Fig. 4

Ino80 is widely expressed in embryonic tissues and is essential for establishing the proximal-distal axis of the post-implantation embryo. a Whole mount RNA in situ hybridization (ISH) of E5.5-, E6.5-, and E7.5-day embryos using either sense or antisense Ino80 riboprobes. All embryos were photographed at identical magnification. Extraembryonic ectoderm (ExE), embryonic ectoderm (EmE) and visceral endoderm (VE) are designated. b Increased magnification showing reduced antisense Ino80 riboprobe staining in the visceral endoderm (VE) in E6.0-day embryos. Embryos were photographed at identical magnification. c Frozen sections of E6.5-day embryos stained whole mount with antisense Ino80 riboprobe. Sagittal and cross sections through the ExE and EPI were obtained for representative embryos. d Whole mount RNA in situ hybridization of E6.5-, E5.5-, and E4.5-day embryos was performed with antisense riboprobes for markers of tissue differentiation. Markers used include the ExE marker (Bmp4), EmE marker (Nodal), mesoderm marker (T), EPI marker (Nanog), VE marker (Hnf4a), and PrE/DVE/AVE markers (Cer1, Lefty1, and Hex1). Embryos were photographed at identical magnification. e Whole mount ISH of E5.5-day embryos was performed with antisense probes to the ExE marker Bmp4 (purple color) and the EmE marker Otx2 (magenta color). Embryos were photographed at identical magnification. f Whole mount analysis of TUNEL positive cells in wild type and Ino80 KO E6.5-day embryos. A representative negative control (- TdT enzyme) is shown. Representative TUNEL positive cells are shown by black arrows. g Quantitation of apoptosis as percentage of TUNEL positive cells from panel f (see methods section). N = 2 wild type (WT) or Ino80 KO embryos. E embryonic day, EPI epiblast, PrE primitive endoderm, DVE distal visceral endoderm, AVE anterior visceral endoderm