*2.2. KITD816V Placentas Show Fewer Proliferating Cells*

Next, we examined whether reduced sizes in KITD816V placentas occurred due to diminished proliferation. Proliferation in placental tissue was assessed by immunohistochemical staining for KI-67—a protein that is present in proliferating cells as well as cells undergoing endoreduplication [30,31]. At both timepoints analyzed, KI-67-positive cells were mainly detectable in the labyrinth and spongiotrophoblast layers of KITD816V and WT placentas. On day E9.5, the labyrinth layer and overall amount of KI-67-positive cells were markedly diminished in KITD816V in comparison to WT (Figure 2A,B). While the labyrinth and spongiotrophoblast layers were enlarged on day E10.5 in comparison to E9.5 in both samples, less KI-67-positive cells were detected in KITD816V placentas than in WT placentas (Figure 2A,B). KI-67-positive P-TGCs can be detected in both KITD816V and WT placentas. These results suggest that the smaller size of KITD816V placentas results from decreased proliferation in the labyrinth and spongiotrophoblast.

*Int. J. Mol. Sci.* **2020**, *21*, x 4 of 18

**Figure 1.** Detection of KITD816V-positive embryos and placentas: (**A**) qRT-PCR for transgene expression of human *KIT* in KITD816V and wildtype (WT) placentas obtained on E10.5 and (**B**) qRT-PCR for endogenous expression of murine *Kit* in KITD816V and WT placentas obtained on E10.5. RNA was obtained from at least three biological repeats. Expression is normalized to the housekeeping gene *Gapdh*. Bars display mean value ± SD. Significance was determined by unpaired t-test and indicated with \*\*\* *p* < 0.001. (**C**) Western Blot detecting KIT and 2A-peptide in placentas at E11.5 in comparison to β-ACTIN and (**D**) photographs showing KITD816V and WT expressing embryos and placentas at E11.5 in brightfield (BF) and GFP fluorescence (scale bar: 1 mm): Experiments were performed in at least tree biological repeats. **Figure 1.** Detection of KITD816V-positive embryos and placentas: (**A**) qRT-PCR for transgene expression of human *KIT* in KITD816V and wildtype (WT) placentas obtained on E10.5 and (**B**) qRT-PCR for endogenous expression of murine *Kit* in KITD816V and WT placentas obtained on E10.5. RNA was obtained from at least three biological repeats. Expression is normalized to the housekeeping gene *Gapdh*. Bars display mean value ± SD. Significance was determined by unpaired t-test and indicated with \*\*\* *p* < 0.001. (**C**) Western Blot detecting KIT and 2A-peptide in placentas at E11.5 in comparison to β-ACTIN and (**D**) photographs showing KITD816V and WT expressing embryos and placentas at E11.5 in brightfield (BF) and GFP fluorescence (scale bar: 1 mm): Experiments were performed in at least tree biological repeats.

#### *2.2. KITD816V Placentas Show Fewer Proliferating Cells 2.3. KITD816V Placentas Show Reduced Labyrinth Layer and Disrupted Formation of Vasculature*

in the labyrinth and spongiotrophoblast.

Next, we examined whether reduced sizes in KITD816V placentas occurred due to diminished proliferation. Proliferation in placental tissue was assessed by immunohistochemical staining for KI-67—a protein that is present in proliferating cells as well as cells undergoing endoreduplication [30,31]. At both timepoints analyzed, KI-67-positive cells were mainly detectable in the labyrinth and spongiotrophoblast layers of KITD816V and WT placentas. On day E9.5, the labyrinth layer and overall amount of KI-67-positive cells were markedly diminished in KITD816V in comparison to WT (Figure 2A,B). While the labyrinth and spongiotrophoblast layers were enlarged on day E10.5 in comparison to E9.5 in both samples, less KI-67-positive cells were detected in KITD816V placentas than in WT placentas (Figure 2A,B). KI-67-positive P-TGCs can be detected in both KITD816V and WT placentas. These results suggest that the smaller size of KITD816V placentas results from decreased proliferation To assess the placental structure, hematoxylin and eosin (H&E) staining was performed on paraffin sections of KITD816V and WT placentas. Starting from day E9.5 in KITD816V placentas, H&E stained sections and quantification of the area showed a reduction in labyrinth size (Figure 3A,B). Also, the spongiotrophoblast layer was slightly reduced (Figure 3A,B). Interestingly, at E10.5, the layer of P-TGCs was increased in KITD816V placentas (Figure 3A,B). Quantification of P-TGCs within this area confirmed a significantly higher total number of P-TGCs in KITD816V sample (Figure 3C). Next, RNA was isolated from KITD816V and WT placentas on days E10.5 and E11.5. Expression of labyrinth TGC markers Placental Lactogen 2 (*Pl2)*, Cathepsin q (*Ctsq)*, and Glial Cells Missing Homolog 1 (*Gcm1)* is diminished in comparison to WT placentas (Figure 3D). This data indicates that the number of *Pl2*<sup>+</sup> and *Ctsq*<sup>+</sup> sinusoidal (S-)TGC and the number of *Gcm 1*<sup>+</sup> labyrinthine cells is reduced.

*Int. J. Mol. Sci.* **2020**, *21*, x 5 of 18

**Figure 2.** KITD816V shows a reduced number of KI-67-positive cells. (**A**, **B**) Immunohistochemical staining of paraffin sections of KITD816V and WT placentas for KI-67 at E9.5 and E10.5: Black arrowheads mark Parietal Trophoblast Giant Cells (P-TGCs) undergoing endoreduplication (biological replicates = 4; scale bar represents 500 μm). **Figure 2.** KITD816V shows a reduced number of KI-67-positive cells. (**A**,**B**) Immunohistochemical staining of paraffin sections of KITD816V and WT placentas for KI-67 at E9.5 and E10.5: Black arrowheads mark Parietal Trophoblast Giant Cells (P-TGCs) undergoing endoreduplication (biological replicates = 4; scale bar represents 500 µm).

*2.3. KITD816V Placentas Show Reduced Labyrinth Layer and Disrupted Formation of Vasculature*  To assess the placental structure, hematoxylin and eosin (H&E) staining was performed on paraffin sections of KITD816V and WT placentas. Starting from day E9.5 in KITD816V placentas, H&E stained sections and quantification of the area showed a reduction in labyrinth size (Figure 3A,B). Also, the spongiotrophoblast layer was slightly reduced (Figure 3A,B). Interestingly, at E10.5, the layer of P-TGCs was increased in KITD816V placentas (Figure 3A,B). Quantification of P-TGCs within this area confirmed a significantly higher total number of P-TGCs in KITD816V sample (Figure 3C). Labyrinthine architecture was further examined by performing anti-CD31 staining. CD31 is expressed in fetal endothelial cells [32]. The staining revealed that at E9.5 there are fewer CD31-positive cells present in KITD816V placentas in contrast to WT placentas (Figure 3E). CD31-positive cells are indicated by red arrowheads. While the vasculature was established from E9.5 to E10.5 in KITD816Vas well as WT placentas, the capillary network in KITD816V remained less prominent than in WT tissue. S-TGCs, indicated by black arrowheads, were lining maternal sinusoids (red asterisks) in both conditions. Surprisingly, maternal blood was detected in between P-TGCs of KITD816V placentas at E10.5, suggesting a disruption of the developing vascular structure (Figure 3E).

Next, RNA was isolated from KITD816V and WT placentas on days E10.5 and E11.5. Expression of

#### labyrinth TGC markers Placental Lactogen 2 (*Pl2)*, Cathepsin q (*Ctsq)*, and Glial Cells Missing *2.4. KITD816V Placentas Show Prominent Di*ff*erentiation into P-TGCs*

(Figure 3E).

Homolog 1 (*Gcm1)* is diminished in comparison to WT placentas (Figure 3D). This data indicates that the number of *Pl2*+ and *Ctsq*+ sinusoidal (S-)TGC and the number of *Gcm 1*+ labyrinthine cells is reduced. Labyrinthine architecture was further examined by performing anti-CD31 staining. CD31 is expressed in fetal endothelial cells [32]. The staining revealed that at E9.5 there are fewer CD31-positive cells present in KITD816V placentas in contrast to WT placentas (Figure 3E). CD31-positive cells are indicated by red arrowheads. While the vasculature was established from E9.5 to E10.5 in KITD816V as well as WT placentas, the capillary network in KITD816V remained less prominent than in WT tissue. S-TGCs, indicated by black arrowheads, were lining maternal sinusoids (red asterisks) in both conditions. Surprisingly, maternal blood was detected in between P-TGCs of KITD816V placentas at E10.5, suggesting a disruption of the developing vascular structure Since the number of P-TGCs is significantly increased in KITD816V placentas (Figure 3C), we next investigated this TGC subtype in more detail by detecting the P-TGC markers PL1 and PLF using in situ hybridization. At E9.5, the number and distribution of cells positive for PL1 and PLF appeared unaltered in KITD816V compared to WT placentas (Figure 4A). At E10.5, however, the number of PL1+/PLF<sup>+</sup> cells was visibly higher in KITD816V placentas than WT placentas (Figure 4B). Further, when comparing PL1 and PLF staining in WT placentas, we observed areas that are PL1 negative but PLF positive (Figure 4B). Such cells are not P-TGCs but rather invading Spiral Artery (SpA-)TGCs (black arrowhead). We assume that the cell clusters indicated by red arrowheads are canal (C-) TGCs (Figure 4B). By contrast, PL1−/PLF<sup>+</sup> cells are not detected in KITD816V placentas, further suggesting a severe reduction in specific TGC subtypes such as SpA-TGC and C-TGC.

*Int. J. Mol. Sci.* **2020**, *21*, x 6 of 18

**Figure 3.** Placental structure is grossly affected by KITD816V. (**A**) Photomicrographs of hematoxylin and eosin (H&E) staining of paraffin sections of KITD816V and WT placentas on E9.5 (biological replicates = 2) and E10.5 (biological replicates = 3): Black lines mark areas of the P-TGCs (P), spongiotrophoblast (S), and labyrinth layer (L). Scale bar represents 500 μm. (**B**) Quantification of P-TGC, spongiotrophoblast, and labyrinth areas at E10.5 compared to the total area of respective placenta (biological replicates = 3) and (**C**) quantification of number of P-TGCs in KITD816V and WT placenta at E10.5 using ImageJ in three biological replicates: Significance was determined by unpaired t-test and indicated with \* *p* < 0.05. (**D**) qRT-PCR analysis of labyrinth-specific TGC marker *Pl2*, *Ctsq*, and *Gcm* in KITD816V and WT placentas at E10.5 and E11.5. RNA was obtained from at least three biological replicates. Expression is normalized to the housekeeping gene *Gapdh*. Bars display mean value ± SD. Significance was determined by unpaired t-test and indicated with \* *p* < 0.05 and \*\* *p* < 0.01; (**E**) Immunohistochemical staining of CD31 on paraffin sections of KITD816V and WT placentas at E9.5 (biological replicates = 2) and E10.5 (biological replicates = 2): Red arrowheads indicate fetal endothelial cells enclosing fetal blood spaces, and black arrowheads indicate S-TGCs. Maternal sinusoids are indicated by red asterisks, and maternal blood in the P-TGC layer is indicated by black asterisks. Scale bar represents 200 μm. **Figure 3.** Placental structure is grossly affected by KITD816V. (**A**) Photomicrographs of hematoxylin and eosin (H&E) staining of paraffin sections of KITD816V and WT placentas on E9.5 (biological replicates = 2) and E10.5 (biological replicates = 3): Black lines mark areas of the P-TGCs (P), spongiotrophoblast (S), and labyrinth layer (L). Scale bar represents 500 µm. (**B**) Quantification of P-TGC, spongiotrophoblast, and labyrinth areas at E10.5 compared to the total area of respective placenta (biological replicates = 3) and (**C**) quantification of number of P-TGCs in KITD816V and WT placenta at E10.5 using ImageJ in three biological replicates: Significance was determined by unpaired t-test and indicated with \* *p* < 0.05. (**D**) qRT-PCR analysis of labyrinth-specific TGC marker *Pl2*, *Ctsq*, and *Gcm* in KITD816V and WT placentas at E10.5 and E11.5. RNA was obtained from at least three biological replicates. Expression is normalized to the housekeeping gene *Gapdh*. Bars display mean value ± SD. Significance was determined by unpaired t-test and indicated with \* *p* < 0.05 and \*\* *p* < 0.01; (**E**) Immunohistochemical staining of CD31 on paraffin sections of KITD816V and WT placentas at E9.5 (biological replicates = 2) and E10.5 (biological replicates = 2): Red arrowheads indicate fetal endothelial cells enclosing fetal blood spaces, and black arrowheads indicate S-TGCs. Maternal sinusoids are indicated by red asterisks, and maternal blood in the P-TGC layer is indicated by black asterisks. Scale bar represents 200 µm.

*2.4. KITD816V Placentas Show Prominent Differentiation into P-TGCs* 

severe reduction in specific TGC subtypes such as SpA-TGC and C-TGC.

Since the number of P-TGCs is significantly increased in KITD816V placentas (Figure 3C), we next investigated this TGC subtype in more detail by detecting the P-TGC markers PL1 and PLF using *in situ* hybridization. At E9.5, the number and distribution of cells positive for PL1 and PLF appeared unaltered in KITD816V compared to WT placentas (Figure 4A). At E10.5, however, the number of PL1+/PLF+ cells was visibly higher in KITD816V placentas than WT placentas (Figure 4B). Further, when comparing PL1 and PLF staining in WT placentas, we observed areas that are PL1 negative but PLF positive (Figure 4B). Such cells are not P-TGCs but rather invading Spiral Artery (SpA-)TGCs (black arrowhead). We assume that the cell clusters indicated by red arrowheads are canal (C-) TGCs

/PLF+ cells are not detected in KITD816V placentas, further suggesting a

**Figure 4.** Increased levels of PL1 and PLF in KITD816V placentas: (**A**) *In situ* hybridization of PL1 and PLF on paraffin sections of KITD816V and control placentas at E9.5 using specific probe for PL1 and PLF (biological replicates = 2). Scale bar represents 1 mm. (**B***) In situ* hybridization of PL1 and PLF on paraffin sections of KITD816V and control placentas at E10.5 (biological replicates = 2): Red arrowheads indicate PL1- /PLF+ cells. Black arrowhead indicates invading Spiral Artery (SpA-)TGCs. Scale bar represents 1 mm. **Figure 4.** Increased levels of PL1 and PLF in KITD816V placentas: (**A**) In situ hybridization of PL1 and PLF on paraffin sections of KITD816V and control placentas at E9.5 using specific probe for PL1 and PLF (biological replicates = 2). Scale bar represents 1 mm. (**B**) In situ hybridization of PL1 and PLF on paraffin sections of KITD816V and control placentas at E10.5 (biological replicates = 2): Red arrowheads indicate PL1−/PLF<sup>+</sup> cells. Black arrowhead indicates invading Spiral Artery (SpA-)TGCs. Scale bar represents 1 mm.
