Tmod3^−/− embryos show hemorrhages with abnormal platelets. (A) Representative images of whole Tmod3^+/+ (left) and Tmod3^−/− (right) embryos at E14.5. Bar, 1 mm. (B) Immunofluorescence staining for CD41 (gray) and Hoechst (blue) in cryosections of blood vessels of Tmod3^+/+ (left) and Tmod3^−/− (right) embryos, revealing circulating CD41⁺ platelets in situ. Bar, 10 µm. Images are single optical sections acquired using a Zeiss LSM 780 confocal laser scanning fluorescence microscope with a ×100 oil-immersion objective (N.A. 1.4), zoom 1. (C) Relative numbers of platelets in peripheral blood of Tmod3^+/+ and Tmod3^−/− E14.5 embryos. Tmod3^+/+, 63 ± 20 (n = 4 fields from 2 Tmod3^+/+ embryos); Tmod3^−/−, 30 ± 7 (n = 5 fields from 2 Tmod3^−/− embryos). *P < .05. (D) Representative images of Tmod3^+/+ (top) and Tmod3^−/− (bottom) platelets stained with CD41. Bar, 4 µm. Images are compressed Z stacks of optical sections acquired using a Zeiss LSM 780 confocal laser scanning fluorescence microscope with a ×100 oil-immersion objective (N.A. 1.4), zoom 3. (E) Platelet sizes in peripheral blood of Tmod3^+/+ and Tmod3^−/− embryos at E14.5, measured from images as in (B). Platelet diameters were determined from line scans across platelets using Volocity 6.3 software. Tmod3^−/− average platelet diameters were ∼×1.5 greater than Tmod3^+/+ platelets. Tmod3^+/+, 1.73 ± 0.46 µm (n = 114); Tmod3^−/−, 2.21 ± 0.76 µm (n = 98). ***P < .001. (F) Representative TEM images of Tmod3^+/+ and Tmod3^−/− platelets (left and middle panels; Bar, 1 µm), with high magnification views of circumferential microtubule rings (red arrows, right panel; Bar, 200 nm). Average WT platelet diameter in TEM images is ∼3 µm, similar to previous studies,¹⁰  but larger than measurements from fluorescence optical sections (B), which include measurements across the short and long axes of the asymmetric platelets (E).