**1. Introduction**

Proper function of the placenta is essential for the development of the embryo as it is responsible for exchanging gases, nutrients, and waste products between the mother and the fetus. Placental insufficiency can result in adverse effects for the embryo including intrauterine growth retardation, embryonic defects, or even fatal outcome. Therefore, placenta development and differentiation are closely regulated [1–3].

The membrane-bound tyrosine receptor kinase KIT is activated by its ligand stem cell factor (SCF), which causes dimerization of the receptor followed by phosphorylation of its tyrosine residues [4–6]. Activation of KIT results in induction of various downstream signaling cascades such as the Mitogen-Activated Protein Kinase (MAPK)/Extracellular-Signal Regulated Kinase (ERK) and Janus Kinase (JAK)/Signal Transducers and Activators of Transcription (STAT) pathways which orchestrate cell proliferation, angiogenesis, cell migration, and cell cycle control [7–10]. During placental development, the KIT protein is detected in the uterine epithelium as well as the maternal and fetal part of the placenta. Importantly, KIT expression has not been detected in uteri of nonpregnant females, indicating a pregnancy-related role of KIT signaling [11–13]. In detail, starting at E9, KIT and SCF are detectable in trophoblast-chorion, ectoplacental cone, and decidua [12]. Placental hematopoietic activity begins around mid-gestation. Hematopoietic stem cells (HSC) are detected in the placenta at E11, peak in numbers at E12 to E13, and disappear thereafter [13,14]. These HSC were shown to be KIT positive, whereas endothelial cells surrounding the HSC niches are SCF positive [13,15]. At E12, KIT can also be found in mesenchymal cells of the chorionic plate and of the labyrinth, in endothelial cells lining the vessels, as well as in few trophoblast giant cells (TGC) [13]. At E14.5, KIT is mainly expressed in maternal decidua cells as well as the labyrinth whereas SCF is found only in labyrinth [11,12]. KIT is restricted to labyrinthine trophoblast cells exposed to maternal blood at E19 and is no longer detected in endothelial or mesenchymal cells [11].

The KIT receptor has been implicated with several disorders such as tumor formation, hematopoietic disorders, and systemic mastocytosis [16–20]. The most common activating mutation in the human KIT receptor encodes for the substitution of the aspartic acid at position 816 with a valine (D816V) causing constitutive phosphorylation of the protein [17,21,22]. Depletion of KIT receptor results in various defects spanning from hematopoietic failure, macrocytic anemia, pigmentation deficiency, and sterility to intestinal dysfunction [23–26]. Mice carrying homozygous KITW/<sup>W</sup> mutation die pre- or perinatally; however, can be rescued by microinjection of wildtype fetal liver hematopoietic cells into placentas of affected fetuses [23,24,26]. Introduction of the viable c-Kit-deficient mouse line allowed for studying the loss of KIT expression in the adult mouse and showed that KIT is essential for adult lymphopoiesis in bone marrow and thymus [26]

Previously, we reported the generation of a humanized KITD816V mouse model [27]. The transgenic KIT receptor consists of murine extracellular and transmembrane domains and the human intracellular domain carrying the D816V mutation. The chimeric KIT receptor is fused to a green fluorescent protein (GFP) and integrated in the ROSA26 locus, allowing for Cre-mediated conditional expression of the protein under control of the endogenous ROSA26 promoter [27,28]. We then examined KIT signaling in fetal liver erythropoiesis and adult hematopoiesis [27,29]. Constitutively active KIT resulted in inhibition of terminal differentiation of erythroid precursors and embryonic death after embryonic day (E) 13.5 [27]. In adult mice, expression of KITD816V led to a polycythemia vera-like myeloproliferative neoplasm with highly increased red blood cells and splenomegaly [29].

While the role of KIT signaling in other tissues is well understood, its role in placental development, however, has so far not been described. Here, we examine the effect of the constitutively active KIT

receptor using the KITD816V mouse model. Interestingly, expression of KITD816V resulted in decreased proliferation of trophoblast cells. As a consequence, placental structure was affected. The labyrinth layer was decreased accompanied by increased differentiation to parietal (P-)TGCs, whereas other TGC subtypes remained underrepresented. Trophoblast Stem Cells (TSC) derived from KITD816V blastocysts showed altered activation of signaling pathways upon differentiation as well as an increased invasive capacity. Due to these effects, the placenta is not able to sustain regular development and the embryos show severe growth retardation and die in utero.
