Primary Bovine Extra-Embryonic Cultured Cells: A New Resource for the Study of In Vivo Peri-Implanting Phenotypes and Mesoderm Formation

Abstract

In addition to nourishing the embryo, extra-embryonic tissues (EETs) contribute to early embryonic patterning, primitive hematopoiesis, and fetal health. These tissues are of major importance for human medicine, as well as for efforts to improve livestock efficiency, but they remain incompletely understood. In bovines, EETs are accessible easily, in large amounts, and prior to implantation. We took advantage of this system to describe, in vitro and in vivo, the cell types present in bovine EETs at Day 18 of development. Specifically, we characterized the gene expression patterns and phenotypes of bovine extra-embryonic ectoderm (or trophoblast; bTC), endoderm (bXEC), and mesoderm (bXMC) cells in culture and compared them to their respective in vivo micro-dissected cells. After a week of culture, certain characteristics (e.g., gene expression) of the in vitro cells were altered with respect to the in vivo cells, but we were able to identify “cores” of cell-type-specific (and substrate-independent) genes that were shared between in vitro and in vivo samples. In addition, many cellular phenotypes were cell-type-specific with regard to extracellular adhesion. We evaluated the ability of individual bXMCs to migrate and spread on micro-patterns, and observed that they easily adapted to diverse environments, similar to in vivo EE mesoderm cells, which encounter different EE epithelia to form chorion, yolk sac, and allantois. With these tissue interactions, different functions arose that were detected in silico and corroborated in vivo at D21–D25. Moreover, analysis of bXMCs allowed us to identify the EE cell ring surrounding the embryonic disc (ED) at D14-15 as mesoderm cells, which had been hypothesized but not shown prior to this study. We envision these data will serve as a major resource for the future in the analysis of peri-implanting phenotypes in response to the maternal metabolism and contribute to subsequent studies of placental/fetal development in eutherians.

Planar polarity of the extraembryonic epithelia in the preimplantation porcine conceptus

“…In the filamentous conceptus, nuclei resulting from a recent division remain coupled, all pairs oriented in the direction of elongation, as observed by scanning electron microscopy (not shown). A syncytial structure of the parietal fusiform E cells is probably the rule in filiform conceptuses; this has also been described in ovine (Fléchon et al, 2007) and bovine conceptuses (Hue et al, 2015). It is difficult to imagin how these cells could migrate; they may just elongate.…”

Dynamic Pattern of HOXB9 Protein Localization during Oocyte Maturation and Early Embryonic Development in Mammals

“…D14 (N = 2) and D17 (N = 2) embryos were co-stained with CDX2 to highlight trophoblastic cells [77], while D15 (N = 1) and D18 (N = 1) embryos were co-stained with VIMENTIN (VIM) and DLX3 to localize embryonic and extra-embryonic mesoderm cells [16, 52, 56] or trophoblastic cells [42], respectively.…”

“…Scotti and Kmita [123] previously demonstrated a Hoxa10 - Hoxa13 expression in the allantois of E7.5 and E8.5 mouse embryos, which is required for proper expansion of the fetal vasculature in the placenta. Ledgard et al [15] and Hue et al [16] also reported HOX expression in the bovine allantois. In the mouse, CDX2 was reported to be essential for allantoic growth and chorio-allantoic fusion [124].…”

“…Antibody used for post-implantation a or pre-implantation b embryos. 1 Blij et al [40]; 2 Goossens et al [41]; 3 Degrelle et al [42]; 4 Hue et al [16]; 5 Papanayotou et al [43]; 6 Plusa et al [44]; 7 Hoshino et al [45]. …”

“…Briefly, embryos were fixed, dehydrated and stored at -20°C until used, as previously described [16]. Embryos were then rehydrated using a decreasing methanol gradient: 75%, 50%, and 25% for 15 min each and for 1 h in PBS.…”