Plakoglobin is a mechanoresponsive regulator of naïve pluripotency

Background

Physical and chemical stimuli from the external environment are key to development; they dictate the processes that transform a single cell into a complex multicellular organism (Heisenberg and Bellaiche, 2013). Even before implantation, during the earliest stages of embryogenesis, naive pluripotent cells must integrate these cues as they are directed towards specific cell fates (lineage specification) (Chan et al., 2017, Vining and Mooney, 2017). But we still lack full understanding of how mechanical signals contribute to this process. How are physical cues, like volumetric compression and spatial confinement, translated into transcriptional changes during naïve pluripotency? Which are the mechanosensitive genes involved?

In their study, Kohler and colleagues tackle these fundamental questions by studying the transcriptional and morphological development of naïve pluripotent embryonic stem cells (ESCs) encapsulated in 3D agarose microgels. Using this approach, they demonstrate that volumetric confinement affects pluripotency and show that expression of Plakoglobin, a vertebrate homologue of β-catenin, is required for naive pluripotency.

Key findings

Kohler et al encapsulated mouse ESCs inside spherical agarose microgels (Fig 1A). This system allows the authors to address their questions because mouse ESCs capture naïve pluripotency in vitro and microgels provide a 3D culture scaffold that mimics the volumetric confinement experienced by cells in the developing embryo. Once generated, microgels were cultured in either: self-renewal conditions in 2i/LIF medium, which sustains naïve pluripotency indefinitely; self-renewal conditions in serum/LIF, which supports a metastable state; or differentiating conditions in N2B27 medium to induce cell differentiation. The naïve (KLF4) and general (OCT4) pluripotency markers were used to confirm these conditions. Accordingly, pluripotency markers were expressed under self-renewal conditions, and upon transfer to differentiating conditions for 2 days, KLF4 and OCT4 were downregulated and absent, respectively. To assess the effects of 3D culture on ESCs, Kohler and colleagues utilized a reporter cell line, Rex1::GFPd2 (hereafter RGd2 cells) as a quantitative and real-time measure of naïve pluripotency in cells. Homogeneous GFP signal in 2i/LIF medium indicated naïve pluripotency whereas loss of expression in N2B27 indicated exit from naïve pluripotency. Thus, 3D agarose microgels are suitable for culture of naive pluripotent ESCs.

To assess how 3D microgels affect transcription, the authors compared gene expression data from naive ESCs in 3D microgels to naïve ESCs in 2D tissue culture. In addition to the global increase of naïve pluripotency markers in 3D, the most differentially expressed genes included components of the Wnt/β-catenin signaling pathways, notably Jup. The gene Jup encodes for the protein Plakoglobin, the vertebrate homologue of β-catenin. Analysis of the genes associated with cell-cell adherens junction (AJs), where β-catenin normally localizes, confirmed that Jup shows the strongest increase at AJs. Likewise, immunofluorescence analysis of the cell adhesion proteins at AJs confirmed that Plakoglobin localized to AJs in 3D microgels but not in 2D, whereas β-catenin was expressed in both but was higher in 2D (Fig 1B).

Figure 1 | ESC encapsulation in 3D microgels promotes expression of β-catenin homologue, Plakoglobin
a. Schematic of the microfluidic technique used to encapsulate ESCs in 3D microgels. b. Immunofluorescence images and quantification for Plakoglobin (left) and β-catenin (right) in 2D culture vs. 3D microgels.

Importantly, the authors also compared their findings to hanging drop cultures, where ESCs grow in a 3D environment without confinement. This culture system would allow them to confirm that the observed effects are due to volumetric confinement from 3D microgels and not just the 3D environment. They found that β-catenin was observed in both conditions, whereas Plakoglobin was upregulated only in 3D microgels. Even in the case where ESCs were released from 3D microgels after 48h and transferred to hanging drop cultures, Plakoglobin expression was lost. This confirmed that volumetric confinement generated by 3D microgels is required for Plakoglobin expression.

To corroborate the link between naïve pluripotency and Plakoglobin in vivo, Kohler and colleagues also analyzed human and mouse embryos. Consistent with their findings in naïve ESCs, they found high expression of Jup in the pre-implantation mouse embryo, when naïve pluripotency is established. At the protein level, the expression of Plakoglobin correlated with expansion of the blastocyst’s cavity. Although harder to assess in vivo, these observations suggest that an increase in pressure from expansion of the cavity may provide the mechanical signal that regulates Plakoglobin.

If 3D microgels promote Plakoglobin expression to maintain pluripotency, could exogenous overexpression of Plakoglobin mimic the effects of 3D microgel culture? To this end, Kohler et al generated Plakoglobin overexpressing cells and derived clonal ESC lines with either low or high Plakoglobin expression levels. Overexpression of exogenous Jup was assessed with mCherry (via Jup-2A-mCherry) and pluripotency with GFP (via Rex1::GFPd2). The use of both low and high Plakoglobin expressing cell lines indicated that under metastable conditions (serum/LIF), a high expression of Plakoglobin is required to retain both the tissue morphology and homogeneous GFP expression associated with naive pluripotency. Thus, high expression of Plakoglobin stabilizes naïve pluripotency in metastable culture conditions.

Finally, Kohler and colleagues wondered if Plakoglobin can maintain naïve pluripotency independently of β-catenin. For this, they analyzed Plakoglobin expressing cells under two conditions: upon XAV treatment, a drug that blocks nuclear signaling of β-catenin, and upon knockout of β-catenin in Plakoglobin expressing cells. In both cases, cells remained naïve pluripotent. Plakoglobin can thus maintain naïve pluripotency independently of β-catenin. This evidence and the encapsulation of mouse ESCs in 3D agarose microgels allows the authors to show that volumetric compression is the upstream mechanical signal that activates molecular signaling via Plakoglobin. This probably occurs in vivo as expansion inside the blastocyst’s cavity generates pressure (the mechanical cue) to regulate naïve pluripotency.

Why I chose this preprint

I chose this preprint because I truly enjoyed reading it but also because it is a comprehensive study on the interplay between forces, a molecular player that is mechanosensitive (Plakoglobin) and the key role of this link during naïve pluripotency. The authors provide thorough (and complementary) evidence to support their main claim, with morphological and transcriptional data in both in vitro 3D culture and in vivo in mouse and human embryos. This study also paves the way (and emphasises the need) for 3D systems to fully replace 2D culture systems in in vitro studies of development.

References

Heisenberg, C.P. & Bellaiche, Y. Forces in tissue morphogenesis and patterning. Cell, 153, 948-62 (2013).

Chan, C.J., Heisenberg, C.P. & Hiiragi, T. Coordination of Morphogenesis and Cell-Fate Specification in Development. Current Biology, 27, R1024-R1035 (2017).

Vining, K., Mooney, D. Mechanical forces direct stem cell behaviour in development and regeneration. Nat Rev Mol Cell Biol 18, 728–742 (2017).

 

doi: https://doi.org/10.1242/prelights.31979

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