Metabolic control of germ layer proportions through regulation of Nodal and Wnt signalling

During embryonic development, cells exit pluripotency to give rise to the three germ layers. Metabolic pathways influence cell fate decisions by modulating the epigenetic, transcriptional, and signalling states of cells. However, the interplay between metabolism and the major signalling pathways that drive the emergence of ectoderm, mesoderm, and endoderm remains poorly understood. Here, we demonstrate an instructive role of glycolytic activity in activating signalling pathways involved in mesoderm and endoderm induction. Using an in vitro model system for mouse gastrulation, we observed that inhibiting glycolysis prevents the upregulation of primitive streak markers, resulting in a significant increase in ectodermal cell fates at the expense of mesodermal and endodermal lineages. We demonstrate that this relationship is dose-dependent, enabling metabolic control of germ layer proportions through exogenous glucose levels. Mechanistically, we found that glycolysis inhibition leads to the downregulation of Wnt, Nodal, and Fgf signalling. Notably, this metabolic phenotype was rescued by Nodal or Wnt signalling agonists in the absence of glycolytic activity, suggesting that glycolytic activity acts upstream of both signalling pathways. Our work underscores the dependence of specific signalling pathways on metabolic conditions and provides mechanistic insight into the nutritional regulation of cell fate decision making.

Tissue Biology and Disease Modelling European Molecular Biology Laboratory (EMBL) Barcelona, Spain. 2 Genomics Core Facility, European Molecular Biology Laboratory (EMBL) Heidelberg, Germany. 3 Developmental Biology, European Molecular Biology Laboratory (EMBL) Heidelberg, Germany. 4 Cluster of Excellence Physics of Life, TU Dresden, Dresden, Germany. 5Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany. 6These authors contributed equally.*Corresponding authors: kristina.stapornwongkul@embl.es, ebisuya@embl.es,trivedi@embl.es,#Lead Contact: trivedi@embl.esdifferentiation [15].Pluripotent stem cells (PSCs) are thought to rely on high glycolytic activity to maintain their characteristic histone acetylation patterns [17].While some studies have suggested that metabolic switching is a prerequisite for epigenetic remodelling and differentiation [17,18], others have found that a shift towards oxidative metabolism is germ-layer specific and only occurs in mesodermal and endodermal cells [19].
In contrast to these findings from directed differentiation of cells using extrinsic signals, a study in the developing tailbud (neuromesodermal progenitors, NMPs) found that inhibition of glycolysis increased the proportion of neuroectoderm at the expense of the presomitic mesoderm via regulation of Wnt signalling [14].Unlike the bipotent NMPs, cells of the early embryo can still give rise to all future cell types.Therefore, the relationship between metabolism and signalling during the earliest stages of differentiation of pluripotent embryonic cells into the three germ layers remains unresolved.Recent studies, both in vitro and in vivo, have reported spatiotemporal restriction of different glucose transporters that accompany germ layer patterning and how metabolism affects signalling particularly during mesoderm specification [20,21].However, the simultaneous control of the three germ layers and their cell type-specific interplay between glycolysis and signalling remains obscure.
In this study, we further elucidate the interplay between glycolysis and the signalling pathways that coordinate germ layer differentiation.Using 3D mouse gastruloids, a stem-cell based model system that allows the co-differentiation of the three germ layers [22], we found that glycolysis plays a crucial role in both endoderm and mesoderm induction by activating Nodal, Wnt and Fgf signalling.Importantly, exogenous glucose (Glc) concentration has a dose-dependent effect on PS marker expression and the development of endodermal and mesodermal cell type derivatives.Thus, we show for the first time that glycolytic activity is not merely permissive but rather acts as an instructive signal which can be used to control germ layer proportions.Moreover, we were able to decouple the metabolic phenotype of glycolysis

Glycolysis is needed for T/Bra expression and symmetry breaking in gastruloids
To address the role of metabolism during germ layer specification, we used gastruloids, aggregates of mouse embryonic stem cells (mESCs) which specify cell types of all three germ layers while establishing an anterior-posterior (AP) axis [22].Between 24 and 48 hours post aggregation (hpa), gastruloids upregulate the PS and early mesoderm marker Brachyury (T/Bra) throughout the tissue (Figure 1A) [23,24].
160 Furthermore, we found an upregulation of the most anterior 161 Hox gene and hindbrain marker Hoxa2 while posterior Hox genes and Cdx2, which marks the posterior embryo, were strongly reduced (Figure 1D, Figure S2E) [34,35].This suggests an anteriorisation of 2-DG treated gastruloids.
The shift in germ layer proportions was further confirmed by in situ hybridisation chain reaction (HCR) showing that in contrast to control gastruloids, cells expressing T/Bra (PS/early mesoderm) and Sox17 (definitive endoderm) transcripts were strongly reduced in 2-DG treated gastruloids (Figure 1E).Instead, Sox2 (ectoderm/pluripotent) was expressed throughout the glycolysis inhibited gastruloids.Further stainings showed that the Sox2 + domain was subdivided in clusters of cells expressing the neuroectodermal marker Sox1 or the pluripotency marker Nanog (Figure S2F) [36,37].Both of these markers were overrepresented compared to control gastruloids, confirming the transcriptomics results (Figure S2C,D).These results show that the inhibition of glycolysis between 24 and 48hpa has long-term effects on gastruloid development and results in a shift of germ layer proportions away from posterior mesodermal and endodermal derivatives towards more anterior neuroectodermal cell fates.

Glucose has a dose-dependent effect on the proportion of T/Bra::GFP expressing cells
The observed effects of 2-DG treatment on gastruloid development raise the question whether glycolytic activity has a permissive or instructive role in T/Bra expression and germ layer induction.A permissive role would suggest the existence of a certain threshold of cellular glycolytic activity needed to allow T/Bra upregulation in gastruloids.If instead the glycolytic activity acted as an instructive signal, one would expect a dose-dependent effect on T/Bra gene expression.To address this question, we aimed to modulate the glycolytic activity in gastruloids to assess the effect on T/Bra::GFP expression and germ layer proportions.Taking advantage of our in vitro system, we varied exogenous Glc levels in the differentiation medium and found that this changes glycolytic activity effectively in 2D cultured cells (Figure 2A).In the absence of Glc, cells relied entirely on OxPhos for ATP production (Figure S3A).Raising Glc levels resulted in increased glycolytic activity which plateaued around 12.5mM Glc.At this concentration, glycolysis and OxPhos contributed approximately evenly to the total ATP production rate (Figure S3A).To test whether Glc and glycolytic activity have a dose-dependent effect on T/Bra::GFP expression, we generated gastruloids in medium containing different amounts of Glc.At 48hpa, when T/Bra::GFP expression is present throughout the gastruloid, a positive correlation between Glc concentration and GFP signal was observed (Figure 2B).However, it was also apparent that gastruloids grown in lower Glc concentrations were strongly reduced in size suggesting that Glc was growth limiting.
To exclude the possibility that size and not glycolytic activity is affecting T/Bra::GFP expression, we generated gastruloids from different initial cell numbers resulting in similar sized gastruloids at different Glc concentrations (Figure 2C, see insets).By assessing the percentage of GFP + cells using flow cytometry at 0.02mM Glc, 0.1mM Glc, 0.5mM Glc and 2.5mM Glc over four different initial cell numbers (350, 700, 1050 and 1400 cells), we found that the percentage of GFP + cells was indeed dependent on Glc concentration and not gastruloid size (Figure 2C, Figure S3B).We further wondered if Glc concentration not only determines the percentage of T/Bra::GFP expressing cells but also cellular T/Bra::GFP expression levels.Based on flow cytometry histograms, we detected no noticeable shift in typical GFP peak intensity levels suggesting that cellular development, especially between 0mM Glc and 0.5mM Glc where dose-dependency is most prominent (Figure S3B).Interestingly, Glc concentration in the uterine fluid has been measured to be around 0.6mM [27].Thus, it is conceivable that the in vivo embryo develops in a regime where Glc can act as an instructive regulator of PS induction.
Glucose has a dose-dependent effect on germ layer Nodal signalling pathway with Activin A (Act-A) during 2-DG treatment between 24 and 48hpa, was able to partially rescue T/Bra::GFP expression at 48hpa (Figure 3B,C).Even though T/Bra::GFP expression was not reaching control levels at 48hpa, symmetry breaking was robust in these rescued gastruloids and elongation was observed in 70% of the cases.We further performed HCR stainings at 96hpa to validate whether cells of all three germ layers can be found in the rescued gastruloids.Quantifications of the relative area of T/Bra and Sox17 expression confirmed that mesoderm and endoderm development was rescued (Figure 3D,E).Further stainings also confirmed the presence of paraxial mesoderm (Meox1) and cardiac precursors (Gata6) in these gastruloids (Figure S4B).These results confirm that Nodal signalling activation during glycolysis inhibition was sufficient to rescue the development of mesodermal and endodermal cell types.Activation of Wnt signalling using CHIR was also able to rescue gastruloid development, albeit in a less reproducible manner (Figure S4C,D).Even though elongation occurred in 34% of gastruloids, endodermal and cardiac cell derivatives were mostly absent, most likely because Wnt signalling strongly promotes differentiation towards posterior mesoderm (Figure S4B,E).We also added Fgf8 simultaneously with 2-DG but did not observe a rescue of T/Bra::GFP induction and polarisation (Figure S4F).Our rescue experiments suggest that Nodal and Wnt signalling activation are downstream of glycolytic activity and can rescue the 2-DG mediated phenotype on gastruloid development.

Glycolytic activity is not rescued by Nodal and Wnt signalling
The signalling, epigenetic and metabolic state of cells are highly integrated through several feedback mechanisms that ensure coordination of cellular behaviour [1,4,6,45].In cancer cells, Wnt signalling is a crucial regulator of metabolic reprogramming resulting in increased Glc uptake and its preferential fermentation to lactate even in the presence of oxygen, known as the Warburg effect [46].Similarly, Wnt, Fgf and Nodal signalling have been suggested to promote glycolysis in the context of normal embryonic development and homeostasis as well as in cancerous tissue [13,[47][48][49][50].
To rule out the possibility that Nodal or Wnt signalling activation rescue the 2-DG mediated glycolysis inhibition, we first analysed gastruloid size that is expected to be affected by 2-DG treatment [51].Indeed, we detected a significant reduction in gastruloid size upon the 24h 2-DG treatment period.In contrast to T/Bra::GFP intensity levels, neither Nodal nor Wnt signalling activation rescued the gastruloid size phenotype suggesting that anabolic metabolism driving proliferation and tissue growth was not restored in these gastruloids (Figure 3F; Figure S4G).To look at a more direct readout of metabolic state, we next measured the ATP production rate from glycolysis (glycoATP production rate).Control cells cultured for two days in differentiation medium displayed a significantly higher glycoATP production rate than cells that had been treated with 2-DG for 24h prior measurement (Figure 3G).We found that the addition of Nodal signalling agonist Act-A did not rescue the glycoATP production rate of 2-DG treated cells.This was also the case when Act-A and CHIR were added simultaneously to cells (Figure S4H).These results were further supported by the finding that T/Bra::GFP expression and symmetry breaking of gastruloids in Glc-free medium (24-48hpa) was also rescued by the addition of Act-A and CHIR (Figure S3I).Here, the absence of Glc abolishes any possibility of reactivating glycolytic activity.Together, these experiments suggest that even though signalling activation can rescue gastruloid development, it does not rescue the metabolic

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In recent years, both in vitro and in vivo studies have contributed to our understanding of the mechanistic link between metabolic pathways and cell fate specification [4,55].Here, we found an important role of glycolytic activity in the induction of endoderm and mesoderm using both a glycolysis inhibitor and Glc-free medium.Most importantly, we demonstrate that this relationship is dose-dependent.As glycolytic activity increases, so does the probability of cells committing to mesodermal and endodermal cell fates.This suggests that glycolysis acts as an instructive cue, rather than merely a permissive factor.Differences in our findings compared to a prior study [19], which indicated that cells differentiating into endoderm and mesoderm required a shift from high glycolysis to high Ox-Phos, might be attributed to the use of directed differentiation protocols involving growth factor-containing germ layerspecific media.In our gastruloid system, cells of all three germ layers co-emerge without the need for exogenous signalling agonists.This allowed us to demonstrate the importance of gly-colytic activity in the induction of mesoderm and endoderm type [21].Contrary to our results, the authors did, however, 434 not observe a phenotype in Glc-free medium and concluded 435 that glycolytic activity is dispensable for mesoderm develop-436 ment in gastruloids.A possible explanation might be that the 437 experiments were conducted in the presence of CHIR, a con-438 dition that is analogous to our rescue experiments.Since even 439 low amounts of Glc are sufficient for Bra induction, residual 440 Glc after washing out high-Glc differentiation media might be 441 another potential explanation for the observed differences.

442
In accordance with our findings, an important role of gly-443 colysis has been suggested for mesoderm development at later 444 stages of embryonic development when bipotent NMPs com-445 mit to either a neural tube or presomitic mesoderm fate [13].

446
In NMPs it is however still disputed whether glycolytic activ- morphogenetic movements in the gastruloid model system are not comparable with in vivo gastrulation, it is however conceivable that a bioenergetic function of glycolysis plays a role in the migration of mesodermal and endodermal cells [7,20].
An open question is how changes in glycolytic activity can regulate Nodal, Wnt and Fgf signalling during germ layer induction.Metabolite-driven posttranslational modifications, moonlighting glycolytic enzymes and metabolite-protein interactions are possible links between metabolism and signalling [4,6,61].For instance, high glycolytic activity promotes acetyl-CoA levels which can be rate-limiting for protein acetylation [62].Interestingly, the activity of Nodal and Wnt signalling transducers is known to be modulated by acetylation [63][64][65].It has been also suggested that glycolysis-driven changes in the intracellular pH may further favour β-catenin acetylation and nuclear translocation during NMP differentiation [14].Glycolytic activity also feeds into the production of the building blocks necessary for glycosylation [66,67].Recently, it was proposed that 2-DG treatment affects mesoderm specification due to reduced glycosylation, based on the knowledge that several proteins involved in Wnt and Fgf signalling transduction are known to be glycosylated [20,21,68].
Besides post-translational modifications, glycolytic activity has also been shown to affect cellular localisation of glycolytic enzymes, thereby allowing them to perform non-canonical functions.For instance, translocation of Pfkl and Aldoa into the nucleus has been hypothesised to modulate Wnt signalling during somitogenesis [12].Moreover, metabolites might also modulate protein activity by direct binding.Sentinel metabolites such as fructose-1,6-bisphosphate (FBP) whose concentration changes with glycolytic activity might be interesting targets for metabolite-protein allosteromes as suggested by Miyazawa and colleagues [12].
Most of the previous work has been focused on Wnt signalling and mesoderm specification.Currently, we know only little about how glycolytic activity modulates the Nodal signalling pathway and endoderm differentiation.Future work will be also important to identify actual changes in acetylation and glycosylation patterns of signalling components and functionally link them to their activity, as well as probe non-canonical glycolytic enzyme function and possible metabolite-protein interactions.

Relevance for in vivo mouse gastrulation
The observation that exogenous Glc concentrations ranging from 0.02mM to 2.5mM have a dose-dependent effect on germ layer proportions, raises the question whether Glc levels within the embryonic environment fall within this regulatory range and whether glycolytic activity may indeed function as an instructive cue during in vivo gastrulation.Notably, measurements of Glc levels in uterine fluid suggest a concentration of approximately 0.6mM [27].Interestingly, dose dependency is almost linear until 0.5mM and starts plateauing afterwards (Figure S3B).Hence, it is conceivable that differential expression of Glc transporters and glycolytic enzymes could account for spatial variations in glycolytic activity, thereby impacting cell fate decision making.This notion gains added significance when considered alongside recent findings that demonstrate the spatiotemporal coordination of Glc transporter expression during mouse gastrula development [20].
Our study provides new insights into the interplay between metabolism and signalling pathways that coordinate the differentiation of pluripotent cells into the three germ layers.The demonstrated instructive role of exogenous Glc concentration on cell fate decision-making represents an initial stride toward establishing nutritional control of cell type composi-tion in complex in vitro model systems.Future work in this emerging research field will further improve our understanding of how metabolism is integrated into cellular behaviour and how metabolic conditions affect embryonic development.

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These findings demonstrate how metabolic activity acts as a 75 regulator of morphogen signalling and cell fate determination 76 and opens new possibilities for metabolic control of cell type 77 proportions in in vitro systems.
An instructive role of glycolytic activity for mesoderm401 and endoderm induction 402 Spatial metabolic patterns observed during amphibian 403 gastrulation in the 1930s sparked early hypotheses about 404 the metabolic regulation of embryonic development [52-54].
447ity has a promoting or inhibitory function on Wnt and Fgf 448 signalling[12,14].Concurrently with our research, another 449 preprint has identified metabolic activity as a key driver of 450 phenotypic variation through integrated molecular-phenotypic 451 profiling of trunk-like structures, an in vitro model system 452 for neural tube and somite formation [56].Specifically, they 453 demonstrate that an early imbalance in OxPhos and glycoly-454 sis leads to aberrant morphology and biases cells towards the 455 neural lineage, consistent with our findings during germ layer 456 formation.457 While the relationship between glycolysis and mesoderm 458 development has been explored in several studies, little is 459 known about the metabolic control of endoderm specification.460 Previous studies have also found that glycolysis can promote 461 endoderm differentiation [57, 58].One suggested mechanism 462 involves the Lin41 protein kinase as a non-canonical phos-463 phorylation target of glycolytic enzyme Pfkp, resulting in 464 the suppression of Sox2 and leading to increased endodermal 465 differentiation of ESCs [57].Here, we show that glycolytic 466 activity is needed for the activation of endoderm-promoting 467 Nodal signalling and therefore introduce an additional layer 468 of metabolic control to our understanding of endoderm vital role in generating the necessary 475 building blocks and energy required for growth and prolifer-476 ation.Differentiating cells might display unique bioenergetic 477 demands which must be met to ensure the proper develop-478 ment of certain cell types [59, 60].For instance, the energetic 479 requirements of prospective mesodermal and endodermal 480 cells migrating through the PS might differ significantly from 481 epithelial cells fated to become neuroectoderm.However, our 482 finding that the metabolic phenotype of glycolysis inhibition 483 can be rescued through the activation of Nodal and Wnt 484 signalling pathways in the absence of glycolytic activity 485 strongly indicates that the phenotype is not related to ener-486 getic constraints.Since glycolysis is not reactivated in these 487 rescued gastruloids, it seems to primarily act as an activator 488 of developmental signalling pathways.This implies that 489 glycolytic activity is not an absolute necessity for mesoderm 490 and endoderm differentiation as long as these signalling 491 pathways are activated to coordinate gastruloid development 492 by inducing T/Bra upregulation between 24-48hpa.Since