Hoxa10 null animals exhibit reduced platelet biogenesis

Abstract

The transcription factor HOXA10 is an important regulator of myelopoiesis. Engineered over-expression of Hoxa10 in mice results in a myeloproliferative disorder that progresses to acute myeloid leukaemia (AML) over time, and in humans over-expression is associated with poor outcomes in AML. Here, we report that loss of Hoxa10 expression in mice results in reduced platelet count and platelet production, but does not affect clotting efficiency. About 40% fewer platelets were found in Hoxa10 null animals in comparison to wild type littermates. We found a nearly 50% reduction in the percentage of reticulated platelets in Hoxa10 null mice, suggesting deficient platelet production. Furthermore, Hoxa10 null animals recovered less efficiently from induced thrombocytopenia, supporting our hypothesis of defective platelet production. This also correlated with reduced colony formation potential of stem and progenitor cells seeded in megakaryocyte-enhancing conditions in vitro. Together, our results indicate that HOXA10 is important for megakaryopoiesis and platelet biogenesis.

Keywords: HOXA10; Hoxa10; Platelets; in vivo; megakaryopoiesis; thrombopoiesis.

Fig 1

Platelet counts in wild type, Hoxa10^+/− and Hoxa10 null mice. Box plots represent median and quartiles of platelet counts, whiskers represent the 95% confidence interval and black dots represent outliers. (A) Platelet counts between genotype cohorts (n=18). Wild type animals exhibited higher platelet counts than Hoxa10^+/− mice (T-test ^(a)p=0.0007) and Hoxa10 null animals (T-test ^(b)p=0.0005). Platelet reduction between Hoxa10^+/− and Hoxa10 null was also significant (T-test ^(c)p=0.03). (B) Platelet counts represented by sex (n=9 males and n=9 females). Females tended to exhibit lower platelet counts in all genotypes. Statistical difference was observed between male and female of Hoxa10^+/− genotype (T-test ^(e)p=0.011), males of wild type vs. Hoxa10 null (T-test ^(d)p=0.017) and females of wild type vs. Hoxa10 null (T-test ^(f) p<0.001). (C) Total platelet volume of animals in the 3 experimental groups (n=18). There was a significant reduction in total platelet volume when comparing Hoxa10 null samples to wild type (^(h)p<0.001) but not Hoxa10^+/− (⁽ⁱ⁾p=0.06), and in Hoxa10^+/− samples in comparison to wild type (^(g)p=0.002). (D) Percentage of thiazole-positive reticulated platelets. Hoxa10 null animals exhibited a reticulated platelet percentage nearly 50% lower than for wild type (^(k)T-test p=0.003) and Hoxa10 ^+/− (^(l)T-test p=0.002) animals, while the difference between wild type and Hoxa10^+/− was not significant (^(j)T-test p=0.45). Values represent the mean ± SEM for n=9.

Fig 2

Recovery from anti-platelet-serum-induced thrombocytopenia in mice. Time course carried out by sampling mouse blood daily for 7 days. Six mice at least 30 weeks of age were randomly selected for wild type, Hoxa10^+/− and Hoxa10 null cohorts. (A) Platelet levels obtained immediately before and daily after thrombocytopenia induction. Wild type mice are represented by black diamonds, Hoxa10^+/− animals by grey squares and Hoxa10 null animals by white triangles. Prior to treatment, wild type mice exhibited significantly higher levels of platelets in comparison to Hoxa10^+/− and Hoxa10 null animals (^*ANOVA p ≤ 0.002, ^*paired T-test p ≤ 0.005 wild type vs. Hoxa10^+/− or Hoxa10 null). Recovery of platelet pool at the end of the time course was most effective in wild type animals (^#ANOVA p ≤ 0.02, ^#paired T-test p ≤ 0.05 wild type vs. Hoxa10^+/− or Hoxa10 null). (B) The average slope of platelet recovery was calculated by taking the average platelet counts for days 2–5 and fitting a regression curve. The regression line for each mouse cohort has an R² ≥0.98. (C) Platelet reticulation spikes at day 1 after induced thrombocytopenia. The percentage of reticulated platelets was significantly different prior to treatment on day 0 (* ANOVA p ≤ 0.001, paired T-test p ≤0.02) for all cohorts. The most significant difference in new platelet production occurred on day 1, with Hoxa10^+/− and Hoxa10 null mice exhibiting a significantly lower percentage of reticulated platelets in comparison to wild type littermates (# ANOVA p ≤ 0.016, paired T-test p ≤0.03). Values represent the mean ± SEM (n=6).

Fig 3

Tail bleed assay and platelet function. Ten mice of each genotype were subjected to tail injury. (A) Bleeding time length was determined by tracking the amount of time it took for clots to form. The assay was discontinued and bleeding was stopped by pressure if bleeding time was longer than 10 minutes. (B) Blood volume loss was determined by weighing the amount of blood collected during the assay and by factoring in the volume of blood gained in the collection tube and measuring the absorbance at an optical density (OD) of 560 nm.

Fig 4

Reduced platelet counts in Hoxa10 null mice may be attributed to lower Mk progenitor proliferation potential. (A) CD41 expression among mononuclear cells freshly harvested from bone marrow for in vitro cultures. Data represents mean ± SEM for n=3. (B) Representative ploidy distributions of bone marrow megakaryocytes for each genotype. The bar graph represents the fraction of high-ploidy bone marrow Mks harvested from mice in the 3 experimental groups. Data represents mean ± SEM for n=6. (C) Total number of colonies in CFU-Mk assay. Data represents mean ± SEM for n=3. Small (3 Mks), medium (4–8 Mks) and large (≥9 Mks) colonies were enumerated and combined. (D) Percentage of medium and large colonies with respect to total colony counts. Larger colonies represent more primitive Mk progenitors and better expansion potential. Statistically significant reduction in the percentage of medium plus large colonies formed was observed for Hoxa10 null lineage-depleted cells (*T-test p=0.02). Data represent mean ± SEM for n=2.

Fig 5

Hoxa10 null mice exhibit increased cortical and trabecular bone area in sternum sections. (A-D) Haematoxylin and eosin stained sternum sections. (A and C) Representative images of wild type mouse sterna at 4x and 20x, respectively. (B and D) Representative images of Hoxa10 null mouse sterna at 4x and 20x, respectively. Images were taken at 4x magnification for 3 mice per genotype with 4 viewing areas per mouse. ImageJ was used to perform area measurements of the bone area (B.Ar) and the tissue area (T.Ar, which includes the bone area) of these two-dimensional images. Scale bar = 500 μm in (A, B) and 100 μm in (C, D). (E) Hoxa10 null sternum sections have increased B.Ar/T.Ar compared to wild type (T-test p=0.03). Values represent mean ± SD.