Defective erythroid maturation in gelsolin mutant mice

Abstract

Background: During late differentiation, erythroid cells undergo profound changes involving actin filament remodeling. One of the proteins controlling actin dynamics is gelsolin, a calcium-activated actin filament severing and capping protein. Gelsolin-null (Gsn(-/-)) mice generated in a C57BL/6 background are viable and fertile.1

Design and methods: We analyzed the functional roles of gelsolin in erythropoiesis by: (i) evaluating gelsolin expression in murine fetal liver cells at different stages of erythroid differentiation (using reverse transcription polymerase chain reaction analysis and immunohistochemistry), and (ii) characterizing embryonic and adult erythropoiesis in Gsn(-/-) BALB/c mice (morphology and erythroid cultures).

Results: In the context of a BALB/c background, the Gsn(-/-) mutation causes embryonic death. Gsn(-/-) embryos show defective erythroid maturation with persistence of circulating nucleated cells. The few Gsn(-/-) mice reaching adulthood fail to recover from phenylhydrazine-induced acute anemia, revealing an impaired response to stress erythropoiesis. In in vitro differentiation assays, E13.5 fetal liver Gsn(-/-) cells failed to undergo terminal maturation, a defect partially rescued by Cytochalasin D, and mimicked by administration of Jasplakinolide to the wild-type control samples.

Conclusions: In BALB/c mice, gelsolin deficiency alters the equilibrium between erythrocyte actin polymerization and depolymerization, causing impaired terminal maturation. We suggest a non-redundant role for gelsolin in terminal erythroid differentiation, possibly contributing to the Gsn(-/-) mice lethality observed in mid-gestation.

Figure 1.

Characterization of gelsolin expression during erythroid maturation. (A) FACS analysis of E13.5 mouse fetal liver cells for expression of c-Kit and Ter119 cell antigens. The three major cell populations are indicated. (B) Real-time PCR on Gsn, GpA and CD44 expression on cDNA from the sorted fetal liver cell populations indicated in (A) FL: cDNA from total fetal liver cells. Histograms show the levels of expression (mean SEM of at least 3 independent experiments) relative to hypoxanthine-phospho-ribosyltransferase (HPRT) considered as 1. Statistical significance is indicated above the chart. (C) Fetal liver cells cells were stained with O-dianosidine (bright orange) and counterstained with hematoxylin/eosin. Immunofluorescence analysis using an anti-Gsn antibody (green), an anti Ter119 antibody (red) and DAPI (blue). Bar: 25 μm. (D) The same analysis was carried out on FACS sorted cell populations. Single cells are shown as an example. Bars: 4 μm.

Figure 2.

Gelsolin null BALB/c mice show defects in embryonic red blood cells. (A-H) Hematoxylin/eosin staining of blood from wt (A, C, E) and Gsn^−/− (B, D, F, G, H) mice embryos showed no gross differences at E13.5 of gestation (panel A-B) and at E.14.5 of gestation (C-D). Some smaller nucleated cells, resembling immature definitive erythroid cells are present (blue arrows, D). At E17.5 of gestation (E-F) mutant (but not wild type) embryos showed a consistent number (about 2.5%) of nucleated definitive erythroid cells in the circulation, together with some primitive nucleated erythroid cells still present in both blood and fetal liver sections (black arrows, G-H). Bars: 24 μm (A-F); 12 μm (G, H).

Figure 3.

Increased number of circulating nucleated cells in Gsn^−/− mice at E17.5. Cytospins from E17.5 wt (A, C, E, G) and Gsn^−/− (B, D, F, H, I) blood samples were stained with Giemsa (A-D) or DAPI (E-F), Alexa Fluor 488–phalloidin (G, H) and βH1 (I). Bars: 20 μm (A, B, E, F); 10 μm (C, D, G, H);(I) 15 μm. (A-D) Blue arrows indicate nucleated cells while green arrows point to binucleated cells; (E, F) DAPI staining emphasizes the increased proportion of nucleated cells in blood samples from E17.5 Gsn^−/− embryos; (G, H) white arrows evidence the contractile actin ring typical of cells undergoing enucleation; (I) red arrows indicate βH1⁺ cells, black arrows indicate βH1⁻ nucleated cells and the blue arrow indicates a nucleated cell weakly positive for βH1 staining.

Figure 4.

(A) FACS analysis on blood cells from E13.5 wt and Gsn^−/−embryos with anti-CD71 and anti-Ter119 antibodies. Three major distinct populations of progressively differentiating cells are present in the circulation: R1: CD71⁺Ter119⁺; R2: CD71⁺Ter119⁺⁺; R3: CD71⁻ Ter119⁺⁺. (B) Hematoxylin/eosin staining of the same samples as in (A) Bar: 10 μm.

Figure 5.

Adult Gsn^−/− mice are unable to recover from PHZ-induced anemia: (A) Morphological analysis of blood from adult Gsn^−/− mice showed the presence of knizocytes. Bar: 5 μm. (B) Osmotic lysis test. X axis: NaCl concentration; Y axis: amount of proteins released upon osmotic lysis, measured as absorbance at 595 nm (Bradford method). (C) The mean weight of the spleen is increased in Gsn^−/− mice under PHZ-induced erythropoietic stress. (D) Morphological analysis of spleen sections from wt and Gsn^−/− mice. Bar: 200 μm. (E) Spleen cells isolated from wt and Gsn^−/− mice and stained with anti CD71-FITC and antiTer119-PE antibodies for FACS analysis. The panel shows dot plots of wt and Gsn^−/− mice. (F) Percentage of positive cells within the four major distinct populations of progressively differentiating cells (CD71⁺⁺Ter119-, CD71⁺⁺ Ter119⁺⁺, CD71⁺Ter119⁺⁺ and CD71⁻ Ter119⁺⁺ cells) were evaluated on total spleen cells. Histograms represent the mean of three wt and three Gsn^−/− mice; significance levels were calculated by a t test for unpaired data.

Figure 6.

Ex vivo Gsn^−/− erythroblasts fail to differentiate properly in hanging drop cultures. (A, B) Fetal liver cells isolated from wt and Gsn^−/− - mice are disaggregated, stained with O-dianosidine (brown) and counter-stained with hematoxylin/eosin. (C, D) At 24 h after cell seeding, a significant proportion of hemoglobinized cells (brown staining) undergoing enucleation (black arrows) or already enucleated (green arrows) was present in wt cultures (C), in contrast with the sharp prevalence of immature cells present in the Gsn^−/− cells (D). (E-H) At 48 h, massive enucleation took place in wt cultures (E, G), whereas the majority of Gsn^−/− derived cells (F, H) became hemoglobinized but failed to undergo proper enucleation. Pyrenocytes are indicated by red arrows (F, H). (I) Relative proportion of cells at different stages of differentiation (> 100 cells scored in 3 independent fields). (L) The addition of Jasplakinolide (JspK) or Cytochalasin D (CytD) to wt cells mimics the accumulation of polynucleated cells and the delay in maturation observed in Gsn^−/− samples. (M, N) Representative fields of wt cells treated with JspK (left) or CytD (right). Bars: 15 μm (A-F and M-N); Bar: 7 μm (G-H).