Conserved roles for Oct4 homologues in maintaining multipotency during early vertebrate development

Abstract

All vertebrate embryos have multipotent cells until gastrulation but, to date, derivation of embryonic stem (ES) cell lines has been achieved only for mouse and primates. ES cells are derived from mammalian inner cell mass (ICM) tissue that express the Class V POU domain (PouV) protein Oct4. Loss of Oct4 in mice results in a failure to maintain ICM and consequently an inability to derive ES cells. Here, we show that Oct4 homologues also function in early amphibian development where they act as suppressors of commitment during germ layer specification. Antisense morpholino mediated PouV knockdown in Xenopus embryos resulted in severe posterior truncations and anterior neural defects. Gastrulation stage embryos showed reduced expression of genes associated with uncommitted marginal zone cells, while the expression of markers associated with more mature cell states was expanded. Importantly, we have tested PouV proteins from a number of vertebrate species for the ability to substitute Oct4 in mouse ES cells. PouV domain proteins from both Xenopus and axolotl could support murine ES cell self-renewal but the only identified zebrafish protein in this family could not. Moreover, we found that PouV proteins regulated similar genes in ES cells and Xenopus embryos, and that PouV proteins capable of supporting ES cell self-renewal could also rescue the Xenopus PouV knockdown phenotype. We conclude that the unique ability of Oct4 to maintain ES cell pluripotency is derived from an ancestral function of this class of proteins to maintain multipotency.

Negative autoregulation of Oct3/4 through Cdx1 promotes the onset of gastrulation

“…The former aimed at maintaining the pluripotent state while the latter promotes gastrulation. The opposed and mutually repressive roles of the Oct3/ 4 and Cdx factors have been previously described in early mouse embryos, embryonic stem cells, and early tailbud frog embryos (Niwa et al, 2005;Morrison and Brickman, 2006). Our results show that this same regulatory module, i.e., the negative cross-regulation between Oct3/4 and Cdx, is employed to regulate the transition into gastrulation.…”

“…These results identify Oct3/4 as a factor contributing to the expression of the Cdx genes and actually activating one of the factors involved in its own repression. Several studies have shown that during gastrulation, the Xenopus Oct3/4 factors function in a manner consistent with the active maintenance of the pluripotent state like their mammalian homologues (Henig et al, 1998;Cao et al, 2006Cao et al, , 2007Morrison and Brickman, 2006;Snir et al, 2006). Thus, Cdx1 acting as a negative regulator of the Pou5f1 genes during gastrulation, would ultimately function to end the undifferentiated state and facilitate the transition into gastrulation.…”

“…The array consists of unique genes identified by whole mount in situ hybridization (Pollet et al, 2005), and additional unique genes selected from Xenopus embryonic EST sequenced clones. Expression of the Xenopus Oct3/4 (Pou5f1) gene family, Oct25, Oct91, and Oct60 (Hinkley et al, 1992;Morrison and Brickman, 2006), was altered as a result of Cdx1 manipulation. These genes were down-regulated beyond two standard deviations in Cdx1-overexpressing embryos compared to control siblings.…”

“…The Xenopus genome has three class V, Oct3/4-like genes, Oct25 (Pou5f1.1), Oct91 (Pou5f1.2), and Oct60 (Pou5f1.3) (Hinkley et al, 1992;Morrison and Brickman, 2006). A number of studies support the functional conservation between mouse Oct3/4 and the Xenopus Pou5f1 genes.…”

“…A number of studies support the functional conservation between mouse Oct3/4 and the Xenopus Pou5f1 genes. The Xenopus Pou5f1 genes perform functions consistent with the maintenance of pluripotency, the regulation of gastrulation, the prevention of germ layer differentiation, and they can even maintain the pluripotency of mouse ES cells to different extents (Henig et al, 1998;Cao et al, 2006Cao et al, , 2007Morrison and Brickman, 2006;Snir et al, 2006). Also, the murine Oct3/4 protein can rescue the developmental malformations resulting from knock-down of the frog Pou5f1 gene activities (Cao et al, 2006).…”