Figure 2: Amot prevents differentiation of the ICM. | Nature Communications

Figure 2: Amot prevents differentiation of the ICM.

From: Angiomotin prevents pluripotent lineage differentiation in mouse embryos via Hippo pathway-dependent and -independent mechanisms

Figure 2

(a) Immunofluorescent confocal images of TE/ICM marker genes of control and Amot-RNAi blastocysts. Control (N=20)/Amot siRNA (N=30) were injected into zygotes and cultured to blastocysts; Amot protein was undetectable in Amot-RNAi blastocysts. In the absence of Amot, TE markers Cdx2 (N=30), Eomes (N=16) and Ck-8 (antibody: Troma-1) (N=16) become upregulated in the ICM and Yap localized to the nuclei of ICM cells (N=12) in contrast to control blastocysts, where Yap is cytoplasmic (N=12). Pluripotency markers Oct4 and Nanog (N=11) are still visible. Yellow areas and dotted lines mark inside cells. Scale bar, 10 μm. (b) Total cell number of Amot-RNAi versus control blastocysts. The total number of cells between Amot-RNAi (46.3±2cells, N=20 embryos) and control blastocysts (43.2±2cells, N=30 embryos) is not significantly different (P=0.51, Student’s t-test). The percentage of Cdx2-positive cells in the ICM of Amot-RNAi blastocysts (61.8±5%, N=434 cells, 30 embryos) is greater than in control blastocysts (5.0±2%, N=311 cells, 20 embryos). Error bars represent s.e.m.. Student’s t-test was used to test significance. (c) qRT–PCR of whole embryos, comparing expression levels of several TE/ICM markers of Amot-RNAi (N=60) versus control (N=60) blastocysts. Error bars represent s.e.m. Student’s t-test was used to test significance. (d) Immunofluorescence of embryos clonally depleted of Amot, showing that Amot governs cell fate in a cell-autonomous manner. One blastomere of two-cell embryos was injected with control/Amot siRNA and Ruby mRNA, and cultured to blastocysts. Ruby-positive cells mark the injected clone. In the ICM of the mosaic Amot-RNAi embryos, only the Ruby-positive cells expressed Cdx2 (N=21 cells) (arrows). Yellow areas mark the ICM. Dotted lines mark Ruby-positive ICM cells. Scale bar, 10 μm. (e) Immunofluorescence of embryos that are clonally overexpressing Amot, showing that Amot is not sufficient to drive cells to ICM. One blastomere of two-cell embryos was injected with Ruby mRNA only or Amot and Ruby mRNA, and cultured to blastocysts (N=10). Ruby-positive cells mark the overexpressing clone. Yellow areas mark the ICM. Dotted lines mark Ruby-positive cells. Scale bar, 10 μm.

Back to article page