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NANOG is required to establish the competence for germ-layer differentiation in the basal tetrapod axolotl [1]

['Luke A. Simpson', 'School Of Life Sciences', 'University Of Nottingham', 'Queens Medical Centre', 'Nottingham', 'United Kingdom', 'Darren Crowley', 'Teri Forey', 'Helena Acosta', 'Zoltan Ferjentsik']

Date: 2023-06

Pluripotency defines the unlimited potential of individual cells of vertebrate embryos, from which all adult somatic cells and germ cells are derived. Understanding how the programming of pluripotency evolved has been obscured in part by a lack of data from lower vertebrates; in model systems such as frogs and zebrafish, the function of the pluripotency genes NANOG and POU5F1 have diverged. Here, we investigated how the axolotl ortholog of NANOG programs pluripotency during development. Axolotl NANOG is absolutely required for gastrulation and germ-layer commitment. We show that in axolotl primitive ectoderm (animal caps; ACs) NANOG and NODAL activity, as well as the epigenetic modifying enzyme DPY30, are required for the mass deposition of H3K4me3 in pluripotent chromatin. We also demonstrate that all 3 protein activities are required for ACs to establish the competency to differentiate toward mesoderm. Our results suggest the ancient function of NANOG may be establishing the competence for lineage differentiation in early cells. These observations provide insights into embryonic development in the tetrapod ancestor from which terrestrial vertebrates evolved.

Given the role of NANOG in programming pluripotency in mammalian embryos [ 14 ], we investigated the role of NANOG during early axolotl development to gain insights into the mechanisms governing pluripotency in basal tetrapods. We show that zygotically expressed NANOG is required for gastrulation. Animal caps (ACs) depleted of NANOG lack the competence to differentiate into embryonic germ layers in response to inductive activin signals. Unlike in zebrafish, we find no evidence that NANOG activates the zygotic genome. In addition, we find that NANOG-depleted embryos lack the active transcription-associated marks H3K4me3 and H3K27ac. Moreover, depletion of NODAL signalling or depletion of the epigenetic modifying enzyme DPY30 both result in a similar loss of H3K4me3 and H3K27ac and an inability to form mesoderm in response to inductive cues. This is reminiscent of findings in human ESC (hESC) whereby NANOG, SMAD2, and DPY30 form a complex to regulate H3K4me3 deposition at SMAD2 target gene loci, suggesting that this mechanism may be conserved between axolotls and humans [ 16 ].

Despite the fact that NANOG, POU5F1, and SOX2 genes appear to be highly conserved throughout vertebrates [ 19 – 22 ], little is known about their function in non-mammals. Axolotl have retained both Nanog and Pou5f1 genes; however, the former has been lost in frogs, while the latter has been lost in zebrafish, frogs, and chicks [ 20 , 21 , 23 ]. Axolotls are therefore a useful model for studying the origins of cell determination in the tetrapod ancestor from which mammals, amphibians, and reptiles evolved [ 4 ].

The pluripotency gene regulatory network (GRN) has been extensively studied in mammals in vitro. It has long been established that the transcription factor NANOG, as well as POU5F1 and SOX2 cooperate to support mammalian pluripotency [ 9 – 14 ]. In the mouse, NANOG itself is absolutely required for the reprogramming of somatic cells back to a pluripotent state in vitro [ 15 ]. Moreover Nanog-null mice fail to establish a pluripotent epiblast and die preimplantation [ 14 ]. NANOG has been implicated in a variety of roles to support pluripotency as an independent transcription factor through regulation of the epigenome [ 16 – 18 ]. In contrast, Nanog is not required for the maintenance of mouse embryonic stem cells (mESC) and Nanog-null mESC retain their competence to form all embryonic cell types in the context of chimeras [ 10 ]. Together, this suggests that NANOG is required for the initial establishment of the pluripotent state in mammals but is not required for its maintenance.

In mammals, pluripotency is defined as the competency of a single cell to contribute to both the soma and germ line [ 1 – 3 ]. Many divergent strategies have evolved throughout the animal kingdom for producing all necessary embryonic cell types. For example, many nonmammalian model organisms such as chicks, frogs, and zebrafish do not form both the soma and the germ line from a common pool of cells, instead, maternally inherited determinants segregate the germ cells. In contrast, mammals, and other amphibians such as the axolotl, induce germ cells and soma from the same cells. This mechanism of germ cell determination, known as epigenesis, appears to be basal while the mechanism observed in chicks, frogs, and zebrafish, known as preformation, appears to be derived [ 4 – 8 ]. It is unclear as to whether the lack of requirement for germ cell competence in animals exhibiting preformation has led to changes in the mechanisms governing pluripotency.

Results

NANOG-depleted ACs cannot respond properly to inductive signals To address if NANOG LOF resulted in a lack of competency to form individual germ layers or simply a lack of proper differentiation cues, we tested the developmental potential of NANOG-depleted AC directly using explants (Figs 3D and S4A–S4F). Explanted caps normally differentiate into epidermis, marked by increased expression of Grhl1 and Cytok, and down-regulation of Nanog and Pou5f1 transcripts. Injection of Activin A (Activin) mRNAs at the 1-cell stage can divert the fate of these cells to other lineages including mesoderm, endoderm, or PGCs in a dose-dependent manner [8,13]. We have previously demonstrated that 1 pg of RNA encoding activin injected at the 1-cell stage induces mesoderm, marked by elongation of the animal cap explant and Tbxt expression [13]. By titration, we established that 200 fg of RNA is insufficient to divert caps from an epidermal fate to mesoderm (Figs 3D and S4D). Higher levels of activin (100 pg) are required to drive caps to endoderm as marked by Sox17 expression and a lack of elongation. We utilised this assay to directly test the ability of NANOG-depleted cells to form tissues. Morphant caps with no exogenous Activin failed to produce epidermis and maintained high expression of Nanog and Pou5f1 mRNA levels (S4B Fig). NANOG-depleted caps injected with sub-mesodermal (200 fg) doses of activin showed a large increase in Sox17, but not Tbxt expression, and 1 pg of activin also induced high levels of endoderm marker Sox17, also without induction of Tbxt (Fig 3D). These data suggest that AC’s depleted of NANOG are hypersensitised to TGF-ß signalling. In amphibians, the foregut derives from organiser endoderm that originates in the animal hemisphere of the embryo, while the hindgut is produced from the yolky cells of the vegetal hemisphere [35–38]. Morphant caps injected with 100 pg of exogenous activin at the 1-cell stage did not show increased expression of foregut marker Nkx2.6 or hindgut marker Hnf4g over uninjected controls, though these caps did express C8b, an endoderm commitment marker expressed post gastrulation (S4E Fig). This suggests that in the absence of NANOG, the cells of the animal hemisphere arrest endoderm development at the progenitor stage. Together, these data suggest that NANOG-depleted AC’s cannot produce mesoderm even in the presence exogenous ACTIVIN signalling. While NANOG KD AC are able to activate some early markers of definitive endoderm, we do not find any evidence of more mature endodermal subtypes. Moreover, NANOG-depleted caps appear to hypersensitised to TGF-ß signalling. The vegetal pole cells of axolotl appear to be NANOG-independent we surmise, therefore that hindgut endoderm expression in morphant embryos is derived from the vegetal pole, which does not express Nanog mRNA [23], while anterior foregut endoderm, as well as mesoderm and ectoderm development from animal hemisphere is arrested by NANOG depletion in line with Nanog’s expression profile.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002121

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