Figure 2.
Hematopoietic-specific inactivation of Hippo kinases results in lethal bone marrow failure with features of del(20q) MDS. Genotypes analyzed for this figure include: Stk4f/fStk3f/f;Vav1-Cre+ (Stk4−/−Stk3−/−), Stk4f/+Stk3f/f; Vav1-Cre+ (Stk4+/−Stk3−/−), Stk4f/fStk3f/f;Vav1-Cre−, or Stk4f/+Stk3f/f;Vav1-Cre− (Stk4+/+Stk3+/+). All mice were analyzed in groups with littermates. Unless otherwise indicated, data are derived from mice of 6 to 9 weeks in age. (A) Western blot showing MST1 (Stk4), MST2 (Stk3), phosphorylated MOB1, and β-actin (loading control) proteins in Lin− hematopoietic cells. Bone-marrow– and spleen-derived mononuclear hematopoietic cells from 2 mice per genotype were pooled before Lin− isolation and protein lysate generation. (B) Kaplan-Meier survival plots for mice of indicated genotypes. (C) Representative image of 7-week-old, sex-matched littermates of mice of the indicated genotypes. (D) Weekly weight measurement (in grams) for multiple cohorts of mice between 3 and 7 weeks of age. n = 6 to 10 mice per genotype. Statistical significance was determined at week 7 by 1-way analysis of variance, followed by comparison against Stk4+/+Stk3+/+ values using the post hoc Tukey test. (E) Representative spleen images for 6- to 8-week-old mice of the indicated genetic backgrounds. (F) Representative hematoxylin and eosin (H&E)–stained bone marrow sections for 6- to 8-week-old mice. Scale bar, 200 μm. (G-J) Peripheral blood measurements for white blood cell (WBC) count (G), platelet count (H), red blood cell (RBC) count (I), and hemoglobin (J). Statistical significance was determined by 1-way ANOVA followed by the post hoc Tukey test for multiple comparisons. (K) Bone marrow frequencies of hematopoietic stem/progenitor populations as measured by flow cytometry in mice of the indicated genotypes. (L) The number of mature morphological megakaryocytes per ×20 magnification field in H&E-stained bone marrow sections. Data are derived from 5 representative sections each of 2 mice per genotype. Statistical significance was determined by 1-way ANOVA, followed by the post hoc Tukey test for multiple comparisons. (M) Frequency (%) of dysplastic megakaryocytes detected in representative H&E-stained bone marrow sections from 3 independent mice of the indicated genotypes. (N) Representative images of megakaryocytes observed in H&E-stained bone marrow sections in mice of the indicated genotypes. A representative dysplastic megakaryocyte with irregular nuclear morphology is indicated (right). Scale bar, 60 µm. Data are presented as mean values with error bars representing SEM and data points for individual mice. *P < .05, **P < .01, ***P < .001. LT-HSC, LSK+CD48−CD150+.

Hematopoietic-specific inactivation of Hippo kinases results in lethal bone marrow failure with features of del(20q) MDS. Genotypes analyzed for this figure include: Stk4f/fStk3f/f;Vav1-Cre+ (Stk4−/−Stk3−/−), Stk4f/+Stk3f/f; Vav1-Cre+ (Stk4+/−Stk3−/−), Stk4f/fStk3f/f;Vav1-Cre−, or Stk4f/+Stk3f/f;Vav1-Cre− (Stk4+/+Stk3+/+). All mice were analyzed in groups with littermates. Unless otherwise indicated, data are derived from mice of 6 to 9 weeks in age. (A) Western blot showing MST1 (Stk4), MST2 (Stk3), phosphorylated MOB1, and β-actin (loading control) proteins in Lin− hematopoietic cells. Bone-marrow– and spleen-derived mononuclear hematopoietic cells from 2 mice per genotype were pooled before Lin− isolation and protein lysate generation. (B) Kaplan-Meier survival plots for mice of indicated genotypes. (C) Representative image of 7-week-old, sex-matched littermates of mice of the indicated genotypes. (D) Weekly weight measurement (in grams) for multiple cohorts of mice between 3 and 7 weeks of age. n = 6 to 10 mice per genotype. Statistical significance was determined at week 7 by 1-way analysis of variance, followed by comparison against Stk4+/+Stk3+/+ values using the post hoc Tukey test. (E) Representative spleen images for 6- to 8-week-old mice of the indicated genetic backgrounds. (F) Representative hematoxylin and eosin (H&E)–stained bone marrow sections for 6- to 8-week-old mice. Scale bar, 200 μm. (G-J) Peripheral blood measurements for white blood cell (WBC) count (G), platelet count (H), red blood cell (RBC) count (I), and hemoglobin (J). Statistical significance was determined by 1-way ANOVA followed by the post hoc Tukey test for multiple comparisons. (K) Bone marrow frequencies of hematopoietic stem/progenitor populations as measured by flow cytometry in mice of the indicated genotypes. (L) The number of mature morphological megakaryocytes per ×20 magnification field in H&E-stained bone marrow sections. Data are derived from 5 representative sections each of 2 mice per genotype. Statistical significance was determined by 1-way ANOVA, followed by the post hoc Tukey test for multiple comparisons. (M) Frequency (%) of dysplastic megakaryocytes detected in representative H&E-stained bone marrow sections from 3 independent mice of the indicated genotypes. (N) Representative images of megakaryocytes observed in H&E-stained bone marrow sections in mice of the indicated genotypes. A representative dysplastic megakaryocyte with irregular nuclear morphology is indicated (right). Scale bar, 60 µm. Data are presented as mean values with error bars representing SEM and data points for individual mice. *P < .05, **P < .01, ***P < .001. LT-HSC, LSK+CD48−CD150+.

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