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. 2010 Dec 15;137(24):4159–4169. doi: 10.1242/dev.056630

Fig. 1.

Fig. 1.

Efficient reduction of Cdx2 mRNA and protein levels in different preimplantation stage mouse embryos by siCdx2 results in phenotypes similar to Cdx2 knockout. (A) Relative Cdx2 mRNA expression levels as a percentage of peak expression in mouse embryos at different preimplantation stages, as assessed by qRT-PCR. The maternal mRNA decreased at the 4-cell stage, followed by upregulation at the 8-cell stage due to the zygotic activation of Cdx2. Embryo collection times (post-hCG): 2-cell stage, 40 hours; 4-cell stage, 52 hours; 8-cell stage, 62 hours; 16-cell stage, 72 hours; morula, 85 hours; early blastocyst, 96 hours; late blastocyst, 115 hours. (B) A comparison of Cdx2 mRNA levels in siControl- and siCdx2-treated zygotes at different preimplantation stages demonstrated robust downregulation of Cdx2 by siCdx2 treatment. KD, knockdown. (C) Confocal images of 8-cell stage embryos immunolabeled with anti-Cdx2 monoclonal antibody illustrate the reduction of Cdx2 protein (green) by siCdx2 treatment. Red, nuclear counterstaining with DRAQ5. (D) Confocal images of blastocyst stage embryos immunolabeled with anti-Cdx2 (green) and anti-Oct4 (red) antibodies. Following siCdx2 treatment, Cdx2 was eliminated, but Oct4 was ectopically expressed in the trophectoderm (TE). (E) DIC images of E4.0 and E6.0 embryos. All siCdx2-treated embryos failed to hatch. Arrows indicate empty zonae pellucidae left by hatched control embryos. Scale bar: 100 μm. (F) Confocal images of ZO-1 (green) and E-cadherin (red) immunohistochemistry show normal E-cadherin distribution, which marks the lateral-basal cell boundary, but disoriented cell polarity of the TE, as indicated by the presence of ZO-1 at both the apical and basal sides (arrowhead and inset). (G) Defective tight junction (arrowhead) in the TE of a Cdx2-deficient blastocyst as shown by electron microscopy. Scale bar: 0.2 μM. (H) Effect of Cdx2 depletion on TE and inner cell mass (ICM) cell numbers. The number of ICM cells was significantly increased (P<0.01), but the total cell number remained unchanged (P=0.64) upon Cdx2 depletion. (I) Quantitation of apoptotic cells shows an increase in apoptotic activity in a Cdx2-deficient E4.0 blastocyst,. MII, metaphase II oocyte; ZP, zona pellucida. Error bars indicate standard deviation.