Additional Supporting Information may be found in the online version of this article.

DVDY_23851_sm_SuppFigS1.tif1124KSupporting Information Figure 1. Preimplantation development of parthenote embryos. A: Representative pictures of normal fertilized (control) and parthenote embryos at different developmental stages between E1.5 and E5.5. At E3.5, arrowheads indicate early blastocysts, and the others are late morulae. Scale bars in A = 50 μm. B: Distributions of developmental stages of embryos at embryonic days E3.5, E4.5, and E5.5. The data values are also included in Table S1.
DVDY_23851_sm_SuppFigS2.tif3400KSupporting Information Figure 2. Levels of Cdx2 and Oct4 and percentages of inner cell mass cells in parthenote blastocysts. A, B: Immunofluorescence images of control and parthenote embryos stained for Oct4 (red), Cdx2 (green), and DAPI (blue) at the early (E3.5) (A) and late blastocyst (E4.5) (B) stages. Note the extensive expression of Oct4 in the trophectoderm (TE) of both control and parthenote early blastocysts (A), which is colocalized with Cdx2 expression (yellow color in A, indicated by arrows). Oct4 expression is restricted to the inner cell mass (ICM) of both control and parthenote late blastocysts (B) and is no longer colocalized with Cdx2 expression (the yellow color in B resulted from overlapping of green Cdx2 immunofluorescence and red Oct4 immunofluorescence on distinct cell layers on serial optical sections). Scale bars in A and B = 20 μm. C: Reduced ICM cell numbers in parthenote embryos (asterisks) compared to controls at both the early and late blastocyst stages (n = 30 for each stage, P < 0.01, Student's t-test). D: Comparable percentages of ICM cells in whole embryos between controls and parthenotes at both the early and late blastocyst stages (n = 30 for each stage, P = 0.15, Student's t-test).
DVDY_23851_sm_SuppFigS3.tif910KSupporting Information Figure 3. Colocalization of Nanog and Sox2. Immunofluorescence images of control and parthenote embryos stained for DAPI (blue), Nanog (red), and Sox2 (green) at the morula (E3.0) (n = 26) (A) and late blastocyst (E4.5) (n = 20) (B) stages. Arrows indicate colocalized Nanog+ and Sox2+ cells in parthenotes. Scale bars in A and B = 20 μm.
DVDY_23851_sm_SuppFigS4.tif1855KSupporting Information Figure 4. Expression of Fgf4 and Fgfr2 in parthenote embryos. A–D: Immunofluorescence images of control and parthenote embryos stained for DAPI (blue), Cdx2 (green), and Fgf4 (red) (A, B) or Fgfr2 (red) (C, D) at the morula (E3.0) (n = 28 for Fgf4; n = 27 for Fgfr2) (A, C) and late blastocyst (E4.5) (n = 21 for Fgf4; n = 22 for Fgfr2) (B, D) stages. The immunostaining intensities of both Fgfr2 and Fgf4 are comparable between controls and parthenotes. A, B: The intensities of Fgf4 signals are higher in inner blastomeres of morulae than outer blastomeres, and higher in the ICM than the TE (trophectoderm). White arrows in A and B indicate that Fgf4 was highly expressed in the perinuclear regions of only Cdx2- blastomeres. On the other hand, Fgfr2 signals are homogeneous throughout whole embryos at both the morula and blastocyst stages (C, D). Scale bars in A–D = 20 μm.
DVDY_23851_sm_SuppFigS5.tif1705KSupporting Information Figure 5. SU5402 does not decrease Fgf3 expression in parthenotes. A–C: Immunofluorescence images for Fgf3 (red), Gata4 (green), and DAPI (blue) in parthenote morulae and blastocysts cultured with the following concentrations of SU5402 for 20 hr: 10 μM (A), 15 μM (B), and 20 μM (C) (n = 20 for each concentration). As the concentration of SU5402 increases, the number of the red-colored Fgf3+ nuclei remains unchanged, whereas Gata4 expression is diminished and becomes restricted to the ICM. Arrows in A–C point to Fgf3+ but Gata4− cells in parthenotes treated with SU5402. D: Average percentages of Fgf3+ and Gata4+ cells in whole embryos at the morula and blastocyst stages, with or without the addition of SU5402. Asterisks indicate significantly decreased percentages of Gata4+ cells in SU5402-treated parthenotes compared to untreated ones (n = 20, P < 0.01, Student's t-test). Note the comparable percentages of Fgf3+ cells between SU5402-treated and untreated parthenotes (n = 20, P = 1.0, Student's t-test). ICM, inner cell mass; TE, trophectoderm. Scale bars in A–C = 20 μm.
DVDY_23851_sm_SuppTables.pdf150KSupporting Information Tables

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