1 and S2 populations (basophilic to orthochro- matic stages) and S3 population (reticulocytes) was decreased, suggesting a further defect at very late stage of erythroid maturation (Figure 2D). Next, we analyzed ery- throblast enucleation in S1-S3 populations using live-cell nuclear staining with Syto-16 (Figure 2E). Most enucle- ation events occurred in the S3 population, and Tmem30a cKO fetal livers showed a significant reduction of enucle- ating efficiency in this population (Figure 2F).
Tmem30a deficiency leads to apoptosis of fetal liver erythroid cells
To explore the underlying mechanisms, we examined proliferation and apoptosis of the fetal liver cells. BrdU incorporation assays showed that the cell cycle profiles were comparable between Tmem30a cKO and control mice (Online Supplementary Figure S3A and B). We then investigated apoptosis, which is usually assessed by Annexin V binding to exposed PS. Tmem30a deficiency lead to increased Annexin V levels (Figure 3A and B). Since
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haematologica | 2019; 104(10)