Figure 6.
Figure 6. Tmod1−/− FL erythroblasts differentiate normally but exhibit a slight enucleation defect and aberrant F-actin organization during polarization and enucleation. (A) Representative flow cytometry gating strategy of mouse FL cells from Tmod1+/+ and Tmod1−/− E14.5 embryos based on CD44 and Ter119 expression levels (left panel). High Ter119 cells are gated for CD44 and forward scatter (FSC) to reveal RII to RV populations (right panel). (B) Quantification of R populations from Tmod1+/+ and Tmod1−/− embryos. (C) Representative flow cytometry gating strategy of FL erythroblasts by forward scatter (FSC) and side scatter (SSC) identifies 2 populations of cells P1 and P2. (D) Enucleation in FL cells determined by Ter119 and Hoechst 33342 staining of P1 (left panel) or P2 population (right panel) to identify nucleated (HoechstHi) vs enucleated (HoechstLo) cells. (E) Quantification of the percent of enucleated cells in each population in FL cells from Tmod1+/+ and Tmod1−/− mouse embryos (N = 13 embryo FLs per group). The smaller P1 population of more mature cells shows impaired enucleation, whereas the larger P2 population representing larger more immature erythroid cells has negligible quantities of enucleated cells that do not change in the absence of Tmod1. (F) Extended focus projections of confocal Z-stacks of Tmod1−/− FL erythroblasts immunostained for Ter119, phalloidin for F-actin, and Hoechst for nuclei. Merges show Ter119 (green), F-actin (red), and Hoechst (blue). Arrows show mislocalized F-actin spots in polarized cells. Open arrowheads show abnormal F-actin accumulation at enucleating erythroblast neck. Protruding nuclear lobes show increased F-actin cables. Images of individual cells are representative of a total of 89 nucleated Ter119-stained erythroblasts, imaged in confocal Z stacks of FL cells obtained from 11 Tmod1−/− embryos (2 different litters). Of the 89 total Ter119-stained polarized and enucleating erythroblasts examined, 28 (∼31%) had abnormal F-actin (multiple foci in polarized cells, mislocalized F-actin spot near constriction, nonpolarized cells with large F-actin aggregates). Bars, 4 µm.

Tmod1−/−FL erythroblasts differentiate normally but exhibit a slight enucleation defect and aberrant F-actin organization during polarization and enucleation. (A) Representative flow cytometry gating strategy of mouse FL cells from Tmod1+/+ and Tmod1−/− E14.5 embryos based on CD44 and Ter119 expression levels (left panel). High Ter119 cells are gated for CD44 and forward scatter (FSC) to reveal RII to RV populations (right panel). (B) Quantification of R populations from Tmod1+/+ and Tmod1−/− embryos. (C) Representative flow cytometry gating strategy of FL erythroblasts by forward scatter (FSC) and side scatter (SSC) identifies 2 populations of cells P1 and P2. (D) Enucleation in FL cells determined by Ter119 and Hoechst 33342 staining of P1 (left panel) or P2 population (right panel) to identify nucleated (HoechstHi) vs enucleated (HoechstLo) cells. (E) Quantification of the percent of enucleated cells in each population in FL cells from Tmod1+/+ and Tmod1−/− mouse embryos (N = 13 embryo FLs per group). The smaller P1 population of more mature cells shows impaired enucleation, whereas the larger P2 population representing larger more immature erythroid cells has negligible quantities of enucleated cells that do not change in the absence of Tmod1. (F) Extended focus projections of confocal Z-stacks of Tmod1−/− FL erythroblasts immunostained for Ter119, phalloidin for F-actin, and Hoechst for nuclei. Merges show Ter119 (green), F-actin (red), and Hoechst (blue). Arrows show mislocalized F-actin spots in polarized cells. Open arrowheads show abnormal F-actin accumulation at enucleating erythroblast neck. Protruding nuclear lobes show increased F-actin cables. Images of individual cells are representative of a total of 89 nucleated Ter119-stained erythroblasts, imaged in confocal Z stacks of FL cells obtained from 11 Tmod1−/− embryos (2 different litters). Of the 89 total Ter119-stained polarized and enucleating erythroblasts examined, 28 (∼31%) had abnormal F-actin (multiple foci in polarized cells, mislocalized F-actin spot near constriction, nonpolarized cells with large F-actin aggregates). Bars, 4 µm.

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