The labyrinth is the highly vascularized part of the rodent placenta that allows efficient transfer of gases, nutrients, wastes, and other molecules between the maternal and embryonic circulations. placenta where the bidirectional transfer of gases, nutrients, wastes, and other molecules between the maternal and embryonic circulations occurs [15]. In the hemochorial mouse placenta, the barrier between the maternal blood and the embryonic vasculature is formed by three layers of embryo-derived trophoblasts, an endothelial BM, and an embryo-derived endothelium (Fig. 1) [16]. The labyrinth is grossly E7080 (Lenvatinib) supplier undervascularized in null mutants, and the vessels that do form are larger caliber compared to control. In addition, fetal placental endothelial cells lose adhesion to the BM, which normally contains LM5. Together with the fact that mutant alleles, as well as Cre, Cre-activated reverse tetracycline transactivator (rtTA), and hLM5 transgenes. Our results suggest that both trophoblasts and endothelial cells normally contribute LM5-containing trimers to the endothelial BM, and that expression by either cell is sufficient for normal placentation. In addition, we confirmed previous tissue grafting studies [18] showing that endothelial LM5 expression is sufficient for vascularization of kidney glomeruli. Results Expression of Laminin Chains in the Placenta Although some classes of endothelial cells have been shown to express LM5, not all do so [19]. To directly investigate whether labyrinth-derived endothelial cells and/or trophoblasts normally express LM5 and other laminin chains found in the placenta [20], we used fluorescence activated cell sorting (FACS) to isolate endothelial (CD31-positive) and non-endothelial (CD31-negative) cell populations from the normal placental labyrinth (schematized in Fig. 1) after its dissociation into single cells (Fig. 2A). RNAs were prepared from these isolated cells and subjected to quantitative real-time RT-PCR for laminin 5, 1, 1, 2, and GAPDH expression (Fig. 2B,C). The results showed that both populations of cells express each of these laminin chains, but that trophoblasts (CD31-negative cells) express more laminin 1 and 1 than 5 and 2, whereas endothelial (CD31-positive) cells express more laminin 5 and 1 than 1 and 2. The fact that null endothelial cells, we took advantage of the selective expression pattern of the Sox2Cre transgene [25]. When this gene is transmitted by the sire, it is expressed in the epiblast (Fig. 3A), which gives rise to the embryo proper and to the allantois, from which originate the extraembryonic endothelial cells of the labyrinth [26]; however, Sox2Cre is not expressed in the trophectoderm (Fig. 3B), which gives rise to the trophoblasts. Figure 3 Mosaic placental labyrinths containing wild-type trophoblasts and females to generate null embryonic phenotypepartially penetrant exencephaly and syndactyly (Fig. 3E; compare to E) associated with a lack of LM5 (Fig. 3C,D; compare to C,D), although BMs were generally positive when immuno-stained for nidogen (Fig. 3D). In contrast, we detected abundant LM5 protein in placental labyrinth BMs, and the overall architecture of the labyrinth was similar to that of control littermates (Fig. TSPAN2 3FCH, FCH); there was an extensive network of PECAM-positive small caliber vessels, and most maternal blood spaces, which are lined by cytokeratin 8-positive trophoblasts, were juxtaposed to embryonic vessels with BMs that stained for LM-111. These results suggest that laminin trimers containing 5 that are synthesized and E7080 (Lenvatinib) supplier secreted by trophoblasts are capable of integrating into the BM and promoting normal vascularization of the placenta, but they are not sufficient to rescue phenotypes within the embryo. In the second approach, we utilized a combination of transgenes and mutations to perform the converse experiment. We used the endothelial cell-specific Tie2Cre transgene to activate expression of the reverse tetracycline transactivator (rtTA), which had been knocked into the locus preceded by a floxed STOP (genotype embryos showed the typical null phenotype (Fig. 4D; compare to D) and lacked mouse LM5 (Fig. 4B,E; compare to B,E). As expected from the approach, hLM5 was detected in embryonic endothelial BMs E7080 (Lenvatinib) supplier (Fig. 4C; compare to C). Human LM5 was also present in placental labyrinth BMs (Fig. 4F; compare to F), and this resulted in apparently normal placentation, as determined from the pattern of LM-111 staining (Fig. 4G and Fig. 5C,C,F,F), which was similar to the control (Fig. 5A,A,D,D). However, this was in stark contrast to the LM-111 staining pattern in the null placenta that did not express hLM5 (Fig. 4G and Fig. 5B,B,E,E). The apparently normal placental labyrinth of transgenic embryos was associated with a larger but not quite normal embryo size at E18.5 (Fig. 6); this could be due to rescue of placental insufficiency, but might also stem from an overall healthier vasculature within the embryo itself..