Axolotl Nanog activity in mouse embryonic stem cells demonstrates that ground state pluripotency is conserved from urodele amphibians to mammals

Abstract

Cells in the pluripotent ground state can give rise to somatic cells and germ cells, and the acquisition of pluripotency is dependent on the expression of Nanog. Pluripotency is conserved in the primitive ectoderm of embryos from mammals and urodele amphibians, and here we report the isolation of a Nanog ortholog from axolotls (axNanog). axNanog does not contain a tryptophan repeat domain and is expressed as a monomer in the axolotl animal cap. The monomeric form is sufficient to regulate pluripotency in mouse embryonic stem cells, but axNanog dimers are required to rescue LIF-independent self-renewal. Our results show that protein interactions mediated by Nanog dimerization promote proliferation. More importantly, they demonstrate that the mechanisms governing pluripotency are conserved from urodele amphibians to mammals.

Fig. 1.

Conservation of Nanog genomic structure, expression profile and transcriptional activity. (A) Intron/exon structures of NANOG (human), Nanog (mouse) and axNanog (axolotl) are aligned. Blue boxes denote protein coding regions. Numbers in blue represent amino acids. Numbers in red are intron lengths; ND, not determined. Black numbers indicate combined length of protein coding region with 5′ and 3′ untranslated regions. (B) Conserved synteny of Nanog genes. Genes linked to axNanog on linkage group 3 (AxLG3) map to human chromosomes 7 and 12 (Hsa7, Hsa12) and chicken chromosome 1 (GG1). (C) Whole-mount in situ hybridization (WISH) showing co-expression of axNanog and axOct4 in the animal cap of blastulae (arrow). Arrowhead points to blastopore. (D) Protein complementation assay (PCA) analyses of axNanog protein-protein interaction. (Top) Vectors express the interactors (cDNA1 and cDNA2) fused to hGL1 and hGL2. (Bottom) PCA shows that axNanog::WR fusions form homodimers, and axNanog monomers interact with axOct4. *P<0.05. (E) Reporter expression from Nanog targets after co-transfection with the indicated constructs (:: indicating substitution with homeodomain, HD). *P<0.05. (F) QPCR quantification of Nanog and Oct4 promoters after immunoprecipitation by anti-myc antibody (mean±s.d.; n=3). Results show fold enrichment of precipitated DNA relative to a 1/100 dilution of input chromatin. *P<0.05.

Fig. 2.

AxNanog dimerization is required to rescue ESC self-renewal. (A) ESC counts of cultures transfected with Nanog variants grown for four passages with or without LIF. (B) Representative plates showing alkaline phosphatase (AP) expression (red) of ESC cultures expressing Nanog or axNanog monomers or homodimers with and without LIF. (C) Representative cultures of Nanog::ZIP- and FKBPv-expressing ESCs cultured with and without LIF or AP20187. (D) QPCR analysis of Oct4 and Tert expression in Nanog variant-expressing ESC cultures with or without LIF (analyzed at P3). Results show fold increase relative to control calibrator sample (mean±s.d.; n=3). (E) PCA showing interaction of axNanog with Nac1, Dax1, Sall4 and Hdac1 (mean±s.d.; n=3). (F) Representative images of cultures and colonies showing reduced AP activity (red) and flattened morphology of Nanog-expressing ESCs cultured for 25 passages without LIF. Scale bar: 200 μm. (G) QPCR showing levels of Oct4, Rex1 and Fgf5 in cultures rescued by Nanog or axNanog dimers (mean±s.d.; n=3).

Fig. 3.

AxNanog enhances fusion-based reprogramming and prevents embryoid body (EB) differentiation. (A) Hybrid colonies from ESC fusions (expressing Nanog and puromycin resistance) with NSCs (expressing Oct4-GFP and Neo). (B) Reprogrammed hybrid colony. (C) Quantification of hybrids colonies (mean±s.d.; n=3). (D) Representative image of EB differentiation assay. QPCR analyses of EBs expressing axNanog at 3, 5 or 10 days of differentiation (mean±s.d; n=3). (E) Oct4-GFP fluorescence is maintained in EBs from ESC:NSC hybrids expressing axNanog. (F) QPCR analyses of Oct4-GFP expression during hybrid EB differentiation (mean±s.d.; n=3). Scale bars: 200 μm in B; 500 μm in D,E.

Fig. 4.

Transformation and proliferation are enhanced in somatic cells by Nanog homodimers. (A) Enhanced proliferation of 3T3 lines expressing Nanog or axNanog dimers. Shown are cell counts of triplicate experiments presented as a percentage of cell numbers from control cell lines (mean±s.d.; n=3). (B) Foci are induced by dimers of Nanog or axNanog. Representative foci (top) and number of foci (bottom) in cultures (mean±s.d; n=3). Scale bars: 100 μm.