Figure 2.

Deletion of Igf2 in the epiblast or endothelium impairs Lz expansion

(A) Left: schematic of Igf2 expression in conceptuses with conditional deletion driven by Meox2^Cre. Right: immunostaining for YFP (green) in a representative fetus and placenta paraffin section at E12 of gestation, double transgenic for Meox2^Cre and Rosa26^flSTOP^flYFP¹⁰ reporter. YFP expression in the placenta is localized to the Lz and Cp (high magnification, inset). Blue—DAPI stain for nuclei; scale bars: 1 mm (low magnification) and 100 μm (high magnification).

(B) Fetal and placental growth kinetics, measured as average wet-weights for each genotype per litter (E12: n = 10 L [n = 41 controls {C} and n = 32 Igf2^EpiKO]; E14: n = 25 L [n = 114 C and n = 88 Igf2^EpiKO]; E16: n = 37 L [n = 154 C and n = 127 Igf2^EpiKO]; E19: n = 37 L [n = 164 C and n = 121 Igf2^EpiKO]).

(C) Absolute volumes of the placental layers (Db, decidua basalis; Jz, junctional zone; Lz, labyrinthine zone; Cp, chorionic plate), measured by stereology (n = 6 per group).

(D) Absolute volumes of Lz components, measured by stereology (LT, labyrinthine trophoblast; MBSs, maternal blood spaces; FCs, fetal capillaries) (n = 6 per group).

(E) Left: schematic representation of Igf2 expression in conceptuses with conditional deletion driven by Tek^Cre. Right: representative confocal microscopy of frozen sections from a fetus and its corresponding placenta, double transgenic for TeK^Cre and Ai9(RCL-tdT) reporter at E16 of gestation. Scale bars: 2 mm (fetus) and 1 mm (placenta).

(F) Fetal and placental growth kinetics (E12: n = 5 L [n = 17 C and n = 16 Igf2^ECKO]; E14: n = 8 L [n = 26 C and n = 34 Igf2^ECKO]; E16: n = 13 L [n = 60 C and n = 46 Igf2^ECKO]; E19: n = 7 L [n = 31 C and n = 27 Igf2^ECKO]).

(G) Absolute volumes of the placental layers measured by stereology (n = 5–7 per group).

(H) Absolute volumes of Lz components, measured by stereology (n = 5–7 per group).

(I) Double immunostaining for EPCAM (epithelial cell adhesion molecule) (red) and MCT1 (monocarboxylate transporter 1) (green) in a representative frozen placental section at E12 of gestation. EPCAM expression is observed as clusters of positive cells within the Lz placenta. Blue—DAPI (4′,6-diamidino-2-phenylindole) stain for nuclei; scale bars: 500 μm (left panel) and 20 μm (right panel).

(J) Analysis of EPCAM^high-positive cells by flow cytometry. Left panel: example of gating used to identify EPCAM^high-positive cells (the viability dye 7-aminoactinomycin D [7-AAD] was used to exclude dead cells). Right: quantification of placental EPCAM^high-positive cells at E12 in conceptuses with conditional Igf2 deletion driven by Meox2^Cre (n = 10 C and n = 9 Igf2^EpiKO from 2 L) or Tek^Cre (n = 8 C and n = 8 Igf2^ECKO from 2 L). For all graphs data are shown as averages; error bars represent SD in (C), (D), (G), (H), and (J) or 95% confidence intervals (95% CI) in (B) and (F); N.S.—statistically not significant; ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001 calculated by a mixed effects model in (B) and (F) (see STAR Methods), two-way ANOVA plus Sidak’s multiple comparisons tests in (D) and (H) or unpaired t tests in (C), (G), and (J). See also Figures S1–S3.