a, Immunohistochemical staining of first trimester placenta (6–8 weeks gestation) for effectors of the following major signalling pathways: (1) WNT signalling through non-phosphorylated-β-catenin (S33/S37/T41); (2) TGFβ signalling through phosphorylated (p)-SMAD2 (S465/467) and p-SMAD3 (S423/425); (3) MAPK signalling through p-ERK1 and p-ERK2 (T202/Y204); and (4) p-STAT3 (Y705) signalling. Scale bars, 50 μm. Representative images from n = 8 for each antibody. BMP signalling through SMAD1, SMAD5 or SMAD8 was not possible to assess by immunohistochemistry. VCT cells and CCCs displayed membrane-localized staining of non-phosphorylated-β-catenin, whereas p-ERK1 and p-ERK2 was mostly found in the cytoplasm in both cell types. Cytoplasmic and nuclear signals for p-ERK1 and p-ERK2 were detected in the EVT. p-SMAD2 and p-SMAD3 staining also showed stronger nuclear signals in the EVT, suggesting a role for TGFβ signalling in differentiation, in accordance with a previous report9. Phosphorylated nuclear STAT3 was detected only in the EVT, again indicating involvement in their differentiation. The SCT was negative for all these signals. b, Summary of findings from a. Trophoblast cells from different regions of the placenta are represented as a circle with a nucleus (small inner circle). Black indicates strong staining, grey indicates faint staining, and white denotes not detected. Thicker circles indicate staining localized to the cell membrane. c, In situ hybridization for LGR5 on first trimester placental villi. LGR5 transcripts are detected in the VCT. Stroma is negative. Positive-control probe is for UBC; negative-control probe is for the bacterial gene dapB. Nuclei are counterstained with haematoxylin. Images are at ×10 magnification. Representative images from n = 2. d, Immunohistochemistry staining for R-spondin 1 in early first trimester (6–8 weeks gestation) and late first trimester (10–12 weeks gestation) decidual samples. Representative images from n = 2 for each tissue type. Images are at ×20 magnification. UG, uterine glands.