*2.3. Plagl1 Is Expressed in Endothelial Cells of the Labyrinthine Layer*

vasculogenesis and labyrinth layer morphology.

*2.3. Plagl1 Is Expressed in Endothelial Cells of the Labyrinthine Layer* To determine where within the mouse placenta *Plagl1* is expressed we performed RNAscope with an e9.5 implantation site. *Plagl1* expression was observed within the developing labyrinth; specifically in the endothelial cells forming the fetal blood vessels (Figure 3a, Supplemental Figure S2a). Positive (*PPIB*) and negative (*DapB*) control probes were used for RNAscope to ensure *Plagl1* signal was not due to noise or unspecific binding (Figure 3b–c, Supplemental Figure S2b–c). Immunohistochemistry for CD34, a marker for vascular endothelial cells [40,41], was also performed and showed a similar staining within the labyrinth (Figure 3d; Supplemental Figure S2d). *Plagl1* is also expressed throughout the allantois, where fetal vasculature begins developing, eventually To determine where within the mouse placenta *Plagl1* is expressed we performed RNAscope with an e9.5 implantation site. *Plagl1* expression was observed within the developing labyrinth; specifically in the endothelial cells forming the fetal blood vessels (Figure 3a, Supplemental Figure S2a). Positive (*PPIB*) and negative (*DapB*) control probes were used for RNAscope to ensure *Plagl1* signal was not due to noise or unspecific binding (Figure 3b–c, Supplemental Figure S2b–c). Immunohistochemistry for CD34, a marker for vascular endothelial cells [40,41], was also performed and showed a similar staining within the labyrinth (Figure 3d; Supplemental Figure S2d). *Plagl1* is also expressed throughout the allantois, where fetal vasculature begins developing, eventually forming the endothelia in the

forming the endothelia in the labyrinth layer of the placenta [42], suggesting a role for PLAGL1 in

labyrinth layer of the placenta [42], suggesting a role for PLAGL1 in vasculogenesis and labyrinth layer morphology. *Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 5 of 21

**Figure 3.** *Plagl1* is expressed within the developing labyrinth layer and allantois of the e9.5 mouse placenta. (**a**) RNAscope shows *Plagl1* RNA in the developing labyrinth as well as throughout the allantois (dark red) in the e9.5 placenta. Box shows zoomed in region (right). Arrowheads indicate *Plagl1* staining of endothelial cells forming fetal blood vessels. (**b**) RNAscope positive control staining *PPIB* within the placenta to determine RNA viability. (**c**) RNAscope negative control staining *DapB* within the placenta to determine nonspecific staining. (**d**) Immunohistochemistry shows CD34 staining of the vascular endothelial cells, displaying a similar pattern to *Plagl1* within the developing labyrinth. Box shows zoomed in region (right). Arrowheads indicate CD34 staining. **Figure 3.** *Plagl1* is expressed within the developing labyrinth layer and allantois of the e9.5 mouse placenta. (**a**) RNAscope shows *Plagl1* RNA in the developing labyrinth as well as throughout the allantois (dark red) in the e9.5 placenta. Box shows zoomed in region (right). Arrowheads indicate *Plagl1* staining of endothelial cells forming fetal blood vessels. (**b**) RNAscope positive control staining *PPIB* within the placenta to determine RNA viability. (**c**) RNAscope negative control staining *DapB* within the placenta to determine nonspecific staining. (**d**) Immunohistochemistry shows CD34 staining of the vascular endothelial cells, displaying a similar pattern to *Plagl1* within the developing labyrinth. Box shows zoomed in region (right). Arrowheads indicate CD34 staining.
