Figure 6
Figure 6. 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.

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.

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