YAP1 Regulates the Self-organized Fate Patterning of hESCs-Derived Gastruloids

Background:  

Gastrulation is the morphogenetic event that gives rise to the three founder lineages of all organs and tissues – ectoderm, mesoderm, and endoderm – from the pluripotent epiblast. Beyond generating these three germ-layers, gastrulation must ensure their proper location and adequate size. This is achieved through the spatio-temporal regulation of the Nodal pathway [1,2]. While Nodal inhibition prompts acquisition of neuroectoderm potential, its activation with cooperative action from BMP and Wnt signaling directs the formation of the other two lineages [3,4].  

Nodal antagonists are secreted by the non-pluripotent cell population in mice and human blastocysts [5,6]. However, the spatial patterning of all three germ layers has been recapitulated by solely culturing Embryonic Stem Cells (ESCs), which are the in vitro counterparts of the pluripotent epiblast. When cultured in defined media conditions, human ESCs (hESCs) undergo morphological and molecular changes reminiscent of gastrulation. Hence, they are referred to as “gastruloids”.  Importantly, fate patterning in both 2D hESC cultures and symmetry breaking in 3D gastruloids were achieved without the precise spatial regulation of the Nodal pathway [7,8]. This hints at the existence of a hitherto unknown cell-intrinsic mechanism which regulates Nodal spatial distribution and thereby governs lineage size and positioning.  

In this preprint, the authors elucidate the molecular logic behind Nodal-mediated fate patterning. They show that the size and location of germ layers and fate choice of hESCs are dictated by the transcriptional regulator YAP1. To achieve this, they employ a robust 2D model of human gastrulation based on micro-patterned hESCs. The key experimental techniques performed include directed differentiation, immunofluorescence, confocal microscopy, western blotting, and genome sequencing analysis. 

Key findings: 

1. YAP1 depletion alters the size and location of 3 discrete germ layers

   Previous work [9] identified the fate restriction role of YAP1 during hESC differentiation. The rationale for focusing on YAP1 also includes its effector function in the Hippo pathway for organ size control. Therefore, the authors surveyed the gastrulation outcome of YAP1 knock-out cells (YAP1-KO). To do so, they confocally imaged hESC 2D-gastruloids immunostained for the putative lineage markers of the 3 germ layers SOX2 (ectoderm), SOX17 (endoderm), and Brachyury/T(Mesoderm). Through image analysis, they observed that YAP1-KO samples had a reduction in the share of SOX2+ve ectodermal cells and a compensatory increase in the individual proportions of mesodermal and endodermal cells. Moreover, the organization of the three germ layers was disrupted: (i) ectoderm located in the center of wild-type (WT) gastruloids versus at the colony periphery in YAP1-KO gastruloids and (ii) thicker mesendoderm in YAP1-KO gastruloids (Figure.1 A&B).
   

   

2. YAP1 promotes ectodermal specification by cytoplasmic retention of SMAD-2.3
   

   Since the percentage of ectodermal cells was reduced upon YAP1 depletion, the authors wanted to test if this was due to an inherent inability of fate decision making. For this, they performed directed differentiation towards individual germ layers. Through transcriptomic analysis, they observed that despite biochemical ectoderm induction, YAP1-KO hESCs continued to express high levels of core pluripotency factors including Pou5f1 (Oct4) and specifically Nanog. The latter is known to repress neuronal differentiation through its functional antagonism of Otx2 [10], an ectoderm specifier. By rescuing YAP1 levels through an inducible expression system, the authors were able to restore Nanog downregulation and Otx2 expression.  

   Having confirmed the repression of Nanog, the authors wanted to identify the molecular framework for YAP1’s inhibitory action. For this, they focused on investigating changes in the levels of SMAD-2.3 and SMAD-1.5 in WT vs YAP1-KO cells. SMAD-2.3 and SMAD-1.5 are respectively the effectors of NODAL and BMP signaling pathways, the two cascades which inhibit ectoderm fate acquisition. More importantly, nuclear SMAD-2.3 drives Nanog expression [11], the phenotypic outcome of YAP1-KO cells in directed differentiation experiment. Therefore, the authors performed western blotting with antibodies targeting the aforementioned SMADs. Additionally, the protein lysates were separated into nuclear and cytoplasmic fractions to observe changes in both overall protein level and subcellular distribution. While SMAD-1.5 had no discernible changes, SMAD-2.3 levels increased in YAP1-KO cells, and it was predominantly localized in the nucleus. The authors tested the relevance of this result through neuronal induction of geometrically confined hESCs. In line with earlier observations, YAP1-KO cells exhibited predominantly nuclear SMAD-2.3 and lacked the expression of the ectodermal marker Sox2 (Figure.2).  

   Thus, the authors proved that ectodermal specification requires YAP1-mediated inhibition of nuclear SMAD-2.3 activity. 

             

3. Direct transcriptional regulation of developmentally relevant genes by YAP1

   The high nuclear levels of SMAD-2.3, a downstream component of the Nodal cascade, prompted the authors to test for expression levels of the Nodal signaling pathway in ectoderm-induced WT and YAP1-KO cells. For this, they utilized the RNA-seq datasets obtained from directed differentiation experiments and performed Signal Pathway Analysis. After filtering the results based on relevance and statistical significance, the authors observed that YAP1-KO hESCs were enriched in genes driven by Nodal signaling. When RT-qPCR was performed for the selected NODAL genes after inducible YAP1 rescue, their levels were found to be reduced. This confirms the transcriptional repression action of YAP1 on NODAL pathway genes.  

   To gain further insight into the regulatory mechanism of YAP1, the authors performed single-nuclei ATAC sequencing. This technique identifies transcriptionally accessible chromatin regions of the genome. The differentially expressed regions can be categorized into proximal and distal which rely on promoter-based and enhancer-based transcriptional regulation, respectively. When the authors probed the loci that gained transcriptional accessibility upon YAP1 depletion, these were found to be upstream of NODAL and FOXH1 genes. ChiP-Seq analysis did not only confirm but also identify those putative-YAP1-binding sites. Importantly, the YAP1-bound region of NODAL was found to be the well-characterized Proximal Epiblast Enhancer (PEE). Further analysis identified a conserved YAP1 binding site of PEE in the mouse genome. This suggests an evolutionarily conserved role of YAP1 for NODAL regulation during gastrulation.  

   Finally, the authors wanted to confirm if the observed gastrulation defects in YAP1-KO hESCs were due to upregulated NODAL signaling. For this, they performed a 2D gastrulation assay using YAP1-KO with NODAL inhibition. With this approach, they were able to rescue the fate patterning defect. Ectoderm was restricted to the center and the other two lineages to the periphery of the colony. Moreover, directed differentiation proved that NODAL inhibition rescued the ability of YAP1-KO cells to be specified towards the ectodermal lineage (Figure.3).

   

Conclusion:

This work proves that YAP1 ensures appropriate fate patterning during hESC gastrulation via its transcriptional repression of NODAL signaling. The transcriptional regulation is expected to be mediated through YAP1 binding to the NODAL enhancer PEE.

What I like about this preprint:

This work approached the process of gastrulation from the viewpoint of cell-intrinsic mechanisms. It showed that when it comes to germ layer specification, epiblast cells are not simply a bystander population obliging to morphogen concentration. Rather, transcriptional regulatory mechanisms allow each cell to be capable of fate decision-making despite the lack of fine-tuned signaling gradients. YAP1, the transcriptional factor in the focus of this study, is mechano-sensitive and has evolutionarily conserved binding sites to key developmental genes. This opens an avenue to study the contribution of in vivo mechanical signaling in the regulation of mammalian gastrulation using mice, thereby circumventing the ethical regulations associated with hESC research. In addition, human-iPSC germ layer specification was shown to be similar to that of hESCs. The knowledge from this work could therefore be adapted to direct differentiation of hiPSCs for effective regenerative therapies.

Questions to the authors: 

1. YAP1 represses mesendodermal lineages. Given that the edge of 2D gastrulating hESC colonies were mesendodermal, did you observe the nuclear exclusion/down-regulation of YAP1 in the edge cells through immunostaining? 
2. Genome topology reorganization by YAP1 is reported to be driven by its phase separation [12]. Having said that, did you observe any phase-separated YAP1 foci in the center of the hESC gastruloids? 
3. YAP1 functions as anti-apoptotic agent in mESCs during differentiation [13]. As cells in YAP1-KO gastruloids appear to be less densely packed, could this be due to increased cell death? Does this reduced density facilitate cell mingling, thereby allowing misplacement of ectoderm lineage?    

References 

1. Brennan J, Lu CC, Norris DP, Rodriguez TA, Beddington RS and Robertson EJ. Nodal signalling in the epiblast patterns the early mouse embryo. Nature. 2001;411:965–9 
2. Brennan J, Norris DP and Robertson EJ. Nodal activity in the node governs left-right asymmetry. Genes Dev. 2002;16:2339–44 
3. Chambers SM, Fasano CA, Papapetrou EP, Tomishima M, Sadelain M and Studer L. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol. 2009;27:275–80 
4. Arnold SJ and Robertson EJ. Making a commitment: cell lineage allocation and axis patterning in the early mouse embryo. Nat Rev Mol Cell Biol. 2009;10:91–103 
5. Vallier L, Mendjan S, Brown S, Chng Z, Teo A, Smithers LE, Trotter MW, Cho CH, Martinez A, Rugg-Gunn P, Brons G and Pedersen RA. Activin/Nodal signalling maintains pluripotency by controlling Nanog expression. Development. 2009;136:1339–49 
6. Xu RH, Sampsell-Barron TL, Gu F, Root S, Peck RM, Pan G, Yu J, Antosiewicz-Bourget J, Tian S, Stewart R and Thomson JA. NANOG is a direct target of TGFbeta/activin-mediated SMAD signaling in human ESCs. Cell Stem Cell. 2008;3:196–206. 
7. Moris N, Anlas K, van den Brink SC, Alemany A, Schröder J, Ghimire S, Balayo T, van Oudenaarden A and Martinez Arias A. An in vitro model of early anteroposterior organization during human development. Nature. 2020;582:410–415 
8. Turner DA, Girgin M, Alonso-Crisostomo L, Trivedi V, Baillie-Johnson P, Glodowski CR, Hayward PC, Collignon J, Gustavsen C, Serup P, Steventon B, P Lutolf M and Arias A. Anteroposterior polarity and elongation in the absence of extra-embryonic tissues and of spatially localised signalling in gastruloids: mammalian embryonic organoids. Development. 2017;144:3894–3906 
9. Hsu HT, Estarás C, Huang L and Jones K. Specifying the Anterior Primitive Streak by Modulating YAP1 Levels in Human Pluripotent Stem Cells. Stem Cell Reports. 2018;11:1357–1364 
10. Su Z, Zhang Y, Liao B, et al. Antagonism between the transcription factors NANOG and OTX2 specifies rostral or caudal cell fate during neural patterning transition. J Biol Chem. 2018;293(12):4445-4455. doi:10.1074/jbc.M117.815449 
11. Vallier L,  Mendjan  S,  Brown  S,  Chng  Z,  Teo  A,  Smithers  LE,  Trotter  MW,  Cho  CH, Martinez  A,  Rugg-Gunn  P,  Brons  G  and  Pedersen  RA.  Activin/Nodal  signalling  maintains pluripotency by controlling Nanog expression. Development. 2009;136:1339-49 
12. Cai, D., Feliciano, D., Dong, P. et al. Phase separation of YAP reorganizes genome topology for long-term YAP target gene expression. Nat Cell Biol 21, 1578–1589 (2019). 
13. LeBlanc L, Lee BK, Yu AC, et al. Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation. Elife. 2018;7:e40167. 

Tags: cell intrinsic mechanisms, fate patterning, gastrulation, human 2d gastruloids, lineage allocation

doi: https://doi.org/10.1242/prelights.29810

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