Increased active Rap1 in Rasa3^−/− megakaryocytes.

<p>Fetal liver cells (FLC) were isolated from E12.5 Rasa3^+/+ and Rasa3^−/− embryos and cultured with TPO. <b>A.</b> Non adherent megakaryocytes on day 6 were analyzed for active, GTP-bound Rap1 by immunofluorescence using GST-RalGDS-RBD and a FITC-conjugated mAb against GST. 3D reconstruction of Rasa3^+/+ (left panels) and Rasa3^−/− (right panels) megakaryocytes, along with the pseudocolor fluorescence intensity scale. The graph represents the intensity of active Rap1 staining, expressed in arbitrary units (A. U.), in Rasa3^+/+ and Rasa3^−/− megakaryocytes. Two independent experiments (quantification of 50 megakaryocytes per experiment,) with mean ± SEM are presented. Statistics (unpaired <i>t</i> test): *: P<0.05 <b>B. and C.</b> Addition of the Rap1 GGTI-298 inhibitor to the culture medium (3 µM) abolished the abnormal adherent phenotype of Rasa3^−/− megakaryocytes. Fetal liver cell (FLC) were isolated from E12.5 Rasa3^+/+ and Rasa3^−/− embryos and cultured with TPO for 3 days. Megakaryocytes were enriched on a BSA-gradient and incubated for 18 hours on Poly-D-Lysine- (PDL) coated plates in medium containing 10% FBS, in the presence or absence of GGTI-298. <b>B.</b> Representative confocal image of Rasa3^+/+ and Rasa3^−/− PDL-adherent megakaryocytes after staining with CD41-APC (green) and DAPI (blue). Scale bar: 500 µm. <b>C.</b> The number of Rasa3^+/+ and Rasa3^−/− adherent megakaryocytes was significantly decreased in GGTI-298 treated cells. Graph represents 2 independent experiments (mean ± SEM). Statistics (unpaired <i>t</i> test): **: P<0.01; ***: P<0.001.</p

Abnormal actin cytoskeleton organization in megakaryocytes derived from Rasa3^−/− fetal liver cell culture.

<p>Fetal liver cell (FLC) were isolated from E12.5 Rasa3^+/+ and Rasa3^−/− embryos and cultured for 6 days with TPO. <b>A</b>. Rasa3^+/+ (left bottom and top panels) and Rasa3^−/− (right bottom and top panels) adherent megakaryocytes in FLC culture after staining with phalloidin-TRITC (actin, red) and DAPI (green). Confocal images were obtained at the bottom and the top of the same megakaryocyte. Graphs represent the fluorescence intensity (FI, scaled from 0 to 256) of phalloidin (actin) and DAPI stainings along the indicated line. The arrowheads on the images indicated the origin of the line. Twenty megakaryocytes were analyzed in duplicate per FLC culture, 6 Rasa^+/+ and 5 Rasa3^−/− FLC cultures. Representative images are shown. <b>B</b>. Quantification of the percentage of adherent Rasa3^+/+ and Rasa3^−/− megakaryocytes with a dotted (ie when F-actin is not detected) or a F-actin (ie when stress fiber can be detected) phenotype (mean ± SEM). Fifty megakaryocytes were analyzed in duplicate per FLC culture, 12 Rasa3^+/+ and 10 Rasa3^−/−. Statistics (unpaired <i>t</i> test): ***: P<0.001.</p

Altered SCID-Rasa3^−/− megakaryocyte motility, adhesion and differentiation into proplatelet forming megakaryocytes.

<p>Femur bone marrow explants were isolated from SCID-Rasa3^+/+ and SCID-Rasa3^−/− mice 2 months after irradiation/reconstitution and cultured under a confocal microscope. Images were taken in bright field every 10 min. <b>A.</b> Number of megakaryocytes released from the explants (left panel), of spreading megakaryocytes (center panel) and of proplatelet forming megakaryocytes (right panel). Results are representative of 3 independent experiments. <b>B.</b> Example of megakaryocytes released from a SCID-Rasa3^+/+ (upper panels) and a SCID-Rasa3^−/− (lower panels) explant. The asterisk indicates the same megakaryocyte that finally develops into a proplatelet forming megakaryocyte (SCID-Rasa3^+/+ explants) or that continuously adheres to the culture plate and fail to form proplatelets (SCID-Rasa3^−/− explants). Insets: higher magnification shows the proplatelet forming megakaryocyte in the SCID-Rasa3^+/+ explants and the adherent megakaryocyte in the SCID-Rasa3^−/− explants. Scale bars: 50 µm. <b>C.</b> Velocity and linear distance covered by individual megakaryocytes 3 hours after release from SCID-Rasa3^+/+ and SCID-Rasa3^−/− explants. Statistics (unpaired <i>t</i> test): *: P<0.05; ***: P<0.001.</p

Decreased survival, splenomegaly, thrombocytopenia and megakaryocyte alterations in SCID-Rasa3^−/− mice.

<p><b>A.</b> Survival of irradiated SCID mice reconstituted with Rasa3^+/+ (SCID-R3^+/+, n = 28 mice), Rasa3^+/− (SCID-R3^+/−, n = 31 mice) or Rasa3^−/− (SCID-R3^−/−, n = 32 mice) embryonic liver cells. <b>B.</b> Spleen weight from age-matched SCID-Rasa3^+/+ and moribund SCID-Rasa3^−/− mice. The mean ± SEM are also presented in each group of mice. A splenomegaly was defined as a spleen weight over 0.168 g (i.e. twice the mean spleen weight of SCID-Rasa3^+/+ mice). <b>C.</b> Blood platelet counts in age-matched SCID-Rasa3^+/+ (black column, n = 9) and moribund SCID-Rasa3^−/− (white column, n = 15) mice. Results represent the mean ± SEM of platelets per µl of blood. <b>D.</b>Mean ± SEM of CD41⁺ megakaryocyte (MK) percentages detected by flow cytometry in the bone marrow isolated from age-matched SCID-Rasa3^+/+ (black column, n = 17) and moribund SCID-Rasa3^−/− (white column, n = 20) femurs. <b>E.</b> vWF-stained bone marrow sections of SCID-Rasa3^+/+ and SCID-Rasa3^−/− femurs 3 months after SCID mice reconstitution. V: vessel; *: megakaryocyte; arrowheads: abnormal vWF deposits. Scale bars: 50 µm. <b>F.</b> Quantification of megakaryocytes (MKs) in the osteoblastic and the vascular niches of SCID-Rasa3^+/+ (black columns) and SCID-Rasa3^−/− (white columns) bone marrow femurs 3 months after irradiation/reconstitution. Results represent the mean ± SEM of the number of megakaryocytes per unit of osteoblastic border, or per vessel. Statistics (unpaired <i>t</i> test): *: P<0.05; **: P<0.01; ***: P<0.001.</p

Abnormal megakaryocyte differentiation from Rasa3^−/− fetal liver cell culture.

<p>Fetal liver cell (FLC) were isolated from E12.5 Rasa3^+/+ and Rasa3^−/− embryos. <b>A</b>. Percentages of CD41⁺ megakaryocytes were determined at day 0 and 2 days after TPO treatment in 6 Rasa3^+/+ and 8 Rasa3^−/− FLC cultures by flow cytometry. Results are expressed as fold of increase of CD41⁺ cells, considering the percentage of Rasa3^+/+ CD41⁺ cells at day 0 as 1. Statistics (unpaired <i>t</i> test): **: P<0.01. <b>B</b>. Increased megakaryocyte ploidy in FLC culture at day 2 after TPO treatment. Representative images of DNA content in Rasa3^+/+ and Rasa3^−/− CD41⁺ megakaryocytes. The table shows a quantification of the percentages of individual ploidy classes of FLC-derived CD41⁺ megakaryocytes (mean ± SEM). Statistics (unpaired <i>t</i> test, n = 6): *: P<0.013; ***: P<0.009. <b>C</b>. CFU-Mk assay from Rasa3^+/+ and Rasa3^−/− FLC. Representative images of a Rasa3^+/+ CFU-Mk with immature megakaryocytes of about 10 µm of diameter, and of Rasa3^−/− mature megakaryocytes of about 30 µm of diameter (arrowheads). Scale bars: 50 µm. The graph represents the number of CFU-Mk formed by 5 Rasa3^+/+ and 3 Rasa3^−/− independent FLC cultures after 3 days (mean ± SEM). Statistics (unpaired <i>t</i> test): **: P<0.01. <b>D.</b> Representative images of FLC Rasa3^+/+ (upper panels) and Rasa3^−/− (lower panels) cultures after 6 days with TPO. A digital magnification (3×) of a proplatelet event in Rasa3^+/+ FLC culture (right upper panel) and of an abnormal adherent megakaryocyte in Rasa3^−/− FLC culture (right lower panel) are presented. In Rasa3^+/+ FLC culture, 8.4±2.2% of megakaryocytes formed proplatelets. No proplatelet forming megakaryocyte was detected in Rasa3^−/− FLC culture. Scale bars: 50 µm. <b>E.</b> Rasa3^+/+ (upper panels) and Rasa3^−/− (lower panels) FLC cultures after 6 days with TPO were stained with a CD41 antibody (green, left panels) or CD41 and DAPI (green and blue, respectively; right panels). The large abnormal adherent cell population detected in Rasa3^−/− FLC culture is CD41-positive. In Rasa3^+/+ FLC culture, 8.4±2.2% of megakaryocytes formed proplatelets. No proplatelet forming megakaryocyte was detected in Rasa3^−/− FLC culture. Scale bars: 50 µm.</p

SCID-Rasa3^−/− mice develop a CD117⁺ CD38⁺ Sca-1⁺ cell preleukemia. A.

<p>Representative images of a hematoxylin/eosin-stained section of a femur from a SCID-Rasa3^+/+ mouse and one of the four SCID-Rasa3^−/− mice with a homogeneous cellular infiltration of the bone marrow and the spleen (upper panels: magnification: ×20; lower panels: magnification: ×100). <b>B</b>. CD117⁺ splenocyte percentages in SCID-Rasa3^+/+ (n = 10) and in the four SCID-Rasa3^−/− mice with a preleukemia. Statistics (unpaired <i>t</i> test): ***: P<0.001. <b>C</b>. Representative flow cytometry analysis of bone marrow cells from a SCID-Rasa3^+/+ mouse (left histogram) and one of the four SCID-Rasa3^−/− (right histogram) mice with a preleukemia, using a CD117 antibody. The histograms show the CD117 fluorescence intensity and the relative number of cells (events). <b>D</b>. Fetal liver cells from Rasa3^+/+ and Rasa3^−/− E12.5 embryos were stained with a CD117 antibody and analyzed for active GTP-bound Ras level by immunofluorescence using GST-Raf1-RBD and a FITC-conjugated mAb against GST. The graph represents the intensity of active GTP-bound Ras staining, expressed in arbitrary units (A. U.), in Rasa3^+/+ and Rasa3^−/− CD117⁺ HSC. Mean ± SEM are presented.</p

Altered inside-out and outside-in integrin signaling in Rasa3^−/− megakaryocytes.

<p>Fetal liver cell (FLC) were isolated from E12.5 Rasa3^+/+ and Rasa3^−/− embryos and cultured with TPO for 3 days. Megakaryocytes were enriched on a BSA-gradient, deprived of serum for 4 hours and used in inside-out and outside-in integrin signaling assays. <b>A.</b> Inside-out αIIbβ3 integrin signaling was investigated in megakaryocytes by quantifying soluble FITC-fibrinogen (FITC-FNG) bound to the CD41⁺ cell surface by flow cytometry. Megakaryocytes were treated with or without 100 ng/ml TPO for 30 min. Specific binding was obtained after subtraction of the amount of soluble fibrinogen bound to the cell surface in the presence of EDTA and was expressed relative to the maximum binding obtained in the presence of MnCl₂. A. U.: arbitrary units. n. s.: non stimulated. <b>B.</b>, <b>C.</b>, <b>D.</b> and <b>E.</b> Megakaryocytes were incubated for 18 hours on Poly-D-Lysine- (PDL), collagen-I- (COL-I) and fibrinogen- (FNG) coated plates in medium containing 10% FBS. Number (<b>B</b>, mean ± SEM) and diameter (<b>C</b>) of adherent megakaryocytes (MKs) was determined in 16 fields. Results are representative of 3 independent experiments. <b>D</b>. The percentage of Rasa3^−/− adherent megakaryocytes with a diameter over 50 µm was significantly increased, as compared with Rasa3^+/+ megakaryocytes (mean ± SEM of 3 independent experiments). <b>E.</b> Rasa3^+/+ and Rasa3^−/− PDL-adherent megakaryocytes were stained with phalloidin-TRICT (actin, red), CD41-APC (magenta) and Talin-FITC (green). Confocal images were obtained from the bottom of the cells. (i–iv): 4× Digital magnification of phalloidin-TRICT and Talin-FITC merge. An increased Talin staining is observed in Rasa3^−/− megakaryocytes (iii and iv), as compared with Rasa3^+/+ megakaryocytes (i and ii). Scale bar: 50 µm. Statistics (unpaired <i>t</i> test): *: P<0.05; **: P<0.01; ***: P<0.001.</p

Rasa3 Controls Megakaryocyte Rap1 Activation, Integrin Signaling and Differentiation into Proplatelet

Rasa3 is a GTPase activating protein of the GAP1 family which targets Ras and Rap1. Ubiquitous Rasa3 catalytic inactivation in mouse results in early embryonic lethality. Here, we show that Rasa3 catalytic inactivation in mouse hematopoietic cells results in a lethal syndrome characterized by severe defects during megakaryopoiesis, thrombocytopenia and a predisposition to develop preleukemia. The main objective of this study was to define the cellular and the molecular mechanisms of terminal megakaryopoiesis alterations. We found that Rasa3 catalytic inactivation altered megakaryocyte development, adherence, migration, actin cytoskeleton organization and differentiation into proplatelet forming megakaryocytes. These megakaryocyte alterations were associated with an increased active Rap1 level and a constitutive integrin activation. Thus, these mice presented a severe thrombocytopenia, bleeding and anemia associated with an increased percentage of megakaryocytes in the bone marrow, bone marrow fibrosis, extramedular hematopoiesis, splenomegaly and premature death. Altogether, our results indicate that Rasa3 catalytic activity controls Rap1 activation and integrin signaling during megakaryocyte differentiation in mouse