Detection of Math6-Expressing Cell Types in Murine Placenta

SIMPLE SUMMARY: The protein, mouse atonal homolog 6 (Math6), is required for correct placental development in mice and causes loss of pregnancy, leading to reproductive insufficiency in females, when it is missing. It is currently unclear which cell types of the placenta predominantly express this protein. With the help of transgenic Math6 Flag-tag mice, we established two methods to characterize the Math6-positive cells: immunohistochemical staining and cell sorting. Using these methods, we discovered that Math6 protein is expressed in multiple layers of the murine placenta in diverse cell types: decidua cells, uterine natural killer cells, macrophages, trophoblast giant cells, and endothelial cells. ABSTRACT: The transcription factor Math6, mouse atonal homolog 6, belongs to the family of highly conserved basic helix–loop–helix transcription factors. It plays an important role in embryonic development and shows a wide expression pattern in murine tissues. The placenta, as a life-sustaining transient organ for the fetus, also depends on the expression of Math6. The adverse effects of deleting Math6 in mice, leading to deficient placental development and pregnancy loss, have already been demonstrated by us. Until now, detailed investigations regarding the specific mechanisms underlying the improper placental development in these murine mutants have failed, as the Math6 expression could not be confined to a specific cell type due to the lack of a highly specific Math6 antibody. To circumvent this problem, we used transgenic mice, where Math6 is marked with a Flag sequence that functions as a specific epitope. Tissues from these transgenic mice were used to establish immunohistochemical staining and fluorescence-activated cell sorting (FACS). The establishment of these methods yielded initial findings pertaining to the identification of Math6-expressing cell types and their localization. Our results reveal that Math6 shows a wide expression pattern in both maternal and fetal components of the murine placenta. It shows expression in various cell types, but predominantly in trophoblast giant cells, endothelial cells and macrophages. The largest subpopulation that we detected in the group of Math6-positive cells were identified as DBA+ uterine natural killer cells. These findings reveal information and a chance for further investigation on the involvement of Math6 in placental development and the molecular pathomechanisms of spontaneous abortion.