1. Tropomodulin3-null mice are embryonic lethal with anemia due to impaired erythroid terminal differentiation in the fetal liver
by Zhenhua Sui, Roberta B. Nowak, Andrea Bacconi, Nancy E. Kim, Hui Liu, Jie Li, Amittha Wickrema, Xiu-li An, and Velia M. Fowler
Blood
Volume 123(5):
January 30, 2014
©2014 by American Society of Hematology

2. Tmod1 expression is upregulated while Tmod3 is downregulated during erythroid differentiation.
Tmod1 expression is upregulated while Tmod3 is downregulated during erythroid differentiation. (A) mRNA levels of Tmods during in vitro erythroid differentiation from human CD34+ cells, determined by qRT-PCR on days 7, 10, and 14, normalized to ACTB mRNA. (B) Western blots of Tmod1 and Tmod3 proteins during in vitro erythroid differentiation of CD34+ cells. Cell pellets were collected on days 3, 6, 8, 10, 13, and 16 after initiation of differentiation, solubilized, and analyzed by western blotting (4.1R was used as positive control for erythroid differentiation, and total actin [C4 antibody] and glyceraldehyde-3-phosphate dehydrogenase proteins were used as loading controls). (C-D) qRT-PCR analysis of Tmod1 and Tmod3 mRNAs in highly pure erythroblasts at distinct developmental stages isolated by FACS from (C) in vitro CD34+cell-erythroid differentiation cultures, or (D) mouse bone marrow, normalized to ACTB mRNA. Stages were: proerythroblasts (Pro-E), early basophilic erythroblasts (early Baso), late basophilic erythroblasts (late Baso), basophilic erythroblasts (Baso-E), polychromatic erythroblasts (Poly-E), and orthochromatic erythroblasts (Ortho-E).
Zhenhua Sui et al. Blood 2014;123:
©2014 by American Society of Hematology

3. Targeted disruption of Tmod3 gene in mice.
Targeted disruption of Tmod3 gene in mice. (A) Tmod3 gene targeting strategy, depicting insertion site of BayGenomics β-geo gene-trap vector into intron 1 of Tmod3, as confirmed by DNA sequencing. Primers A, B, and C were designed for genotyping wild-type and mutant alleles, as shown. (B) PCR analysis of genomic DNA from mouse embryonic yolk sacs derived from Tmod3+/− intercrosses. Primers A and B detect the expected 729 bp band in the wild-type allele, while primers A and C detect the predicted 313 bp band in the mutant allele. (C) RT-PCR of Tmod3 transcripts in E14.5 fetal liver mRNA, using Tmod3 exon 1 and 2 primers. (D) Tmod mRNA levels in Tmod3+/+, Tmod3+/−, and Tmod3−/− fetal livers from E14.5 embryos, determined by qRT-PCR. (E) Western blot analysis of Tmods and erythroid membrane proteins bands 3, and 4.1R in Tmod3+/+, Tmod3+/−, and Tmod3−/− fetal livers from E14.5 embryos. Each lane is from a single embryo. Purified Tmod proteins (5 ng/lane) were loaded as controls for antibody specificity (hTmod1, Tmod1 from Homo sapiens; mTmod3 and mTmod4, Tmod3 and Tmod4 from Mus musculus; rTmod2, Tmod2 from Rattus norvegicus).43 (F) Calculation of Tmod1, Tmod3, bands 3, and 4.1R protein levels, normalized to glyceraldehyde-3-phosphate dehydrogenase. Band intensities were analyzed by ImageJ. Values in panels D and F are means ± SD from triplicate individual fetal livers from different embryos. **P < .005; ***P < β-geo, fusion of β-galactosidase and neomycin phosphotransferase II; En2, engrailed 2-splice acceptor sequence; PolyA, polyadenylation.
Zhenhua Sui et al. Blood 2014;123:
©2014 by American Society of Hematology

4. Tmod3−/− mice are embryonic lethal with anemia.
Tmod3−/−mice are embryonic lethal with anemia. (A) Viability of Tmod3−/− embryos. Timed matings of Tmod3+/− intercrosses were performed and embryos were harvested at stages E13.5 up to E18.5. The percentages reflect the numbers of live Tmod3−/− embryos with respect to all embryos harvested in litters at each gestational stage. (B) Gross morphology of Tmod3+/+ and Tmod3−/− embryos at E14.5. Scale bar, 1 mm. (C) Wright-Giemsa staining of peripheral blood cytospins from Tmod3+/+ and Tmod3−/− embryos at E14.5. Large primitive enucleated RBC (arrowhead); smaller definitive enucleated RBC (arrow). Note that sizes of enucleated cells from Tmod3−/− embryos are variable and do not fall into clear size categories. Scale bar, 20 µm. (D) The percentage of enucleated RBCs in peripheral blood cytospins from Tmod3+/+ and Tmod3−/− embryos at E13.5, E14.5, and E15.5. At least 300 cells were counted for each sample. Values are means ± SD (n = 5 to 13). ***P < (E) Gross morphology of representative fetal livers from Tmod3+/+ and Tmod3−/− embryos at E15.5. (F) Total number of fetal liver cells from Tmod3+/+ (n = 7), Tmod3+/− (n = 18), and Tmod3−/− (n = 5) embryos at E14.5. Values are means ± SD. ***P < (G) Hematoxylin and eosin staining of paraffin sections from Tmod3+/+ and Tmod3−/− fetal livers fixed in Bouin’s solution. Normal orthochromatic erythroblast nuclei in Tmod3+/+ fetal liver (arrowheads). Abnormal multilobular orthochromatic erythroblast nuclei in Tmod3−/− fetal liver (arrows). Scale bar, 10 µm (i-ii); 5 µm (iii-iv). Panels C and G were acquired with a Zeiss Axioskop microscope with AxioCam ICc3 color camera using a ×20 objective (N.A. 0.5) at a zoom of 2 (Gi-ii) or a ×100 oil-immersion objective (N.A. 1.3) at a zoom of 1 (C and Giii-iv).
Zhenhua Sui et al. Blood 2014;123:
©2014 by American Society of Hematology

5. Tmod3−/− embryos are defective in definitive erythropoiesis with reduced progenitors, impaired terminal differentiation, and enucleation.
Tmod3−/−embryos are defective in definitive erythropoiesis with reduced progenitors, impaired terminal differentiation, and enucleation. (A) BFU-E per fetal liver. (B) CFU-E per fetal liver. Values in both are means ± SD of at least 3 independent embryos. (C) Representative flow cytometry profiles of R1-R5 erythroblast populations labeled with CD71/Ter119 in fetal livers from Tmod3+/+ mice at E14.5. The percentage of cells in each R population with respect to total PI-negative cells is indicated for a representative Tmod3+/+ fetal liver. (D) Percentage of cells in each R population, normalized to the number of total viable fetal liver cells from each embryo (Tmod3+/+, n = 6; Tmod3+/−, n = 19; Tmod3−/−, n = 8). (E) Representative CD71/FSC profile of Tmod3+/+ Ter119hi cells sorted into 3 populations (S1, S2, and S3) according to cell size. The percentage of cells in each S population with respect to total Ter119hi cells is indicated for a representative Tmod3+/+ fetal liver. (F) Percentage of cells in each S population, normalized to the number of total Ter119hi fetal liver cells from each embryo. (G) Representative flow cytometry of enucleated cells in the S3 population of Tmod3+/+ fetal livers, using Syto-16 for nuclei and SytoX for cell viability. The percentage of enucleated and nucleated cells with respect to total S3 cells is indicated for a representative Tmod3+/+ fetal liver. (H) Percentages of enucleated cells in S1, S2, and S3 populations. All values are means ± SD with at least 3 replicates. *P < .05, **P < .005; ***P < .001 for Tmod3−/− vs Tmod3+/+.
Zhenhua Sui et al. Blood 2014;123:
©2014 by American Society of Hematology

6. Tmod3−/− fetal liver erythroblasts display increased apoptosis throughout terminal differentiation.
Tmod3−/−fetal liver erythroblasts display increased apoptosis throughout terminal differentiation. (A) Percentage of apoptotic cells based on Annexin V-positive cells as a proportion of total 7-AAD–negative fetal liver cells at E14.5. (B) Percentage of apoptotic cells within each R population. The 7-AAD–negative cells were first gated on CD71/Ter119 to identify R populations as in Figure 4C, and percentages for each R was calculated as the number of Annexin V-positive cells as a fraction of the total cell number in that R population. All values are means ± SD with at least 3 replicates with samples prepared from different individual embryos. *P < .05. (C) Cytospins of fetal liver cells stained with lamin B (red) for nuclear envelope, Ter119-Alexa488 (green) for a GPA-associated antigen to identify late erythroblasts, and Hoechst (blue) for nuclei. (D) Cytospins of fetal liver cells stained with active-caspase3 (red) for apoptosis, Ter119-Alexa488 (green), and Hoechst (blue). (E) Cytospins of fetal liver cells stained with Annexin V (green) for apoptosis, Ter119-PE (red), and Hoechst (blue). Panels C-E were acquired with a Zeiss LSM 780 laser scanning confocal fluorescence microscope using a Zeiss 100× oil-immersion objective (N.A. 1.4) at a zoom of 2. Scale bar, 4 µm.
Zhenhua Sui et al. Blood 2014;123:
©2014 by American Society of Hematology

7. Cell-cycle progression and actin cytoskeleton structures are impaired during erythropoiesis in Tmod3−/− fetal liver.
Cell-cycle progression and actin cytoskeleton structures are impaired during erythropoiesis in Tmod3−/−fetal liver. (A) Representative flow cytometry profiles of cell-cycle analysis of fetal liver cells at E14.5 after PI staining. (B) Percentages of fetal liver cells in sub-G1, G0/G1, S, and G2/M phase. (C) Percentages of Ter119hi fetal liver cells in sub-G1, G0/G1, S, and G2/M phase. All values are means ± SD with at least 3 replicates. **P < .005; ***P < .001 for Tmod3−/− vs Tmod3+/+. (D) Confocal fluorescence microscopy images of enucleating Tmod3+/+ or Tmod3−/− erythroblasts stained with Ter119-Alexa488 (green), Ki67 (red), and Hoechst (blue), and imaged in cytospins. Scale bar, 4 µm. (E) Percentages of enucleating Ki67+ and Ki67- erythroblasts in Tmod3+/+ or Tmod3−/− fetal livers. (F) Confocal fluorescence microscopy images of enucleating Tmod3+/+ (i-ii) and Tmod3−/− (iii-vii) erythroblasts, imaged in cytospins of fetal liver cells stained with Ter119-Alexa488 (green), rhodamine-phalloidin (red), and Hoechst (blue). Enucleating erythroblasts were identified in both genotypes by membrane sorting of Ter119 staining, and a nuclear constriction at the transition between the bright and dim Ter119 membrane staining. F-actin assembles into a contractile actin ring at the neck region in Tmod3+/+ enucleating erythroblasts and in bright foci in the cytoplasm of the incipient reticulocyte (i-ii). Tmod3−/− enucleating erythroblasts occasionally have an F-actin contractile ring (iii), but not always (iv-vii), and sometimes have F-actin enrichment on the dim Ter119 membrane overlying a protruding nuclear lobe (v-vi). Similar to wild-type, Tmod3−/− enucleating erythroblasts also contain F-actin foci in the cytoplasm (iii-vii). Scale bar, 6 µm. Panels D, Fiii-vii were acquired with a Zeiss LSM 780 laser scanning confocal microscope using a Zeiss 100× oil immersion objective (N.A. 1.4) at a zoom of 2, and panels Fi-ii with a Bio-Rad Radiance 2100 confocal microscope using a Zeiss 100× oil-immersion objective (N.A. 1.2) at a zoom of 3.
Zhenhua Sui et al. Blood 2014;123:
©2014 by American Society of Hematology

8. Tmod3 is required in both erythroblasts and macrophages for erythroblast-macrophage island formation in the fetal liver.
Tmod3 is required in both erythroblasts and macrophages for erythroblast-macrophage island formation in the fetal liver. (A) Representative images of native islands isolated from fetal livers at E14.5. Cells were stained with F4/80-Alexa647 (red) for macrophages, Ter119-Alexa488 (green) for erythroblasts, and Hoechst (blue) for nuclei. (B) Numbers of erythroblasts bound per macrophage at E13.5 and E14.5. Islands were prepared from 3 independent litters and 30 to 90 islands were counted for each genotype. (C) Representative images of reconstituted islands from Tmod3+/+ or Tmod3−/− erythroblasts (Ery) incubated with Tmod3−/− macrophages (Mac). (D) Numbers of erythroblasts bound per macrophage. For each cell combination, 3 independent litters with a total of 80 to 100 islands were counted (9.9 ± 0.7 Tmod3+/+ erythroblasts, but only 2.5 ± 0.2 Tmod3−/− erythroblasts bound to each wild-type macrophage). (E) Representative images of reconstituted islands from Tmod3+/+ erythroblasts (Ery) incubated with Tmod3+/+ or Tmod3−/− macrophages (Mac). (F) Numbers of erythroblasts bound per macrophage. For each cell combination, 3 independent litters with a total of 70 to 80 islands were counted; 14.6 ± 0.9 wild-type erythroblasts were attached to each Tmod3+/+ macrophage, while only 5.2 ± 0.5 were attached to each Tmod3−/− macrophage. All values are means ± SD. ***P < Panels A, C, and E were acquired using a Bio-Rad Radiance 2100 confocal microscope with a Zeiss 63× oil immersion objective (N.A. 1.4). Scale bar, 10 µm. ND, not determined.
Zhenhua Sui et al. Blood 2014;123:
©2014 by American Society of Hematology