Distinct sequential cell behaviours direct primitive endoderm formation in the mouse blastocyst

Abstract

The first two lineages to differentiate from a pluripotent cell population during mammalian development are the extraembryonic trophectoderm (TE) and the primitive endoderm (PrE). Whereas the mechanisms of TE specification have been extensively studied, segregation of PrE and the pluripotent epiblast (EPI) has received comparatively little attention. A current model of PrE specification suggests PrE precursors exhibit an apparently random distribution within the inner cell mass of the early blastocyst and then segregate to their final position lining the cavity by the late blastocyst. We have identified plateletderived growth factor receptor alpha (Pdgfrα) as an early-expressed protein that is also a marker of the later PrE lineage. By combining live imaging of embryos expressing a histone H2B-GFP fusion protein reporter under the control of Pdgfra regulatory elements with the analysis of lineage-specific markers, we investigated the events leading to PrE and EPI lineage segregation in the mouse, and correlated our findings using an embryo staging system based on total cell number. Before blastocyst formation, lineage-specific factors are expressed in an overlapping manner. Subsequently, a gradual progression towards a mutually exclusive expression of PrE-and EPI-specific markers occurs. Finally, cell sorting is achieved by a variety of cell behaviours and by selective apoptosis.

Pluripotency in the Embryo and in Culture

“…Genetic manipulations in ESCs suggested that the two transcription factors Nanog and Gata6 may act in a mutually antagonistic manner to determine the two lineages. In the embryo, however, Nanog and Gata6 both begin to be expressed in an apparently stochastic manner at around the eight-cell stage (Dietrich and Hiiragi 2007), and they can be colocalized in some cells of the ICM at least until the 32-cell stage (Plusa et al 2008). Hypoblast markers, such as platelet-derived growth factor receptor a (PDGFRa) and Sox17, also initially show heterogeneous expression in the early blastocyst.…”

“…Nanog protein is initially found at varying levels throughout the ICM (Dietrich and Hiiragi 2007;Plusa et al 2008). By the late blastocyst stage, however, Nanog is precisely restricted to the new-formed epiblast, where it is present at similar levels in all cells.…”

The molecular underpinnings of totipotency

“…B 369: 20130549 mutually exclusive expression of these two transcription factors [40,41]. These cells then physically sort based on a variety of cell behaviours, including differential adhesion, apoptosis and downregulation of the PE programme in inside cells [41], resulting in the segregation of Epi and PE layers by E4.5. In addition to Nanog, a number of other Epi markers such as Rex1 [42] are expressed in early blastomeres and are later restricted to the pluripotent Epi.…”

“…The expression of these canonical lineage markers begins with OCT4, GATA6, NANOG and CDX2, all apparent by the 8-cell stage [38,41]. Many of these transcription factors continue to be coexpressed into blastocyst stages ( figure 1a).…”

From blastocyst to gastrula: gene regulatory networks of embryonic stem cells and early mouse embryogenesis

“…First, they support the notion that the divergence of TE and PrE involves reciprocal genetic changes within GRNs shared, at least in part, with the ICM and Epi. These changes ultimately derive from dynamic and reinforcing cellular interactions, in which cell polarity and positional cues appear to trigger events separating TE from ICM, while cell division orientation, migration and apoptosis drive the divergence of PrE and Epi [114][115][116]. Secondly, these studies provide evidence that pluripotency factors function in networks regulating lineage-specific differentiation in the embryo.…”