Abstract
Formation of the hatching mouse blastocyst marks the end of preimplantation development, whereby previous cell cleavages culminate in the formation of three distinct cell lineages (trophectoderm, primitive endoderm and epiblast). We report that dysregulated expression of Wwc2, a genetic paralog of Kibra/Wwc1 (a known activator of Hippo-signaling, a key pathway during preimplantation development), is specifically associated with cell autonomous deficits in embryo cell number and cell division abnormalities. Division phenotypes are also observed during mouse oocyte meiotic maturation, as Wwc2 dysregulation blocks progression to the stage of meiosis II metaphase (MII) arrest and is associated with spindle defects and failed Aurora-A kinase (AURKA) activation. Oocyte and embryo cell division defects, each occurring in the absence of centrosomes, are fully reversible by expression of recombinant HA-epitope tagged WWC2, restoring activated oocyte AURKA levels. Additionally, clonal embryonic dysregulation implicates Wwc2 in maintaining the pluripotent epiblast lineage. Thus, Wwc2 is a novel regulator of meiotic and early mitotic cell divisions, and mouse blastocyst cell fate.
Highlights
Following fertilization of metaphase II arrested (MII) mouse oocytes, a series of asynchronous cleavage divisions leads to the formation of blastocyst embryos that comprise three distinct cell lineages
Immuno-fluorescent (IF) confocal microscopy analysis of treated/microinjected embryos did not reveal any ectopic CDX2 expression within the inner-cell mass (ICM) of either Kibra or Wwc2 KD embryos, that could be expected if activation of the Hippo-signaling pathway was blocked or impaired (Figure 1C); suggesting neither gene is required for activation of Hippo-signaling in inner cells to supress Cdx2 expression
CDX2 expression was observed in the outer cells of Wwc2 KD embryos but was sometimes observed to be significantly less robust or absent when compared to the levels observed in either the control or Kibra-specific siRNA microinjected groups, imaged under the same conditions (Figure 1C – see blastomeres marked with asterisks, often associated with presence of nuclear abnormalities such as micronuclei, denoted by magenta arrows – expanded below)
Summary
Following fertilization of metaphase II arrested (MII) mouse oocytes, a series of asynchronous cleavage divisions leads to the formation of blastocyst embryos (at E4.5) that comprise three distinct cell lineages. Eventual EPI specification by the late blastocyst stage, requires ICM cell YAP redistribution to the nucleus (implying suppression of Hippo-signaling) in an inherently heterogeneous process that causes competitive apoptotic elimination of EPI progenitors of reduced naïve pluripotency (Hashimoto and Sasaki, 2019). These data illustrate the important and integral nature of Hippo-signaling in regulating key cell fate events in preimplantation mouse embryo development. We hypothesize they indicate potential roles for other functionally upstream, uncharacterised and potentially novel factors (related to the core Hippo-pathway machinery) that may be functionally important during early mouse embryogenesis
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