FIG. 2.
Failure of the NS−/− blastocyst cells to proliferate normally. (A and B) number of nuclei detected by DAPI staining of blastocysts from NS+/− intercrosses. (A) Mean cell number of wild-type and heterozygous embryos combined and of homozygous NS embryos at E3.0 and E3.5. Error bars denote standard errors. There is a significant difference between the mean numbers by Student's t test at both stages of development (E3.0, P = 6.10−13; E3.5, P = 1.10−11). (B) Representative images of E3.5 NS+/+ or NS+/− and NS−/− mutant embryos stained with DAPI (blue) and anti-NS (red) antibodies. Magnification, ×200. (C and D) Number of cells positive for phospho-Ser-histone H3 (P-H3, the M-phase specific marker) among the total number of blastocyst cells (determined by DAPI staining of nuclear DNA). (C) The percentage of P-H3-positive cells in wild-type and heterozygous embryos combined and in homozygous NS embryos at E3.0. Error bars denote standard errors. There is a significant difference between the mean numbers by Student's t test (P = 0.03). (D) Representative images of E3.0 NS+/+ or NS+/− and NS−/− mutant embryos stained with DAPI (blue), anti-P-H3 antibody (green), and anti-NS antibody (red). Magnification, ×200. (E and F) NS-deficient blastocysts fail to incorporate BrdU. Blastocysts were collected and cultured for 16 h in ES cell medium containing 50 μM BrdU. (E) The percentage of BrdU-positive cells among the total number of blastocyst cells (determined by DAPI staining of nuclei) in wild-type and heterozygous embryos combined and in homozygous NS embryos at E4.0. Error bars denote standard errors. There is a significant difference between the mean numbers by Student's t test (P = 2.10−10). (D) Representative images of E4.0 NS+/+ or NS+/− and NS−/− mutant embryos stained with DAPI (blue), anti-BrdU antibody (green), and anti-NS antibody (red). Magnification, ×200. n, number of embryos analyzed.