April 1, 2011;
Negative autoregulation of Oct3/4 through Cdx1 promotes the onset of gastrulation.
Gastrulation marks the onset of germ layer formation from undifferentiated precursor cells maintained by a network including the Pou5f1 gene, Oct3/4. Negative regulation of the undifferentiated state is a prerequisite for germ layer formation and subsequent development. A novel cross-regulatory network was characterized including the Pou5f1 and Cdx1
genes as part of the signals controlling the onset of gastrulation. Of particular interest was the observation that, preceding gastrulation, the Xenopus Oct3/4 factors, Oct60
, and Oct91
, positively regulate Cdx1
expression through FGF signaling, and during gastrulation the Oct3/4 factors become repressors of Cdx1
negatively regulates the Pou5f1 genes during gastrulation, thus contributing to the repression of the network maintaining the undifferentiated state and promoting the onset of gastrulation. These regulatory interactions suggest that Oct3/4 initiates its own negative autoregulation through Cdx1
up-regulation to begin the repression of pluripotency in preparation for the onset of gastrulation and germ layer differentiation.
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Figure 1. Cross-regulation between Cdx1 and the Pou5f1 genes during the onset of gastrulation. The expression levels of the Xenopus Oct3/4 genes, Oct25, Oct91, and Oct60, and Cdx1 were studied by qPCR in embryos with manipulated levels of Cdx1 or the Pou5f1 gene activity. A–C:Cdx1 gain-of-function was induced by capped RNA injection (1.6 ng/embryo) and the embryos were analyzed during blastula stages (st. 9 and st. 9.5) or Cdx1 activity knockdown was induced by injection of the specific Cdx1 antisense morpholino oligonucleotide (Cdx1MO; 5 ng/embryo). Embryos were analyzed during late blastula (st. 9.5) and early gastrula (st. 10). The effect on Oct25 (A), Oct91 (B), and Oct60 (C) was determined by qPCR. D: Analysis of the regulatory effect of the Oct3/4 factors on Cdx1 expression was performed by qPCR. Oct3/4 gain-of-function was induced by mouse Oct3/4 mRNA injection (1.28 ng/embryo), Oct60 overexpression was induced by RNA injection (0.8 ng/embryo), while partial loss-of-function was induced by co-injection of Oct25MO and Oct60MO (0.25 ng each/embryo). RNA samples were collected during late blastula (st. 9.5) and early gastrula (st. 10.25). The positive regulatory effect of the Oct3/4 genes on Cdx1 expression was also studied by whole mount in situ hybridization. Control (E) and Oct3/4 RNA injected (F) were analyzed for changes in the pattern of Cdx1 expression during late blastula stages in cleared embryos. *P < 0.05 and **P < 0.01 relative to control embryos. ***P < 0.01 relative to Oct3/4 or Oct60 overexpressing embryos at st. 9.5.
Figure 3. Cdx1 activity is required for the normal progression of gastrulation. To study the role of Cdx1 in the regulation of gastrulation, the activity of this gene was manipulated and different aspects of gastrulation were studied. A–C: Embryos were injected with Cdx1MO (5 ng/embryo) to induce a knockdown of Cdx1 activity (see also Supp. Fig. S1), or co-injection of Cdx1MO and the chicken Cdx1 RNA (cCdx1; 400 pg/embryo) to rescue the Cdx1 knockdown. The progression of gastrulation was studied by analyzing blastopore size. A: Control embryos. B: Embryos with delayed gastrulation resulting from reduced Cdx1 activity (Cdx1MO). C: Embryos with reduced Cdx1 activity were rescued by overexpression of cCdx1. D–F: Analysis of the effect of Cdx1 knockdown on cell proliferation was performed taking advantage of the cyclinB-GFP chimeric protein to identify proliferating cells. Embryos were injected in all four blastomeres at the 4-cell stage with the expression plasmid encoding the cyclinB-GFP protein. D: Control, E: Cdx1MO-injected embryos. The arrowheads mark the proliferating cells. F: Embryos with reduced Cdx1 activity and overexpressing the chicken Cdx1 mRNA. The dorsal marginal zone of the embryos was photographed. G–I: Analysis of cell proliferation based on BrdU incorporation. Embryos were injected unilaterally and then stained for BrdU incorporation. G: Control. H: Cdx1MO-injected embryos. I: Embryos co-injected with Cdx1MO and Oct3/4 RNA.
Figure 4. Cdx1 activity is required for germ layer differentiation. To study the effect of Cdx1 manipulation on germ layer differentiation, embryos injected with Cdx1MO to induce an activity knockdown (B,E,H,K) or overexpressing Cdx1 (C,F,I,L) were analyzed during late blastula (st. 9.5) to study the earliest expression of the germ layer markers. The embryos were processed for whole mount in situ hybridization with probes for the early mesodermal markers eomesodermin (A–C) and Xbra (D–F) or the endodermal markers Sox17α (G–I) and mixer (J–L), and cleared for photography
Figure 6. Oct3/4 contributes to germ layer differentiation. To determine whether Oct3/4 contributes to the transition into gastrulation, the effects on the early mesodermal markers, Xbra (A–D) and eomesodermin (E–H) were studied. (A,E) Control, (B,F) Oct3/4, (C,G) dnFGFR1 and (D,H) Oct3/4 and dnFGFR1. The injected embryos were incubated to late blastula and then processed for whole mount in situ hybridization and cleared at the end of the process.