Selective utilization of glucose metabolism guides mammalian gastrulation

Cao, Dominica; Bergmann, Jenna; Zhong, Liangwen; Hemalatha, Anupama; Dingare, Chaitanya; Jensen, Tyler; Cox, Andy L.; Greco, Valentina; Steventon, Benjamin; Sozen, Berna

doi:10.1038/s41586-024-08044-1

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Published: 16 October 2024

Selective utilization of glucose metabolism guides mammalian gastrulation

Nature volume 634, pages 919–928 (2024) Cite this article

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Abstract

The prevailing dogma for morphological patterning in developing organisms argues that the combined inputs of transcription factor networks and signalling morphogens alone generate spatially and temporally distinct expression patterns. However, metabolism has also emerged as a critical developmental regulator^{1,2,3,4,5,6,7,8,9,10}, independent of its functions in energy production and growth. The mechanistic role of nutrient utilization in instructing cellular programmes to shape the in vivo developing mammalian embryo remains unknown. Here we reveal two spatially resolved, cell-type- and stage-specific waves of glucose metabolism during mammalian gastrulation by using single-cell-resolution quantitative imaging of developing mouse embryos, stem cell models and embryo-derived tissue explants. We identify that the first spatiotemporal wave of glucose metabolism occurs through the hexosamine biosynthetic pathway to drive fate acquisition in the epiblast, and the second wave uses glycolysis to guide mesoderm migration and lateral expansion. Furthermore, we demonstrate that glucose exerts its influence on these developmental processes through cellular signalling pathways, with distinct mechanisms connecting glucose with the ERK activity in each wave. Our findings underscore that—in synergy with genetic mechanisms and morphogenic gradients—compartmentalized cellular metabolism is integral in guiding cell fate and specialized functions during development. This study challenges the view of the generic and housekeeping nature of cellular metabolism, offering valuable insights into its roles in various developmental contexts.

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Main

The formation of a body plan from a simple multicellular structure occurs during gastrulation, the essential process of embryogenesis. Localized morphogen signals guide cell-fate decisions and behaviours to shape the embryo, but the precise mechanisms by which these critical signals integrate at the correct time and place to mediate gastrulation morphogenesis are not fully understood. The remarkable fidelity of this process indicates that multiple regulatory layers ensure robust embryonic patterning.

To explain how an organism can establish positional identity during development and regeneration, a ‘gradient theory’—whereby graded metabolism along an embryonic axis directs tissue patterning and morphogenesis—was first experimentally introduced in 1915 (refs. ^11,12. Although this model had been challenging to incorporate into a mechanistic framework, it is now supported by the recent conceptualization of metabolic signalling, in which metabolic enzymes and metabolites themselves function beyond bioenergetics to actively modulate or instruct cellular and developmental programmes¹³. Studies have shown regionalized glycolytic gradients in chick and mouse embryos, indicating that glycolysis can function independently of energy production during the development of various tissues^1,2,3,6,9,10 and stem cell differentiation^4,5. Despite these insights, questions remain about whether the mammalian embryo indeed processes nutrients uniformly, how metabolic signalling might be linked to morphogen signalling pathways, and how these signals are integrated to modulate broader cell function during early post-implantation morphogenesis. Moreover, although it is known that glycolysis is enhanced after implantation, high glucose uptake does not necessarily lead to increased utilization in the glycolytic pathway; instead, it can also redirect glucose into parallel, glycolysis-independent pathways.

Challenges to simultaneously probe metabolic and morphogen signalling kinetics at the single-cell level in live early embryos has hindered efforts to understand how these pathways might work together to support complex differentiation and morphogenetic events. Here we delineate the instructive role of cellular metabolic activity in the temporal and spatial coordination of mouse gastrulation in developing embryos, with support from in vitro stem cell and embryo-derived tissue explant models. We find that a single nutrient, glucose, is used selectively through different metabolic pathways to instruct both local and global embryo morphogenesis, in a cell-type and stage-specific manner. Specifically, we identify two spatiotemporally resolved waves of glucose uptake that start in early post-implantation epiblast cells at the onset of mouse gastrulation. This graded metabolic activity extends distally in epiblast tissue as development progresses, and re-emerges within the mesenchymal cells as they exit the primitive streak and migrate laterally within the mesodermal wings. We find that these glucose-mediated events are coupled to high ERK activity, but are distinctly regulated in each wave, suggesting a critical connection between glucose metabolism and ERK signalling that drives successful tissue patterning during mammalian gastrulation. Collectively, our integrated examination of metabolic and signalling axes provides a comprehensive framework for understanding the mechanisms that guide gastrulation, one of the most fundamental and evolutionarily conserved stages of animal life.

Regional glucose waves in embryos

During implantation, the mammalian embryo adopts a heavy reliance on glucose to support rapid morphological changes, proliferation and differentiation¹⁴. How this metabolic shift aligns with the evolving landscape of post-implantation embryogenesis remains unclear. We first analysed in utero-dissected mouse embryos for expression of the canonical glucose transporter GLUT1, and performed a glucose-uptake analysis using the fluorescent glucose analogue (2-NBDG). Both GLUT1 protein expression and the ratiometric 2-NBDG signal revealed compartmentalized glucose uptake in two distinct regions: first within the small population of posteriorly positioned epiblast cells destined to form the primitive streak that we term ‘transitionary epiblast’, and second within the lateral mesodermal wings, which develop as cells exit the primitive streak to form migratory mesenchyme (Fig. 1a–c and Extended Data Fig. 1a–c). Notably, cells within the primitive streak exhibited no or minimal glucose (2-NBDG) uptake, concomitant with a gradual reduction in GLUT1 expression as cells entered the streak (Fig. 1b and Extended Data Fig. 1a–c).

**Fig. 1: A wave of glucose activity precedes mouse gastrulation.**

To analyse how this regionalized surge of glucose uptake evolves, we analysed the temporal sequence of gastrulation through three streak stages: early streak (ES; embryonic day 6.25 (E6.25)–E6.5), mid-streak (MS; E6.5–6.75) and late streak (LS; E6.75–7.25) (Fig. 1d). This spatiotemporal analysis revealed two distinct waves of glucose activity. In the first ‘epiblast wave’, we observed an anteroposterior gradient of glucose uptake, originating in the posterior-proximal-most transitionary epiblast cells at gastrulation onset. As gastrulation proceeded and the primitive streak developed distally, this pattern of glucose activity expanded within the epiblast tissue towards the anterior-distal axis, directly preceding primitive streak elongation (Fig. 1d,e and Extended Data Fig. 2a–d). Cells switch back to a glucose-dependent programme after exiting the primitive streak, with high metabolic activity observed in mesenchyme as they expanded laterally, marking the onset of the second ‘mesodermal wave’ of glucose activity (Fig. 1b,c and Extended Data Fig. 1a,b). This spatial distribution was also reflected in the expression pattern of another canonical glucose transporter, GLUT3 (Fig. 1f and Extended Data Fig. 2b,c).

We then performed label-free live imaging of NADH and NADPH (collectively referred to as NAD(P)H), an endogenous auto-fluorescent readout of glycolytic activity¹⁵, via multiphoton microscopy in TCF/LEF:H2B-GFP-reporter developing gastrulas (Methods). This showed that NAD(P)H intensity was also intrinsically graded over the course of gastrulation and localized to epiblast cells anterior to the expanding primitive streak population of mesoderm progenitors (Fig. 1g). This NAD(P)H intensity gradient overlapped with regions of 2-NBDG uptake, confirming the specificity of this assay in imaging the metabolic activity of live gastrulas (Extended Data Fig. 2e).

Finally, we probed a spatial transcriptome dataset of the mouse gastrula¹⁶ for key genes involved in glucose metabolism. Transcripts such as Slc2a1, Gpi1, Pfkb and Ldhb from the glycolysis pathway, as well as Ogt and Gnpnat1 from the hexosamine biosynthetic pathway (HBP; a branch of glucose metabolism), were graded within epiblast and mesodermal wings during progressive stages of gastrulation (Extended Data Fig. 2f).

These observations suggested finely tuned spatiotemporal metabolic regulation during gastrulation, potentially influencing both cell-fate determination and morphogenetic processes.

HBP guides fate transition in epiblast

The spatiotemporal pattern of glucose uptake observed in epiblast cells preceding primitive streak expansion led us to investigate the role of glucose metabolism in preparing these cells for streak entry. To assess this, we treated ex vivo developing mouse embryos with inhibitors that target different enzymatic steps of glucose metabolism (Fig. 2a). In these perturbation experiments, gastrulas were collected at the ES stage (around E6.5), at which point embryos had already broken symmetry and initiated primitive streak formation (see pre-culture control in Fig. 2b), and subsequently cultured for 12–18 h with selective inhibitors to define effects on primitive streak progression.

**Fig. 2: Glucose metabolism via HBP is necessary for mesoderm fate acquisition and maintenance.**

First, to block all glucose-dependent pathways, we used 2-deoxy-d-glucose (2-DG) and 3-bromopyruvate (BrPA), competitive inhibitors of hexokinase and glucose phosphate isomerase (Fig. 2a). After 18 h, control embryos advanced through post-gastrulation stages, forming double mesoderm wings, whereas gastrulas treated with 2-DG and BrPA were significantly delayed, with most not progressing past the LS stage (Extended Data Fig. 3a–c). Shorter (12 h) treatments revealed that inhibiting the entirety of glucose metabolism by treatment with 2-DG and BrPA significantly impaired distal elongation and development of the primitive streak, suggesting that epiblast cells need glucose metabolism for mesodermal transition (Fig. 2b).

We next targeted each major branch of glucose metabolism to clarify the specific metabolic pathway(s) responsible for the primitive streak-specific phenotype. This effect was recapitulated with azaserine, an inhibitor that blocks the conversion of fructose-6-phosphate to glucosamine-6-phosphate, the rate-limiting step that links glucose metabolism to HBP (Fig. 2a,b). Concentration-response analysis, wherein the degree of pathway inhibition is indirectly assessed using the primitive streak developmental phenotype as a readout, demonstrated a dose-dependent effect under 2-DG or azaserine inhibition (Extended Data Fig. 3d). Azaserine yielded a bimodal response, indicating heterogeneous sensitivity among the treated embryos, potentially owing to differences in cell state or temporal developmental dynamics (Extended Data Fig. 3d). By contrast, blocking glycolytic enzymes that bypass the HBP branch—which we refer to as ‘late-stage-glycolysis’ components—with YZ9 (targeting PFKFB3) or shikonin (targeting pyruvate kinase M2) had no effect on primitive streak progression (Fig. 2a,b and Extended Data Fig. 3e). Similarly, inhibitors of lactate dehydrogenase (galloflavin), pentose phosphate pathway (6-aminonicotinamide (6-AN)) and ATP synthase (oligomycin) also did not affect streak development (Fig. 2b and Extended Data Fig. 3e).

To further confirm on-target effects of the inhibitors and to functionally test glucose dependency in the epiblast, we performed rescue experiments by culturing embryos in various nutrient-supplemented or nutrient-sparse media. Some embryos cultured in medium devoid of glucose, pyruvate and glutamine showed defects in primitive streak development, similar to those treated with 2-DG and BrPA (Extended Data Fig. 3f). Selectively reintroducing glucose or glutamine improved primitive streak elongation, whereas pyruvate did not, suggesting the importance of non-glycolytic glucose metabolism branches (Extended Data Fig. 3f). Although all groups maintained a SOX2⁺ epiblast, SOX2 expression levels remained lower in the glutamine-reintroduced condition¹⁷ (Extended Data Fig. 3f). Additionally, galactose supplementation, which bypasses inhibition by 2-DG and BrPA by conversion to glucose-6-phosphate and restoring carbon flux to HBP (Fig. 2a), rescued primitive streak development in both inhibitor-treated and nutrient-sparse conditions (Fig. 2b). Finally, neither 2-DG and BrPA nor azaserine treatments impeded cell proliferation within developing embryos, confirming stable growth and bioenergetics (Extended Data Fig. 3g).

As the highly conserved HBP metabolic branch generates substrates that are important for protein post-translational modifications, such as glycosylation and the attachment of N-acetylglucosamine^18,19 (GlcNAc), this points to the possibility that glucose uptake in epiblast cells during early primitive streak induction might have critical roles that transcend bioenergetics. Overall, these findings underscore the critical importance of glucose metabolism in the developing epiblast, functioning through the HBP, in supporting cell-fate transitions required for primitive streak formation during early mouse gastrulation.

Embryo models link HBP to fate change

To support these in vivo findings, and to confirm the primary metabolic effect on epiblast patterning during mesodermal fate acquisition in isolation from interactions with other tissues, we utilized stem cell-based embryo models. Gastruloids (3D aggregates of mouse embryonic stem cells^20,21) treated with 2-DG did not specify mesoderm-like cells, and exhibited reduced expression of T (which encodes Brachyury) and incomplete elongation (Extended Data Fig. 4a). Sox2 was retained across groups, whereas Etv4, a fibroblast growth factor (FGF)–ERK target gene involved in mesoderm specification²², was downregulated under 2-DG treatment (Extended Data Fig. 4b). Gastruloids under YZ9 treatment, which blocks late-stage-glycolysis, developed normally, highlighting that the role of glucose metabolism in mesoderm specification is independent of the late-glycolysis branch.

We also treated mouse extraembryonic endoderm (mXEN) cells for 24 h with the same metabolic inhibitors used on embryos (Fig. 2a). None of these inhibitors affected endodermal identity (indicated by GATA6 expression) or proliferation (marked by histone H3 phosphorylation) (Extended Data Fig. 4c). Although potential differences between in vitro XEN cells and in vivo visceral endoderm cannot be ruled out, these results suggest that glucose metabolism does not have tissue-autonomous roles in endoderm, despite GLUT1 expression and 2-NBDG uptake in the visceral endoderm (Extended Data Fig. 2d). Oligomycin-treated mXEN cells showed reduced proliferation (Extended Data Fig. 4c), and active mitochondrial labelling in live gastrulas revealed significant accumulation within the visceral endoderm layer (Extended Data Fig. 4d). This indicates a preference for oxidative phosphorylation endodermal cells, in line with previous evidence²³.

We next performed mesoderm-directed differentiation of mouse embryonic stem cells. We applied selective inhibitors from day 1 to day 4 to target epiblast-to-mesoderm transition state, or from day 3 to day 4 to target cells that had predominantly already reached the mesodermal state (Extended Data Fig. 5a). Consistent with our findings in embryos and gastruloids, 2-DG and BrPA or azaserine treatments significantly disrupted the transition of mouse embryonic stem cells from epiblast-like stem cells (EpiLSCs) to mesoderm states (days 1–4), whereas late-stage glycolysis-specific inhibition with YZ9 had no effect (Extended Data Fig. 5a,b). Between day 1 and day 4, mouse embryonic stem cell colonies treated with 2-DG and BrPA or azaserine grew in size over time but maintained high expression of pre-patterned epiblast genes (Sox2, Rex1 and Klf2) and low expression of mesoderm genes (T, Eomes, Mesp1 and Pdgfra) compared with controls (Extended Data Fig. 5a,b,d). Conversely, treatment duration targeting cells that had already reached the mesodermal state (days 3–4) did not yield significant phenotypic differences, and mesoderm-differentiated cells continued to blanket the plates on which they were cultured, similar to control conditions (Extended Data Fig. 5a,b). None of the inhibitors or treatment durations induced cell death (Extended Data Fig. 5c).

FGF signalling via ERK activation has a vital role in mouse gastrulation for the specification of mesoderm^24,25, and has been linked to glucose activity in the presomitic mesoderm of developing chick embryos late in gestation⁶. To compare the effects of glucose metabolism inhibition with that of signalling inhibition on the early mesoderm fate transitions, we applied the fibroblast growth factor receptor (FGFR) inhibitor SU5402 or the ERK inhibitor PD0325901 during mesoderm-directed-differentiation assays. This revealed overlapping phenotypes between inhibition of these signals and inhibition of HBP, with significantly low levels of mesodermal genes and high levels of pre-differentiation epiblast genes (Extended Data Fig. 5a–d), suggesting an interplay between glucose metabolism via HBP and cellular signalling in mesoderm specification during gastrulation.

These results support the idea that glucose metabolism via the HBP is essential for epiblast cells to acquire a stable mesodermal identity. Although glucose uptake may have additional non-tissue-autonomous roles in in vivo endodermal lineages, our 3D gastruloid and mesoderm-directed-differentiation assays suggest that the observed embryo phenotypes result primarily from the effect on epiblast and primitive streak derivatives.

HBP links to EMT in posterior epiblast

Given the high level of glucose activity in epiblast cells before primitive streak entry (Fig. 1b), we investigated whether a metabolic regulation of the epithelial-to-mesenchymal transition (EMT) occurs during mouse gastrulation. In utero-dissected embryos exhibited GLUT1-rich posterior epiblast cells adjacent to the intact laminin⁺ basement membrane, with strong MMP14 co-expression, suggesting that glucose uptake may be involved in basement membrane breakdown, which is essential for EMT and epiblast cell ingression into the primitive streak (Fig. 2c and Extended Data Fig. 6a,b). Transitionary epiblast cells, in which E-cadherin (also known as ECAD) expression was weaker, exhibited GLUT1 and GLUT3 activity, indicating that enhanced glucose uptake spans the early-stage EMT (Extended Data Fig. 6c). Additionally, reanalysis from single-cell RNA sequencing of E6.5 to E8.5 mouse embryos²⁶ revealed upregulation of genes encoding rate-limiting glycolytic enzymes (Eno1, Gapdh, Ldha, Slc2a1 and Slc16a3) at the onset of EMT, whereas expression of Krebs cycle genes (Aco2, Mdh2, Sdha and Sdhb) remained stable (Extended Data Fig. 6d).

We next treated embryos with 2-DG and BrPA or azaserine. These treatments inhibited basement membrane breakdown, an outcome that, notably, could be significantly rescued by supplementation with GlcNAc (the end product of HBP) (Fig. 2d). By contrast, late-stage glycolysis inhibitors, ATP synthase blockers, lactate dehydrogenase inhibitors and pentose phosphate pathway inhibitors, had no such effect on basement membrane (Fig. 2d).

We then utilized an in vitro proteolytic activity assay using GFP-tagged DQ gelatin, a substrate that fluoresces at sites of proteolytic digestion²⁷. To correlate our assay with the post-implantation epiblast state that undergoes EMT, we examined differentiating-EpiLSC colonies during their transition to mesoderm (schematic in Extended Data Fig. 5a). In control groups, the presence of many GFP⁺ EpiLSC clusters indicated regions on the plate where cells were actively transitioning to mesoderm via the local release of matrix metalloproteases²⁷ (Fig. 2e). Treatment with 2-DG and BrPA or azaserine resulted in an absence of GFP signal, indicating that aberrant glucose metabolism, specifically HBP branch inhibition, functionally impairs the process of EMT (Fig. 2e)—a result similar to that of ERK inhibition (PD0325901 treatment)²⁵ (Extended Data Fig. 7a). Finally, quantitative PCR (qPCR) analyses during embryonic stem cell-to-mesoderm differentiation showed a significant reduction in the expression of distinct EMT genes after treatment with 2-DG and BrPA, including Cdh2 (which encodes NCAD), Mmp2 and Vim (Extended Data Fig. 7b).

Together, these findings show that early EMT in the mouse epiblast is closely linked to glucose metabolism through HBP, demonstrating how intracellular shifts in glucose flux can directly modulate cellular and molecular regulatory networks to primarily control fate decisions of epiblast cells and, secondarily, allow their subsequent ingression into the primitive streak. Anterior epiblast cells are influenced by the anterior visceral endoderm, which prevents posteriorization and inhibits EMT. Thus, the shift of glucose activity to anterior regions at later stages (Fig. 1d) is likely to reflect a move toward ectoderm fate, as these cells show prolonged glucose metabolism⁴.

HBP mediates ERK in mesoderm transition

ERK (ERK1 and ERK2) signalling—a critical downstream pathway activated by various receptor tyrosine kinases (RTKs)—has an essential role in mesoderm specification and gastrulation cell movements in vertebrates, including mice^{24,25,28,29,30,31}. It has been suggested that blocking protein-modifying enzymes that utilize substrates from glucose metabolism can impair RTK function^32,33. Thus, we next investigated the effect of the glucose-uptake waves on ERK signalling during mouse gastrulation.

We found high levels of diphosphorylated ERK (dpERK) in transitionary epiblast cells and the newly formed mesodermal layer (Extended Data Fig. 7c), correlating with glucose-uptake patterns (Fig. 1b). The amount of dpERK was reduced in embryos treated with 2-DG and BrPA, indicating a functional link between glucose metabolism and ERK signalling (Extended Data Fig. 7c). Western blots confirmed decreased pERK levels in EpiLSCs treated with 2-DG and BrPA or azaserine, whereas total ERK levels remained unchanged (Fig. 3a and Supplementary Fig. 1). PD0325901- or SU5402-treated embryos mimicked the developmental delays seen with 2-DG and BrPA treatment (Extended Data Fig. 7d,e), suggesting a potential coupling of glucose activity and signalling.

**Fig. 3: Glucose metabolism modulates ERK signalling in the epiblast.**

To obtain a quantitative real-time readout for ERK signalling activity, we used ERK kinase translocation reporter (KTR^mClover) mouse embryonic stem cells^34,35,36 in a tetraploid complementation assay^37,38 (Fig. 3b and Extended Data Fig. 8a). Live imaging showed a graded pattern of increased cytoplasmic localization of KTR along the anteroposterior axis in the epiblast, indicative of active ERK (Fig. 3b and Extended Data Fig. 8a–c) that resembled our previously defined glucose-uptake pattern (see Fig. 1). Simultaneous label-free live imaging of ERK-KTR^mClover alongside NAD(P)H activity in embryos with multiphoton microscopy showed a skewing of NAD(P)H signal anteriorly to the site of active KTR^mClover, supporting our hypothesis that the wave of glucose metabolism precedes ERK signalling dynamics within the gastrulating embryo (Extended Data Fig. 8c).

Expectedly, inhibition of ERK (PD0325901) or FGFR (SU5402) each decreased cytoplasmic ERK signal with no clear nuclear and cytoplasmic distinction, or higher nuclear signal, both indicative of low ERK activity (Extended Data Fig. 8d). HBP inhibition, using either 2-DG and BrPA or azaserine, also reduced cytoplasmic ERK signal in epiblast cells, again showing no clear distinction between nuclear and cytoplasmic regions (Fig. 4c and Extended Data Fig. 8e). Notably, GlcNAc supplementation into the azaserine-containing culture medium restored active ERK signal in the epiblast, as indicated by increased cytoplasmic KTR localization (Fig. 4c and Extended Data Fig. 8e). These results collectively support the idea that the wave of glucose uptake and HBP flux in epiblast tissue may be essential for functional ERK-mediated signal transduction.

**Fig. 4: Glucose metabolism is necessary for proper mesoderm migration.**

Having observed the capability of GlcNAc, an HBP-specific metabolic intermediate, to rescue both primitive streak formation and ERK signalling in azaserine-treated embryos (Figs. 2d and 4c and Extended Data Fig. 8e), we next aimed to gain a mechanistic insight into how this regulation occurs and its roles within the epiblast. RTK signalling in mammalian cells, including embryos, relies on the presence of heparan sulfate proteoglycans (HSPGs), which function as co-receptors by regulating morphogen retention, stability and extracellular diffusion^32,33,39. These HSPGs are composed of core proteins and their tethered heparan sulfate chains, which are synthesized from repeating disaccharide units of glucuronic acid and, notably, GlcNAc. To assess HSPG formation in HBP-inhibited embryos, we first examined expression of the heparan sulfate chain 10E4. Control embryos showed expected 10E4 expression within the basal side of epiblast cells, visceral endoderm and mesoderm²⁶, and after HBP inhibition, embryos had globally reduced, albeit not eliminated, levels of 10E4 compared with controls (Extended Data Fig. 8f). The HSPG cell surface-tethered core protein syndecan-1 showed higher expression within cell surface areas of epiblast cells that seemingly undergo mesenchymal transition, as well as visceral endoderm and mesoderm²⁶, and it was also globally decreased under treatment with 2-DG and BrPA or with azaserine (Extended Data Fig. 8f).

We next treated embryos with sodium chlorate (NaClO₃)—a proteoglycan sulfation inhibitor that is known to affect HSPG and morphogen binding^39,40. In NaClO₃-treated embryos, expression of 10E4 and syndecan-1 was reduced (Extended Data Fig. 8f). Notably, these embryos showed a reduced ERK signal (Fig. 4c and Extended Data Fig. 8e) and mirrored the HBP-inhibited embryo morphology of defective primitive streak development (Extended Data Fig. 8h). Finally, we confirmed these observations in ERK-KTR^mClover differentiating-EpiLSC colonies, which revealed a significant reduction in ERK activity in cells treated with PD0325901, azaserine, 2-DG and BrPA or NaClO₃ (Fig. 4d and Extended Data Fig. 8g). Finally, we attempted to rescue the HBP-inhibited embryo phenotype with supplementation with FGF2, FGF4 and FGF8, with the logic that ligand supplementation would only be able to restore normal development if ERK signalling can function independently of HSPGs. FGF ligand supplementation did not rescue this phenotype in both embryos and cells (Fig. 4d and Extended Data Fig. 8h). Although this does not rule out the possibility that ERK activity in the epiblast is responsive to a RTK other than FGFR, it reflects similar observations from previous studies^32,39.

Together, these findings provide compelling evidence for a role of the direct link between HBP, HSPG and ERK activity within epiblast during gastrulation to initiate the mesodermal fate acquisition.

Committed mesoderm does not rely on HBP

Our observations reveal that both epiblast and mesodermal wings exhibit compartmentalized glucose uptake as the embryo progresses through temporal sequences of gastrulation (Fig. 1 and Extended Data Fig. 1). After establishing the role of the first glucose-uptake wave in the epiblast to specify primitive streak, we next examined the function of glucose metabolism in mesenchymal cells that are exiting the primitive streak.

Consistent with our findings on the second wave of glucose metabolism in the mesenchyme (Fig. 1b), we observed high GLUT3 expression in the NCAD⁺ mesoderm wings (Extended Data Fig. 9a). We then examined whether there is still a relationship between glucose metabolism and ERK signalling in the differentiated mesoderm, as seen in the epiblast. To investigate this, we first differentiated ERK-KTR^mClover mouse embryonic stem cells to mesoderm and inhibited glucose metabolism (with 2-DG and BrPA), HBP (with azaserine) and ERK signalling (with PD0325901). Inhibitor treatments were limited to 2.5 h on day 4 of differentiation, when cells were in a stable mesoderm state to avoid confounding the role of HBP in the transition (Extended Data Fig. 5a). Live imaging revealed the expected significant ERK inactivation with PD0325901 treatment (Fig. 4e,f). Notably, inhibition of glucose metabolism (2-DG and BrPA) also caused significant ERK inactivation, but this was not attributed to the HBP, as azaserine-treated cells retained ERK activity (Fig. 4e,f). We functionally validated this by maintaining directed-differentiating mesoderm cells in glucose, pyruvate and glutamine-free medium for 6 h, after which ERK signal was only reactivated after the re-addition of glucose (Fig. 4g).

This suggests that glucose metabolism is differentially regulated in epiblast and mesodermal tissues across the embryo. Just as the activity of a morphogen can vary in various developmental contexts, we speculate that glucose utilization may also vary to guide distinct processes during gastrulation. Our data suggest that whereas initial glucose utilization through HBP is crucial for ERK signalling in the epiblast, indicating hierarchical regulation, glucose metabolism appears to correlate with ERK signalling through a different, perhaps less direct, mechanism once cells are committed to the mesodermal fate.

Late glycolysis in lateral mesoderm

During gastrulation, FGF–ERK activity occurs in two temporal waves: first in posterior epiblast cells before primitive streak ingression, and then during mesenchymal migration^24,28,29. Activated through various RTKs, this pathway controls fate transitions in the posterior epiblast and localizes within the mesodermal precursors to facilitate mesodermal commitment and their anterior migration away from the primitive streak. The FGFR1 mutant phenotype of mouse gastrulas was previously shown to display posterior build-up of cells in the primitive streak, interpreted as a migratory failure of mesoderm progenitors to exit the streak^25,31 or an impaired mode of cell ingression^41,42.

In line with these observations, when we treated mouse gastrulas with SU5402 (FGFR inhibitor) or PD0325901 (ERK inhibitor) around E6.75, we observed a similar primitive streak-specific phenotype (Extended Data Fig. 9b). Notably, our data support a close interaction between HBP and ERK signalling in the epiblast tissue, however, embryos under HBP inhibition did not phenocopy the primitive streak build-up observed in SU5402- or PD0325901-treated embryos (Extended Data Fig. 9b). Whereas embryos, gastruloids and mesoderm-directed-differentiation assays comprehensively showed no specific effect of YZ9 treatment (to inhibit rate-limiting enzymes of the latter steps of glycolysis) on mesoderm specification and primitive streak elongation (Fig. 2b and Extended Data Figs. 4a and 5a,b), quantification of the primitive streak–mesoderm surface area in sagittal sections of YZ9- or PD0325901-treated gastrulas showed a significant increase in these embryos (Extended Data Fig. 9b–e). These results suggest that whereas the HBP is involved in guiding cell-fate transitions within the epiblast, the HBP is not critical for cells once they ingress into the streak and specify toward mesoderm. Instead, late-stage glycolytic enzymes may become important when cells adopt a stable mesodermal fate and begin their lateral migration away from the midline of the embryo (Extended Data Fig. 9c). Increased numbers of phospho-histone H3-expressing cells within the primitive streak were observed in YZ9- or PD0325901-treated embryos (Extended Data Fig. 9f,g), but not in those treated with both drugs (Extended Data Fig. 9g,h). Finally, we observed that ERK inhibition via PD0325901 treatment led to increased glucose uptake into mesodermal wings at late-stage gastrulation (E7.5) (Extended Data Fig. 9i). Although this seemingly mesoderm-specific mechanism that suggests a relationship between ERK signalling and late-stage glycolysis would support previous observations in chick development⁶, future work examining the precise connection between the latter steps of glycolysis and ERK signalling would be beneficial to understand these phenomena.

Glycolysis supports mesoderm migration

Guided by these data, we next investigated whether later glycolysis steps might influence mesoderm-specific cellular behaviours, such as motility. Indeed, YZ9- or PD0325901-treated embryos demonstrated reduced migration distance of SNAI1⁺ lateral mesoderm compared with controls (Extended Data Fig. 10a).

To study this further, we isolated mesoderm explants from E7.25/7.5 embryos at the late streak-to-no bud stage using TCF/LEF:H2B-GFP reporter mice, and cultured them on fibronectin-coated dishes for 24 h ex situ (Fig. 4a). The explant surface expansion during ex situ culture showed a significant decrease with global glucose metabolism inhibition (2-DG and BrPA), indicating impaired cell migration from the explant centre (Extended Data Fig. 10b). However inhibition of ERK (PD0325901), late glycolysis (YZ9) or HBP (azaserine) did not result in a significant difference in the explant surface area expansion compared with controls (Extended Data Fig. 10b). Quantifying cell movements through time-lapse live imaging revealed an increase in proliferation rate, but decreased cell displacement after PD0325901 and YZ9 treatments (Fig. 4b and Extended Data Fig. 10c). This indicates that explant expansions in PD0325901 and YZ9 groups were driven primarily by increased cell divisions, rather than actual migration. This further confirms the expanded primitive streak sizes of PD0325901 and YZ9-treated embryos (Extended Data Fig. 9b–g) are likely to be a combined result of increased proliferation and aberrant migration. Azaserine-treated explants did not show these effects, suggesting again that HBP is not required for mesodermal cell migration post-streak (Fig. 4b and Extended Data Fig. 10c). Explants treated with 2-DG and BrPA showed reduced migration without increased proliferation (Fig. 4b), possibly because the combination of 2-DG and BrPA affects multiple branching glucose metabolism pathways (Fig. 2a), which may mitigate proliferative effects.

To functionally test whether glycolytic pathway inhibition affects mesodermal cell migration, we explanted mesoderm tissue isolated from wild-type embryos on FITC–fibronectin, a fluorescent substrate that reveals fluorescence-devoid regions where migratory mesenchymal cells have extended their protrusions to degrade the underlying extracellular matrix⁴³ (Fig. 4c). This invadopodia assay showed significantly less substrate degradation in explants treated with 2-DG and BrPA, YZ9 or PD0325901, but no difference in those treated with azaserine, indicating that late glycolysis and ERK signalling affect mesoderm migration, whereas the HBP is decoupled from this process (Fig. 4c). Immunolabelling under late glycolysis inhibition demonstrated upregulation of the adhesion marker ECAD (Fig. 4d) with retention of the mesenchymal markers NCAD, EOMES and LEF1 (ref. ⁴⁴) (Extended Data Fig. 10d,e), similar to previous observations in FGF-mutant embryos²⁵.

Finally, we performed RNA sequencing on mesoderm explants treated with PD0325901 or YZ9 ex situ. Pathway enrichment analysis identified differentially expressed genes and revealed a downregulation of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway terms, including ‘focal adhesion’, that have direct roles in cell migration⁴⁵, as well as ‘ECM–receptor interaction’ and ‘MAPK signalling pathway’ (Fig. 4e). Differential GO (Gene Ontology) term analysis showed that significantly downregulated processes were similar between late-stage glycolysis and ERK-inhibited groups, including essential aspects of mesoderm function such as focal adhesion and integrin signalling (Extended Data Fig. 10f).

Collectively, these data support the hypothesis that late-stage glycolysis in established mesoderm regulates the cell movements crucial for successful triploblastic development of mouse embryos.

Discussion

Mammalian embryos undergo a conserved metabolic shift to glucose metabolism at the time of implantation, which was historically assumed to present a uniform metabolic phenotype across spatial and temporal scales with the same metabolic pathways employed over different tissues. However, our findings reveal that this glucose shift is critically fine-tuned to drive distinct cell differentiation and morphogenetic processes that control gastrulation. Our data demonstrate a preference for sugar processing and differential metabolic reliance within specific tissues of the embryo—when these specific metabolic requirements are not met, crucial morphogenetic processes are hindered (Fig. 4f). Increasing in vitro evidence links metabolism with cell fate. Our work now demonstrates that metabolic changes can both phenocopy and rescue cell signalling defects that impair key developmental processes in vivo, revealing a novel perspective that glucose metabolism is not only ‘life-sustaining’, but also ‘life-directing’ during early embryogenesis.

Indeed, advances in experimental methods such as carbon tracing and improved ex vivo embryo culture have highlighted the potential instructive role of metabolism in embryogenesis^8,10. Consequently, the concept of metabolic guidance during development has gained momentum, and recent findings have revitalized interest in the ‘gradient theory’ that was first proposed in the early twentieth century¹¹. Our work expands on this theory to explain how metabolic gradients shape developmental processes. In a broad context, we demonstrate how the selective use of a single nutrient interacts with molecular signalling networks during development, which supports a more integrative, metabolite-guided, view of embryogenesis. ‘Metabolic signalling’ offers a useful paradigm for understanding how the entire body plan is established during early development, a time when a semblance of organismal identity is first acquired.

Mechanistically, our study uncovers a link between distinct glucose metabolism branches and signalling regulation. HBP, a nutrient-sensing pathway, produces UDP-GlcNAc, which is essential for HSPG biosynthesis and post-translational modifications that are vital for cell-fate transitions^32,33,46,47. Our observations that ERK signalling follows glucose uptake in epiblast cells and is rescued by GlcNAc supplementation, alongside HBP inhibition reducing HSPG expression, underscore the role of UDP-GlcNAc in signalling transduction in epiblast tissue. Indeed, while this initially preprinted manuscript⁴⁸ was under revision, a study using mouse gastruloids as an in vitro system revealed that mannose, a glucose epimer, is essential for glycosylation during mesoderm specification and axial elongation⁷. Two additional recent studies that utilized 2-DG treatment in mouse gastruloids and derivative trunk-like structures^49,50 have also highlighted the role of glucose metabolism in modulating cellular signalling^7,49 and in facilitating proper neural and mesodermal formation⁵⁰. These findings support our conclusions regarding the effect of glucose on epiblast cell-fate transitions.

We provide a holistic view of early embryonic development in a living organism, demonstrating that compartmentalized glucose usage varies depending on the developmental context and the cell types involved. The specifics of how ERK signalling interacts with glucose metabolism steps, whether through post-transcriptional modifications by HBP^18,19, regulation of glycolytic transcripts⁶ or a combination of both, remain to be explored further. Future studies could explore crosstalk between ectoderm, endoderm and mesoderm lineages during late gastrulation or after gastrulation, especially considering the global reduction in SDC1 and 10E4 expression in HBP-inhibited embryos in our data. Although our experiments show that general mesenchymal migratory dynamics are HBP-independent, HBP may have other roles in mesoderm, such as migration directionality or final migratory location, that we did not investigate. It is also conceivable that carbon sources from other substrates, such as glutamine or fatty acids (both of which feed into HBP) could become preferred by committed mesenchymal cells to influence cellular behaviour.

Signalling ligands such as FGF8 and FGF4 that localize to the primitive streak, node and mesodermal tissues during the post-implantation period are crucial for regional cell-type specification and gastrulation patterning. Our findings suggest that compartmentalized activity of glucose metabolism provides necessary intermediates for cells to respond to key morphogens, indicating a synergistic relationship between glucose metabolism and signalling activities in embryonic regions. The differentiation from epiblast to mesoderm is likely to be driven by the combined influence of the cells’ metabolic status and the presence of specific signalling ligands, which together induce mesodermal fate. Thus, the interplay between localized metabolic activity and signalling not only supports, but actively directs, the epiblast to mesodermal transition.

Future research will benefit from current advancements and certain limitations of our study. Chemical inhibitors offer rapid control, mitigating embryonic lethality typically linked with genetic metabolic perturbations. However, predicting system-level effects of specific metabolic reaction inhibition remains challenging. Our study lacks quantitative data that precisely measures the degree of inhibition achieved by these inhibitors, primarily owing to difficulties in conducting metabolic flux analyses in post-implantation mammalian embryos at cell- and tissue-level resolution. Potential partial inhibition might trigger compensatory cellular responses that are not present under complete inhibition, possibly explaining some variability in our results. Despite these challenges, our study establishes dose–response relationships that demonstrate different degrees of the same phenotype, reinforcing the view that the observed effects are consistent and significant regardless of inhibition level. Our findings align with existing literature validating the efficacies of these inhibitors, and we demonstrate their specificity through in vivo functional rescue experiments and identify tissue-specific effects using stem cell and tissue explant models. As single-cell and spatial metabolomics become more feasible, future research is likely to provide deeper insights into metabolic processes and their implications for developmental biology and related fields.

As the saying goes, the best-laid plans of mice (and humans) can often go awry. In the context of developmental biology, our findings can offer crucial insights into how the body plan is created during early life, and the mechanisms behind developmental lethality that arise from deregulated signals and perturbed metabolism. These results underscore the importance of metabolism for embryonic patterning and developmental success, and are highly applicable to other contexts in which cellular transitions and migration take place, such as cancer biology and regeneration.

Methods

Data reporting

No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.

Mouse embryo recovery

Mice were maintained in accordance with national and international guidelines. All experimentation involving animal subjects was approved by the Institutional Animal Care and Use Committee at Yale School of Medicine and conducted following the approved animal handling protocol (protocol no: 2023-20352). All experimental mice were maintained in specific pathogen-free conditions on a 12 h:12 h light:dark cycle temperature-controlled facility (between 20–26 °C and humidity 30–70%) with free access to water and food, and used from 6 weeks of age. All mice were bred to a mixed CD1 albino background. V. Greco provided the H2B-GFP:Tcf-LEF⁵¹ reporter mouse line.

To collect in vivo embryos at gastrulation stage, 5- to 7-week-old female CD1 mice were naturally mated with 12- to 24-week-old male CD1 mice and sacrificed 6.5, 6.75, 7.0 or 7.25 days post coitum. Uteri were recovered and embryos were dissected from deciduae in DMEM medium containing 5% FBS, 20 mM HEPES, and 1× Penicillin-Streptomycin (Gibco) warmed up to 37°. The sex of the embryos was not determined.

Embryo culture at gastrulation

Embryos were cultured in a 1:1 ratio of DMEM F/12 (Gibco) to rat serum (Envigo), with 1× Glutamax (Gibco), 0.2× Penicillin-Streptomycin (Gibco), and 0.2× MEM NEAA (Gibco) in 37 °C at 20% O₂ and 5% CO₂. Medium was equilibrated in 5% CO₂ for ≥15 min prior to culture.

Tetraploid complementation

Mouse embryos at the pre-implantation 2-cell stage (E1.5) were recovered in KSOM medium by flushing the oviduct, from 5- to 6-week-old CD1 females that were superovulated by injection of 10 IU pregnant mare serum gonadotropin (ProSpec) followed by 10 IU human chorionic gonadotropin (Sigma) after 48 h and were mated with CD1 males. Two-cell-stage embryos were then fused to induce tetraploidy using BTX Embryo Manipulation Electro Cell Fusion System. Fused embryos were transferred to advanced KSOM (Sigma) covered with mineral oil (FUJIFILM) and cultured to blastocyst stage until embryonic stem cell injection. Each host tetraploid blastocyst was injected with 10–15 mouse ES cells, the injected blastocysts were transferred into uterus of the prepared day 2.5 CD1 pseudopregnant surrogates (6 weeks old) which were plugged by vasectomized CD1 male mice. These mouse embryonic stem cell-derived embryos were collected at the certain developmental timeline for subsequent experiments by uterine dissection (as described above).

Metabolism and signal modulation experiments

Chemical inhibitors were administered as follows except where otherwise stated: 2 mM 2-DG⁵² (Santa Cruz Biotechnology), 20 µM BrPA (Santa Cruz Biotechnology), 10 µM PD0325901 (StemCell Technologies), 10 µM SU5402 (StemCell Technologies), 200 µM galloflavin⁵³ (Cayman Chemical), 100 µM oligomycin⁵⁴ A (Santa Cruz Biotechnology), 5 µM 6-AM⁵⁵ (Cayman Chemical), 10 µM YZ9⁸ (Cayman Chemical), 5 µM shikonin⁵⁶ (Cayman Chemical) and 5 µM azaserine⁵⁷ (Cayman Chemical or Santa Cruz Biotechnology). Embryos were treated for 4 h, 7 h, 12 h or 18 h. Mesoderm explants were treated for 3 h, 12 h or 16 h. Stem cells were treated for the duration of the experiment as indicated in the figure captions. The proteoglycan sulfation inhibitor NaClO₃ (Sigma) was applied at 20 mM. FGF2, FGF4 and FGF8 (all Peprotech) were applied at 50 ng ml⁻¹.

Nutrient-sparse and rescue experiments

Nutrient-sparse medium was prepared using an Advanced DMEM/F-12 medium devoid of d-glucose, l-serine, l-glutamine and sodium pyruvate (Caisson Laboratories) with the addition of 25 mM sodium bicarbonate (Sigma), 0.2× penicillin-streptomycin (Gibco) and 0.2× MEM NEAA (Gibco). This was supplemented with the following nutrients, according to each experimental condition: 17 mM glucose (Gibco), 0.5 mM sodium pyruvate (Gibco), and/or 1× Glutamax (Gibco). For galactose rescue experiments 20 mM galactose (Sigma) supplemented to the culture medium. For GlcNAc rescue experiments 1 mM or 2 mM GlcNAc (Sigma) supplemented to the culture medium. All embryos were cultured in a 1:1 ratio of customized DMEM and rat serum (Envigo).

Cell culture, gastruloids and in vitro mesoderm-directed-differentiation assay

All mouse embryonic stem cells were cultured at 37 °C in 20% O₂ and 5% CO₂ and passaged once they had reached 80% confluency. Cells were routinely tested for mycoplasma contamination by PCR. Mouse embryonic stem cells were cultured on gelatinized tissue-culture–grade plates in FBS-containing DMEM medium with 2i/LIF (1 μM MEK inhibitor PD0325901, 3 μM GSK-3 inhibitor CHIR99021 and 10 ng ml−1 LIF). FBS-containing DMEM (Gibco) medium comprised of: 18% inactivated FBS (Gibco), 1.2× penicillin-streptomycin (Gibco), 1.2× Glutamax (Gibco), 1.2× MEM NEAA (Gibco), 1.2 mM sodium pyruvate (Gibco), and 120 µM 2-mercaptoethanol (Gibco). The ERK-KTR mouse embryonic stem cells used in this study were sourced from Simon et al.³⁴ (JAX 035333).

For in vitro mouse gastruloid experiments, we followed the protocol as described^20,21. Gastruloids were treated with indicated drugs culture day between 3 and 4 and fixed for subsequent HCR staining or qPCR assay, as described in Dingare et al.⁷.

For in vitro mesoderm-directed differentiation experiments, we followed the protocol as described⁵⁸, with some modifications. Cells were passaged (day 0) after reaching 80% confluency into 6-well culture plates or 8-well µ slides (Ibidi) at a density of 20,000 per cm² in FBS-containing DMEM medium with 10 ng ml⁻¹ Fgf2 (R&D Systems). Cells were treated at day 1 with 10 ng ml⁻¹ FGF2, at day 2 with 10 ng ml⁻¹ FGF2 and 5 µM CHIR99021 (StemCell Technologies), and at day 3 and 4 with 5 µM CHIR99021. Drug treatments added to the culture at least 12 h after FGF2 treatment was started on day 1, or on when cells transitioned to mesoderm state on day 3.

In situ mesoderm explant assay

For mesoderm isolation from E7.25 mouse embryos, we followed a modified protocol as described⁵⁹. Embryos were collected as described above. Extraembryonic tissue proximal to the amnion was removed, and the cup-shaped embryo was transferred to a dissociation medium consisting of 0.5% Trypsin EDTA (Gibco) and 2.5% Pancreatin (Thermo Scientific Chemicals) for 15 min at 4 °C. Embryos were washed with collection medium (described earlier) and mesoderm tissue was dissected using insect pins (Roboz) attached to syringes. Explants were transferred to fibronectin-coated 18-well µ slides (Ibidi) plates and left to adhere for 3–4 h in medium containing a 1:4 ratio of rat serum to embryo culture medium (described earlier), prior to downstream experiments and/or chemical inhibitor treatments. For live imaging of migration dynamics, explants were imaged every 5 min up to 3 h. For immunofluorescence staining, explants were cultured up to 17 h and fixed in PBS containing 4% paraformaldehyde (PFA) for 20 min. For RNA sequencing, explants were cultured to 27 h then scraped off with sterile insect pins and flash-frozen.

Glucose-uptake assay

Mouse embryos were collected at early streak, mid streak and late streak stages of gastrulation and cultured with 1 mM 2-NBDG⁶⁰ (Cayman Chemicals) for 2 h. For multiphoton microscopy, embryos were immediately live-imaged. For confocal microscopy, embryos were fixed in PBS containing 4% PFA for 15 min then immediately imaged after a 5 min wash in PBS-T (PBS with 0.05% Tween-20).

DQ gelatin assay

Cells underwent mesoderm-directed differentiation on 8-well µ slides (Ibidi) as previously described. On day 2.5 when cells are at the EpiSC stage, cells were cultured with differentiation medium that included 50 µg ml⁻¹ DQ gelatin (Invitrogen), then were fixed on day 3.5 in PBS containing 4% PFA for 20 min, protected from light. Cells were incubated in blocking buffer (described below) with DAPI (overnight at 4 °C or 20 min room temperature) prior to imaging.

Invadopodia assay

18-well µ slides (Ibidi) were prepared with FITC–Fibronectin (Sigma-Aldrich) and 0.1% gelatin following the manufacturer’s 2-day protocol and protected from light. Explants were cultured on these plates for 16 h prior to fixation in PBS containing 4% PFA for 20 min. Explants were incubated in blocking buffer (described below) with DAPI (overnight at 4 °C or 20 min room temperature) prior to imaging.

Labelling active mitochondria

Mouse gastrulas were collected at ES stage and cultured with either 200 nM MitoTracker Deep Red or 100 nM tetramethylrhodamine, methyl ester, perchlorate (TMRM). Embryos were cultured in the respective dyes for 30 min and then imaged in a humidified chamber with 37 °C and 5% CO₂ according to the same parameters described below for live imaging.

Immunofluorescence staining

Samples were fixed in PBS containing 4% PFA for 20-45 min room temperature, or in methanol for 20 min at 4 °C where stated. After fixation, samples were washed twice with PBS-T (PBS with 0.05% Tween-20) and permeabilized in PBS with 1 mM glycine and 0.3% Triton X-100 for 20–60 min at room temperature. Primary antibody incubations took place overnight at 4 °C in blocking buffer (PBS containing 10% fetal bovine serum (FBS), 10% Tween-20). Samples were washed twice with PBS-T prior to secondary antibody incubations at 4 °C in blocking buffer. On the final day, samples were washed twice with PBS-T, then transferred into PBS-A droplets (PBS with 0.75% Bovine Albumin Fraction V (Gibco)) and covered with mineral oil (Sigma-Aldrich) in 35 mm glass-bottom dishes (MatTek) before confocal imaging. All PBS-T washes were done for 10 min room temperature, and all room temperature incubations or washes took place on a rocking platform. All antibodies used in this study are listed in Supplementary Table 1.

Image data acquisition and processing

Samples were imaged with the Leica STELLARIS 5 microscope using a HC PL APO CS2 40×/1.10 or a HC FLUOTAR L 25×/0.95 W VISIR 0.17 water objectives, a z-spacing of 0.75 µm to 5 µm and appropriate laser and filters for Alexa 405, Alexa 488, Alexa 546 and Alexa 633 or combinations thereof. To correct for fluorescence decay along the z axis during embryo imaging, ‘z-compensation by AOTF and PMT’ was defined in a control embryo and applied across all experimental conditions during that imaging session, so that changes in laser power and gain across the z-stack were equivalent across conditions, and only adjusted to each embryo’s size. Raw data were processed using open-source image analysis software Fiji/ImageJ2 2.9.0 or AIVIA 10.5.1 AI Image Analysis Software and assembled in Photoshop 2021 22.3.1 (Adobe) or Illustrator 2024 28.6 (Adobe). Transverse views were generated from 1 µm z-spaced images using ImageJ’s orthogonal viewer. Digital quantifications and immunofluorescence signal intensity graphs were obtained using plot profile measurements in Image J and visualized in GraphPad Prism10.2.3. software.

Time-lapse live imaging

Confocal time-lapse imaging of embryos, mesoderm explants, and mesoderm-differentiated cell cultures were performed using Leica STELLARIS 5 microscope using a 25× (HC FLUOTAR L 25×/0.95 W VISIR 0.17) or 40× (HC PL APO CS2 40×/1.10) water objective and appropriate laser/filters for Alexa 488, Alexa 546 and Alexa 633 or combinations thereof. Samples were imaged under a humidified chamber with 37 °C and 5% CO₂. Explants were imaged at 5 min intervals in 2 µm z-spaced planes for up to 4 h on pre-treated ibidi dishes (Ibidi). Images were processed using AIVIA, described below under ‘Image analysis’.

Multiphoton live imaging for NAD(P)H autofluorescence

Multicolour two-photon microscopy was used for live-embryo imaging of NAD(P)H dynamics. Embryos kept in ex vivo culture medium (described earlier) with the addition of 20 mM HEPES. Images were acquired with LA Vision TriM Scope II (LaVision Biotec) laser scanning microscope equipped with a Chameleon Vision II and Discovery ultrafast lasers (Coherent) for different wavelengths imaged sequentially after each z-section¹⁵. Wavelengths of 750 nm were used for NAD(P)H and 940 nm for H2B-GFP:Tcf-LEF or ERK-KTR^mClover reporter. Although excitation ranges of NAD(P)H and GFP overlapped, their emission was separated by band pass filters: blue range (425–475 nm; NAD(P)H) and green range (500–550 nm; FAD). Exclusion of nuclear GFP signal (in H2B-GFP:Tcf-LEF embryos) from NAD(P)H channel validated separation of emission signals of NAD(P)H and GFP. Embryos were imaged using a 40× water immersion lens (Nikon; NA 1.15) at 400 Hz with pixel size of 0.2 µm or 0.3 µm and a z-step of 1 or 1.5 µm. For optimal signal-to-noise ratio, Line averaging of 2 was done for all NAD(P)H images. The fluorescence detected from the reduced metabolites through this method captures both NADH and NADPH (thus called NAD(P)H)⁶¹. It should be noted that the intracellular concentrations of the non-phosphorylated NADH and NAD+ is much higher than NAD(P)H and NADP⁺ (ref. ⁶²). We further validated that NAD(P)H fluorescence signal in the embryos closely co-localized and followed 2-NBDG uptake.

Image analysis

All embryos were positioned exactly along the A–P axis (refer to figures) during imaging for easier quantifications.

Angle of GLUT expression in the epiblast

Mid-embryo sagittal sections of embryos stained for GLUT1 and GLUT3 and with DAPI were used for glucose-uptake quantification in Fiji/ImageJ2. For each embryo, the angle vertex was allocated at the proximal-most boundary between epiblast and extraembryonic ectoderm, at the mid-point between posterior-most (0°) and anterior-most (180°) epiblast. Two angle values were calculated for every embryo (‘GLUT start’ and ‘GLUT end’) to capture the range of observable GLUT expression in the epiblast, along with the primitive streak distal length of the embryo to allocate Theiler staging (ES, MS or LS, as described above). ‘GLUT start’ and ‘GLUT end’ means were calculated for each stage and visualized in a rose diagram with RStudio.

Primitive streak distal elongation percentage

Mid-embryo sagittal sections of DAPI-stained embryos were used for primitive streak distal elongation quantification in Fiji/ImageJ2. For each embryo, the 0% cut-off was marked by anterior epiblast morphology, the 100% cut-off was assigned to the distal-most epiblast cell, and ‘PS’ (primitive streak) was marked at the distal-most point where the primitive streak morphology extends (Fig. 2b). Measurements were obtained for the vertical distance between 0% and 100% and between 0% and ‘PS’, so that each embryo’s primitive streak elongation percentage (0-to-PS divided by 0-to-100) is adjusted to its size. Thus, a control embryo’s primitive streak distal elongation percentage of 95% (Fig. 2b) can be interpreted as ‘this embryo has elongated its primitive streak to 95% of its epiblast height’. Theiler stages could be assigned with this measurement: ≤50 for ES; between 50 and 100 for MS; >100 for LS.

Basement membrane breakdown

Mid-embryo sagittal sections of laminin and DAPI-stained embryos were used for basement membrane calculations in Fiji/ImageJ2. For each embryo, the 0% cut-off was marked by anterior epiblast morphology, the 100% cut-off was assigned to the distal-most epiblast cell, and ‘intact BM’ was marked at the distal-most point where laminin was still intact (Fig. 2d). Measurements were obtained for the vertical distance between 0% and 100% and between 0% and ‘BM’, so that each embryo’s basement membrane breakdown percentage (0-to-BM divided by 0-to-100) is adjusted to its size. Thus, a control embryo’s basement membrane breakdown readout of 96% (Fig. 2d) can be interpreted as ‘this embryo has broken down 96% of its vertical basement membrane length’.

FITC–fibronectin invadopodia assay

z-sections of DAPI-stained mesoderm explants imaged with a 40× objective were used for fibronectin perforation calculations, with ≥3 images captured per explant. For every image, cell number and perforation number were calculated. Invadapodia degradation is represented as a percentage (perforation number divided by cell number).

ERK-KTR nuclear:cytoplasmic ratio quantification

Sagittal sections of ERK-KTR embryos were used to quantify ERK activity. Manual segmentations were drawn for each cell in Fiji/ImageJ2 to delineate nuclear and cytoplasmic areas, using ERK-KTR and brightfield channels to verify cell morphologies (Extended Data Fig. 8b). For every cell, the following measurements were quantified: nuclear area n^a, cytoplasmic area c^a (including the nucleus), nuclear ERK-KTR intensity nⁱ, and nuclear-subtracted cytoplasmic ERK-KTR intensity cⁱ. To calculate the nuclear:cytoplasmic ratio, the average nuclear intensity (nⁱ/n^a) was divided by the average cytoplasmic intensity (cⁱ/(c^a − n^a)), such that a ratio ≥1 indicates no ERK activity.

Proliferation quantification

Live-imaged videos captured with a 40× objective were used for proliferation counts of TCF-LEF reporter mesoderm explants, quantified manually in Fiji/ImageJ2. For each explant, an overall cell count for ‘starting population’ was calculated on the first frame, and a ‘cell division’ event (TCF-LEF telophase observation) was also assigned by careful frame-to-frame assessment over the course of the video. A proliferation index (cell division number/starting population) was then obtained for each explant, such that a highest index of 1 can be interpreted as ‘every cell at the beginning of the video has divided by the end of the video’. This was then adjusted to the total frame length of the video so that explants across different experimental replicates and video lengths could be compared. Thus, a control explant with a readout of 16.5% (Fig. 4b) can be interpreted as ‘16.5% of the mesoderm cells at the beginning of the video have divided by the end of the video’.

AIVIA-based image analysis

AIVIA 10.5.1 AI image analysis software was used to examine mesoderm explant migration dynamics. Nuclear segmentations were generated using a ‘cell-tracking’ recipe, applying a pixel classifier that was trained on TCF-LEF nuclear fluorescent channels from videos of each treatment group in every experimental replicate (Fig. 4b). Parameter values were modified between rounds of pixel classifier ‘previewing’ and training to ensure nuclear segmentation accuracy. Every track was manually examined against the brightfield channel to verify detection accuracy. Incorrect lineages were corrected in the ‘track editor’ or discarded from the final dataset, and parent and daughter tracks were treated independently. Every track measurement of interest was exported to Excel and normalized to its detection length (‘first frame’ subtracted from ‘last frame’) so that lineages of different detection lengths could be compared. Thus, all data points are plotted in µm min⁻¹ or µm s⁻¹.

Directed-differentiated mesoderm cell counting

Imaris 10.0.1 software was used to count T-positive, total, and pyknotic cells. Nuclear segmentations were generated using the ‘Surfaces’ function. Parameters such as background subtraction and morphological splitting were adjusted using the Creation Wizard tool to identify the optimal algorithm settings. Pyknotic cells were counted in the DAPI channel using an increased threshold for background subtraction and a smaller diameter for segmentation than when counting healthy cells using the DAPI signal. Surfaces were examined relative to the original staining throughout creation and in their final state to ensure accuracy.

Western blot

Protein was extracted using RIPA buffer (Thermo Scientific 89900). The reagents were supplemented with 1× protease inhibitor (Thermo Fisher Scientific, 87786) and 1× phosphatase inhibitor (Phos Stop Roche 4906845001) to prevent protein degradation and dephosphorylation during extraction. The protein concentrations were quantified using Pierce BCA. protein assay kit (ThermoFisher, 23225) following the manufacturer’s instructions. The protein was denatured at 95–100 °C for 5 min in 1× NuPAGE LDS sample buffer (ThermoFisher, NP0007) containing 10% v/v β-mercaptoethanol (Millipore Sigma, M6250). The protein electrophoresis was performed using 4–20% precast polyacrylamide gels (Bio-Rad, 4568095) in 1× Tris/Glycine/SDS buffer (diluted from Bio-Rad, 1610732) at 110 V for 1 h for all proteins. The proteins were transferred from the gel onto a nitrocellulose membrane (ThermoFisher, IB301032) by wet transfer for 1 h at 100 V using the Bio-Rad Mini Trans-Blot Cell following the manufacturer’s instructions. Wet transfer buffer was 20% methanol, 200 mM glycine, and 250 mM Tris. The membrane was blocked in 5% milk in TBST for 1 h at room temperature with gentle shaking. Primary antibodies were incubated with the membrane at recommended concentrations at either 1 h room temperature or 4 °C overnight with gentle shaking. The membrane was washed three times in TBST for 5 min each, followed by incubation with horseradish peroxidase-conjugated secondary antibodies diluted at 1:5,000 ratio in 5% non-fat milk in TBST for 1 h at room temperature. The blots were then washed three times in TBST for 5 min each. Blots were treated with SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher, 34094) for 2–5 min at room temperature. The blots were imaged using a CCD camera-based imager ProteinSimple FluorChem E system.

qPCR with reverse transcription

Total RNA was extracted from cells using an RNeasy Micro Kit per the manufacturer’s instructions (Qiagen). cDNA synthesis was performed with 1 μg of total RNA using a High-Capacity cDNA Reverse Transcription Kit according to the manufacturer’s instructions (Applied Biosystems). The amounts of mRNA were measured using the PowerUp SYBR Green PCR Master Mix (Applied Biosystems). Relative levels of transcript expression were assessed by the ΔΔC_t method, with Gapdh as an endogenous control. For primers used in qPCR with reverse transcription, see Supplementary Table 2.

RNA-sequencing sample collection and data analysis

Total RNA was extracted from mesoderm explants using a PicoPure RNA Isolation Kit per the manufacturer’s instructions (Applied Biosystems). RNA samples were submitted to the Yale Center for Genome Analysis for quality assessment, library preparation and sequencing. Each RNA sample contained explants from 2 embryos, and 2 samples were submitted per condition. Paired-end reads were aligned using Star (v2.7.9a) to the mouse genome (GRCm38) using the Ensembl transcriptome (release 109)⁶³. Analysis of differential gene expression was performed using DESeq2 (v1.40.1). To identify differentially regulated genes between samples for downstream analyses, we selected genes with log₂-transformed fold change greater than 0.7 or less than −0.7 and an adjusted P value < 0.01. Gene ontology analysis was performed using topGO (v2.52.0) using the ‘weight01’ algorithm for Fisher’s exact tests on R studio (version 2023.12.1 + 402). KEGG pathway analysis was performed using DAVID (https://david.ncifcrf.gov/tools.jsp)^64,65.

Reanalysis of single-cell sequencing data

Previously published single-cell RNA sequencing data from gastrulating mouse embryos were accessed using MouseGastrulationData (v1.12.0) (https://github.com/MarioniLab/MouseGastrulationData). Data were subset to include only ‘epiblast’, ‘primitive streak’ and ‘nascent mesoderm’ cell states from all samples staged E6.5 to E8.5 (samples 1–10, 12– 20 and 23– 37; samples 11, 21 and 22 were omitted from our analysis because their staging was ambiguous, for example, a sample labelled as ‘mixed gastrulation’). Counts were log-normalized using Seurat (v. 4.3.0)^{66,67,68,69,70}. Cells were first randomly down-sampled for visualization purposes to 400 cells per cell type. Pseudotime and trajectory interference analyses were performed using Slingshot (v2.8.0)⁷¹ for principle curve calculation; SingleCellExperiment (v1.22.0)⁷² and scater (v.1.28.0)⁷³ were used for visualization of gene expression over pseudotime. The principle curve was then traced over the first two principal components to infer pseudo-temporal organization.

Statistics and reproducibility

Statistical tests were performed on GraphPad Prism 10.2.3 software. For comparison of two groups, two-sided unpaired t-tests were applied. For comparisons of three or more groups, one-way ANOVAs were used, followed by Dunnett’s multiple comparison tests for comparisons to control or Tukey’s multiple comparison tests for comparisons between all groups, unless otherwise stated. P values are displayed on figure panels or in legends. All the experiments were performed at least in three biological replicates unless specifically described in the methods and the figure legends. Figure legends indicate the number of embryos, cells, explants or stem cell structures and, when relevant, the number of experiments performed for each analysis. Statistical power calculations were not used to determine sample size.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The RNA-seq data for mesoderm explants generated in this study have been deposited in the Gene Expression Omnibus under accession number GSE271973. Published mouse embryo single-cell RNA-seq datasets used in this study were obtained from Pijuan-Sala et al.²⁶ under accession number E-MTAB-6967 (processed data available at https://github.com/MarioniLab/EmbryoTimecourse2018). Source data are provided with this paper.

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Acknowledgements

The authors thank K. Sumigray for support with confocal microscopy; Z. Smith and Smith laboratory members for support with microinjection and preparation of pseudopregnant mice for tetraploid complementation assays; Yale Genome Editing Center for support in tetraploid complementation experiments; T. Finkelstein for support with mouse experiments and illustrations shown in Extended Data Fig. 1c; members of the Sozen laboratory for discussions throughout the project; and Z. Smith for helpful feedback on the manuscript. J.B. is supported by the NIH NRSA F31 pre-doctoral fellowship. A.H. is a NYSCF-Druckenmiller Fellow and is supported by The New York Stem Cell Foundation. V.G. is supported by NIH grants number 1R01AR063663, 1R01AR067755 and DP1AG066590. B. Steventon is supported by a Wellcome Trust Discovery Award (225360_Z_22_Z). Work in the Steventon laboratory was funded by MRC Research grant MR/ V009192/1 jointly held by C.D. and B. Steventon. The Sozen laboratory is funded by NIH Early Innovators Award (DP2HD112040), the Richard and Susan Smith Family Foundation, Reprogrants and American Society of Reproductive Medicine (ASRM). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Author information

These authors contributed equally: Dominica Cao, Jenna Bergmann

Authors and Affiliations

Department of Genetics, Yale School of Medicine, Yale University, New Haven, CT, USA
Dominica Cao, Jenna Bergmann, Liangwen Zhong, Anupama Hemalatha, Tyler Jensen, Andy L. Cox, Valentina Greco & Berna Sozen
Department of Genetics, University of Cambridge, Downing Site, Cambridge, UK
Chaitanya Dingare & Benjamin Steventon
MD–PhD Program, Yale University, New Haven, CT, USA
Tyler Jensen
Yale Stem Cell Center, Yale University, New Haven, CT, USA
Valentina Greco & Berna Sozen
Howard Hughes Medical Institute, Yale School of Medicine, New Haven, CT, USA
Valentina Greco
Department of Obstetrics, Gynecology and Reproductive Sciences, Yale School of Medicine, Yale University, New Haven, CT, USA
Berna Sozen

Authors

Dominica Cao
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Jenna Bergmann
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Liangwen Zhong
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Anupama Hemalatha
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Chaitanya Dingare
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Tyler Jensen
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Andy L. Cox
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Valentina Greco
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Benjamin Steventon
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Berna Sozen
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Contributions

D.C. and J.B. performed experiments and analysed data on mouse embryos and stem cells, with the help of B. Sozen. D.C. performed mesoderm explant experiments. J.B. performed analyses of RNA-seq datasets. L.Z. performed mouse tetraploid complementation assays. A.H. and D.C. established and performed multiphoton live imaging of NAD(P)H in embryos and A.H. and V.G. helped with data interpretation. C.D. and B. Steventon performed gastruloid experiments and provided critical discussions. T.J. performed western blot experiments and analysed the data. A.L.C. provided critical discussions and illustrations, and helped with manuscript writing. B. Sozen conceived and supervised the project, and wrote the manuscript with the help of D.C. and J.B. All authors reviewed the manuscript.

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Correspondence to Berna Sozen.

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Extended data figures and tables

Extended Data Fig. 1 Mouse gastrulation is preceded by compartmentalised glucose activity.

(a-c) Visualizing glucose uptake in early-streak (a) and mid-to-late-streak (b, c) mouse gastrulas via live-imaging of 2-NBDG (fluorescent glucose analog). Illustrations demonstrate developmental stage and plane of imaging. 15 independent experiments. (a) 2-NBDG signal is high in the transitionary epiblast on the posterior side of the embryo distal to the primitive streak. (b, c) 2-NBDG remains high in the transitionary epiblast distal to the primitive streak (purple cells in illustration, wave 1), is low in the primitive streak itself (grey cells in illustration), and is high in the lateral mesoderm that has exited the primitive streak as mesenchyme (yellow cells in illustration). Scale bars, 20 µm.

Extended Data Fig. 2 Characterisation of compartmentalised glucose activity in mouse embryos during gastrulation.

(a-d) Representative z-sections of GLUT1 (a, d) and GLUT3 (b, c) protein expressions (heatmap intensity colours via Fiji’s LUT) in relation to cell types of interest in mid- (MS) or late-streak (LS) stage mouse gastrulas. Zoom insets of the epiblast and primitive streak border shown on right. A, Anterior; P, Posterior. (a (n = 21 embryos), b (n = 7 embryos), c (n = 8 embryos), d (n = 14 embryos). Scale bars represent 40 µm. (e) Representative z-section of NAD(P)H and 2-NBDG in MS stage mouse gastrula. Zoom insets of the epiblast and primitive streak border shown on the right. Scale bars represent 40 µm. (f) Top: Schematic of mouse embryo transverse planes showing laser capture microdissection transcriptome spatial coordinates of Bottom: corn plots of primitive streak gene T, glycolytic genes Slc2a1, Gpi1, Pfkp, Ldhb, and HBP genes Ogt and Gnpnat1 generated by querying genes of interest from the online e-gastrulation Geo-seq database (Peng et al.¹⁶). A, Anterior; P, Posterior; AE, Endoderm Anterior; EP, Endoderm Posterior; M, Mesoderm; L, Left; R, Right.

Extended Data Fig. 3 Glucose metabolism via HBP controls mesoderm fate acquisition.

(a) Left: Schematic of glucose metabolism and its three branches: light-grey = Hexosamine Biosynthetic Pathway (HBP); pink = core/late-stage glycolysis; green = Pentose Phosphate Pathway (PPP). Red text indicates chemical inhibitors and their metabolic enzyme targets in blue. (b) Representative z-sections showing the expression pattern of SOX2, T/BRA, and LEF1 proteins in embryos treated with 2-DG+BrPA for 18 h ex vivo and (c) their Theiler developmental outcomes: Control (n = 17), 2-DG+BrPA (n = 24) across 6 experimental replicates; early-streak (ES), mid-streak (MS), late-streak (LS), no bud (OB), early bud (EB), late-bud (LB), early head fold (EHF). Scale bars represent 40 µm. Plots show mean ± SEM. On average, ~53% of control embryos develop to OB stage gastrulation, while ~73% of 2-DG+BrPA treated embryos only develop to MS stage gastrulation. (d) Dose response curves demonstrating efficacy of the inhibitors 2-DG (left; pre-culture, n = 7; control, n = 42; 0.5 mM, n = 5; 1 mM, n = 4; 2 mM, n = 7; 3 mM, n = 4; 5 mM, n = 5 embryos) and Azaserine (right; pre-culture, n = 7; control, n = 42; 1 μM, n = 3; 3 μM, n = 4; 5 μM, n = 12; 7 μM, n = 4; 10 μM, n = 3 embryos). Plots show mean and SD. Tukey’s multiple comparison tests following ordinary one-way ANOVAs. ****P < 0.0001 whenever indicated. Pre-culture vs. 1 mM 2-DG **P = 0.0066, pre-culture vs. 5 mM 2-DG **P = 0.0036, 1 mM 2-DG vs. 2 mM 2-DG **P = 0.0016, 1 mM 2-DG vs. 3 mM 2-DG *P = 0.0173, 2 mM 2-DG vs. 5 mM 2-DG *P = 0.0152, 3 mM 2-DG vs. 5 mM 2-DG *P = 0.0217. Pre-culture vs. 1 μM Azaserine *P = 0.0522, pre-culture vs. 3 μM Azaserine *P-0.0435, control vs. 5uM Azaserine ***P = 0.0006, 1 μM Azaserine vs. 10 μM Azaserine ***P = 0.0001, 5 μM Azaserine vs. 7 μM Azaserine ***P = 0.0001, 5 μM Azaserine vs. 10 μM Azaserine ***P = 0.0009. Please note that the “pre-culture” condition refers to the embryos at the beginning of the experiment, when they already exhibit some level of primitive streak formation. Drug concentrations that overall compromised embryo viability are considered toxic. Concentrations used in this study (boxed in red) altered primitive streak elongation without compromising embryo viability. (e) Representative images show mouse gastrula developmental outcomes following 12 hr metabolic inhibitor treatment. Also see Fig. 2b. A, Anterior; P, Posterior. Scale bars represent 40 µm. (f) Mouse gastrulas cultured in nutrition-sparse media (free of glucose, pyruvate, and glutamine), and rescue conditions (selective reintroduction of the excluded nutrients) to functionally validate the specific effects of chemical inhibitors. A, Anterior; P, Posterior. Scale bars represent 40 µm. Graph on the left shows mean ± SEM of SOX2 expression intensity (normalised to DAPI). Dunnett’s multiple comparison tests following ordinary one-way ANOVAs. Control (n = 4 embryos) compared to no nutrients (n = 3 embryos), glucose (n = 5 embryos), pyruvate (n = 5 embryos), glutamine (n = 4 embryos), and glucose + glutamine (n = 5 embryos for anterior epiblast, n = 3 embryos for PS + mesoderm). Graph on the right shows PS distal elongation % (Control, n = 7 embryos; no nutrient, n = 6 embryos; glucose, n = 8 embryos; pyruvate, n = 9 embryos; glutamine, n = 7 embryos). Plot shows mean + SD. (g) Representative z-sections showing similar PHOSPHO-HISTONE H3 localizations (cyan) in 2-DG-BrPA and Azaserine treated embryos compared to control embryos at the MS stage of gastrulation. Scale bars represent 40 µm. Plots show mean + SD. Ordinary one-way ANOVA. P-values shown.

Source Data

Extended Data Fig. 4 Stem cell models shows the direct impact of glucose metabolism on mesoderm fate acquisition.

(a) Representative images of mouse gastruloids treated with 2-DG and YZ9. 2-DG treated gastruloids fail to elongate and do not express T. YZ9 treated gastruloids elongate and have T expression comparable to control. (b) Quantitative RT-PCR demonstrates significantly reduced T and Etv4 in 2-DG treated gastruloids. Plots show mean + SD. Dunnett’s multiple comparisons test following one-way ANOVA. P-values shown. N = 3 replicates. (c) Representative images from mXEN cells treated with 2-DG+BrPA, Azaserine, YZ9, Galloflavin, and 6AN for 24 h and treated with Oligomycin for 6 h, all shown to maintain expression of endoderm marker GATA6 (red). N = 3 independent experimental replicates examined for each condition. (d) Representative images from live imaging of early-streak stage gastrulas incubated with dyes for active mitochondria (MitoTracker, top) and mitochondrial membrane potential (TMRM, bottom). Zoomed-in panels visualizing the epiblast-VE boundary. Heaptmap intensity colours via Fiji’s LUT. N ≥ 3 embryos examined for each dye.

Source Data

Extended Data Fig. 5 Mesoderm directed-differentiation reveals the direct impact of glucose metabolism on mesoderm fate acquisition and maintenance.

(a) Top Left: Schematic of mesoderm directed-differentiation steps from mESCs. Top Right: Brightfield image of starting mESC population and control cells fixed on Day (D)4 stained for T (red). Bottom: Representative images from D1-D4 2-DG+BrPA, Azaserine, PD, and SU-treated groups show an absence of T/BRA (red). Scale bars represent 200 µm. Boxes in bottom-left corner are 2X magnified from each image. Graphs show the number of T/BRA-expressing cells in (b), and number of pyknotic cells, indicating cell death, in (c), both relative to total cell number for control treatment conditions: Control (n = 12 from 6 independent experiments), 2-DG+BrPA (n = 3 per treatment length from 2 independent experiments), Azaserine (n = 5 per treatment length from 2 independent experiments), YZ9 (n = 3 per treatment length from 2 independent experiments), PD (n = 3 per treatment length from 2 independent experiments), and SU (n = 4 for D1-D4 treatment, n = 3 for D3-D4 treatment from 2 independent experiments). Graphs show mean ± SEM. Dunnett’s multiple comparison test following ordinary one-way ANOVA. Figure shows P-values. (d) qPCR analyses of directed-differentiation experiments, querying transcript changes in mesodermal pluripotency genes (treatment from D1-4 or D3-4) across 3 independent experimental replicates (4 replicates for all pluripotency genes in D1-4 Control and Azaserine, 4 replicates for Sox2 and Klf2 in D1-4 2-DG+BrPA and D1-4 PD, 2 replicates for Pdgfra in D3-4 Azaserine). Plots show mean ± SEM. Dunnett’s multiple comparisons tests following one-way ANOVAs. ****P < 0.0001. Day 1-4 compared to control: Mesp1 2-DG *P = 0.0309, Mesp1 Azaserine *P = 0.0103, Rex1 PD *P = 0.0326, Klf2 PD *P = 0.0395. Day 3-4 inhibition compared to control: Pdgfra1 PD **P = 0.0052, Sox2 PD *P = 0.0354, Klf2 PD ***P = 0.0003.

Source Data

Extended Data Fig. 6 Epiblast glucose metabolism is associated with the epithelial-to-mesenchymal transition (EMT) program.

(a) Representative montage of sagittal z-sections through the MS stage embryo shows epiblast GLUT1 (heatmap intensity colours via Fiji’s LUT) expression co-localizing to regions of intact basement membrane, as identified via LAMININ (magenta) immunostainings (n = 28 embryos). A, Anterior; P, Posterior. Scale bar represents 100 µm. (b) Anterior and posterior epiblast (Epi) insets from the same embryo stained for GLUT1 and MMP14 (heatmap intensity colours via Fiji’s LUT for both protein expressions). In the posterior region, GLUT1 expression co-localises to regions of MMP14 high activity (n = 3 embryos), particularly in Epi cells bordering the primitive streak (PS) (white asterisks). Scale bars represent 40 µm. (c) Z-section of a posterior MS stage embryo showing GLUT1 and GLUT3 co-localizations to CADHERINs, especially within epiblast and PS cell neighbours (ECAD, white asterisk). Heatmap intensity colours via Fiji’s LUT. Scale bars represent 40 µm. P-values shown. (d) Principal curves of EMT, glucose metabolism, and Krebs Cycle genes over pseudo-time (Epiblast to Primitive Streak to Nascent Mesoderm states) in gastrulating in vivo mouse embryos.

Extended Data Fig. 7 Epiblast glucose metabolism is associated with the epithelial-to-mesenchymal transition (EMT) program.

(a) Representative outcome of a PD-treated in vitro EMT assay with DQ Gelatin (magenta). Quantification shows the number of DQ+ clusters identified in each imaging field (n = 25 fields quantified over 2 independent experimental replicates). Two-tailed parametric t-test. **P = 0.0011. (b) qPCR results of directed-differentiation experiments, show a downregulation of key EMT transcripts upon 2-DG+BrPA treatment. Plots show mean + SD. Two-tailed parametric t-tests. N = 3 experimental replicates for all groups. (c) Left: Single z-sections of sagittal, frontal, and transverse views of control mouse embryo’s epiblast (green-dotted line) show that GLUT1 expression (heatmap intensity colours via Fiji’s LUT) is anterior and distal to dpERK expression (green). Plot profile shows expression intensities in the epiblast across the anterior-posterior axis of the orthogonal image above. Right: 2-DG+BrPA treatment abrogates dpERK activity in cells within the epiblast (green-dotted line): Control (n = 6 embryos), 2-DG+BrPA (n = 5 embryos). Graph shows mean expression intensities in the epiblasts of each embryo. Scale bars represent 40 µm. Plots show mean + SD. Two-tailed parametric t-test. P-value shown. (d) Illustration shows experimental design. Embryo phenotypes after ERK (PD) and HBP inhibition (2DG+BrPA or Azaserine) for 12 h in culture. T/BRA marks the primitive streak. (e) Mouse gastrulas cultured with 2-DG+BrPA or PD for 18 h ex vivo show equivalent developmental delay: Control (n = 21), 2-DG+BrPA (n = 24), PD (n = 16). Plots show mean ± SEM.

Source Data

Extended Data Fig. 8 HBP mediates ERK activity in the epiblast during mesodermal transition.

(a) Left: Tetraploid complementation assay generates embryos where cells of the embryo proper are only derived from ERK-KTR^mClover 2 N mESCs. Scale bar represents 40 µm. Right: Nuclear and cytoplasmic manual segmentations of an ES stage embryo (generated by tetraploid complementation) were used to quantify nuclear-to-cytoplasmic ratios (N:C) of ERK-activity, quantified in order of anterior (A) to posterior (P) location in the epiblast. Scale bar represents 40 µm. (b) Quantifications of ERK activity by calculating N:C ratio in the epiblast tissue. As development proceeds, ERK activity becomes graded across the anterior-posterior axis. Plots show epiblast N:C ratio in representative ES (R² = 0.24) and MS (R² = 0.40). (c) Multi-photon live imaging of NAD(P)H confirms that glucose metabolism activity occurs anterior to ERK activation in the epiblast (nuclear-excluded regions). n = 3 embryos. Heatmap intensity colours via Fiji’s LUT. (d) Representative images of ERK-KTR embryos collected at E6.5 and cultured for 7hrs with SU or PD show nearly uniform cytoplasmic and nuclear reporter levels in ERK-inhibited conditions. (e) 2-DG+BrPA, NaClO3, Azaserine, Azaserine+GlcNAc rescue treatments of representative MS gastrula stage embryos decrease the strength of the nuclear-excluded phenotype of ERK activity in posterior epiblast regions. Heatmap intensity colours used for better visualization of ERK-KTR nuclear exclusion. (f) SYNDECAN1 (SDC1) (upper panel) and 10E4 (lower panel) expressions within the embryonic region of mouse gastrulas after 12 h culture with indicated conditions. N = minimum of 6 embryos per condition. (g) Confirmation of EpiLSC state with upregulated OTX2 protein expression in culture. Naïve mESC pluripotency condition is indicated with 2iLif, which lacks OTX2 expression (n = 2 independent experiments). (h) Mid-embryo sagittal z-sections show PS elongation (dotted line, T/BRA expression) under indicated conditions for 12 h culture. Graph shows comparison of PS distal elongation lengths in embryos grown under indicated conditions displaying mean ± SEM. Dunnett’s multiple comparison test following ordinary one-way ANOVA. Figure shows P-values. Azaserine (n = 13), NaClO₃ (n = 9), Azaserine+FGFs (n = 7) each compared to Control (n = 9) across 3 independent experiments.

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Extended Data Fig. 9 Metabolic response within the committed mesoderm.

(a) Transverse section of E6.75 mouse embryo labelled for GLUT3 and NCAD+ lateral mesoderm (n = 10 embryos) across 3 independent experiments. (b) Representative sagittal sections of mouse gastrulas treated with inhibitors (independent experiments: Azaserine (n = 11), YZ9 (n = 10), SU (n = 4) or PD (n = 13)) for 12 h ex vivo. A, Anterior; P, Posterior. Scale bar represents 40 µm. (c) Embryo illustration depicts spatiotemporal mesodermal migration upon exiting the primitive streak (PS) (d) Quantification shows NAD(P)H signal intensity for control (n = 12), YZ9 (n = 18) and 2DG+BrPA (n = 12) treated embryos. Graph shows mean as black bar. Dunnett’s multiple comparison test following ordinary one-way ANOVA. Figure shows P-values. Data points indicate different regions measured within epiblast, with each embryo colour coded. (e) PS surface areas calculated from mid-embryo sagittal z-sections show a significant size increase in PD (n = 18 embryos) and YZ9 (n = 8 embryos) treated groups compared to control (n = 35 embryos). Plots show mean + SD. Dunnett’s multiple comparison test following ordinary one-way ANOVA. Figure shows P-values. (f) Representative single z-sections show that PS surface areas (marked by T/BRA, in magenta) are expanded in PD & YZ9 treated embryos (magenta-dotted regions), with high PHOSPHO-HISTONE H3 (cyan) expression in those regions: Control (n = 7), PD (n = 6), YZ9 (n = 7). Scale bars represent 40 µm. (g) Transverse sections confirm expanded PS phenotype and PHOSPHO-HISTONE H3 localizations in the PS (red arrows). Scale bars represent 40 µm. Quantification shows Phospho-Histone H3 counts in the PS (indicated via morphology or T expression) are significantly increased in PD (n = 7) and YZ9 (n = 7) treated embryos compared to control (n = 8) and Azaserine (n = 3) treated. No significant differences are seen in SU (n = 7) and PD + YZ9 (n = 7) treated embryos. Plots show mean ± SEM. Tukey’s multiple comparison test following ordinary one-way ANOVA. Figure shows P-values. (h) Representative image from PD + YZ9 dual-treated embryo. (i) Late streak stage (E7.5) embryos with 2NBDG uptake after 4 h control and PD treatment.

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Extended Data Fig. 10 Glycolytic pathway is necessary for proper mesoderm migration.

(a) Representative surface view images from control, PD and YZ9 treated embryos stained for lateral mesoderm marker SNAI1 to visualise migration distance. (b) Surface area changes of mesoderm explants following 17 hr ex situ culture show that only 2-DG-BrPA treatment (n = 3 explants) results in a significant size decrease compared to other groups (Control, n = 4; PD, n = 3; YZ9, n = 3; Azaserine, n = 3 explants). Plots show mean ± SEM. Two-tailed parametric t-test. Dunnett’s multiple comparison test following ordinary one-way ANOVA. Figure shows P-values. (c) AIVIA “cell tracking” software allows for automated segmentations of live-imaging videos to track the migration rate (graph on the left) and velocity (graph on the right) of TCF/LEF-GFP mesodermal explants across different treatment groups. Individual data points represent unique cell tracks (Control, n = 104; 2-DG+BrPA, n = 41; PD, n = 85; YZ9, n = 60; Azaserine, n = 39 cells). 3 independent experiments. Plots show mean ± SEM. Dunnett’s multiple comparison test following ordinary one-way ANOVA. Figure shows P-values. (d) NCAD expression (heatmap intensity colours via Fiji’s LUT) in mesoderm explants is retained across different treatment groups. 3 independent experiments for all groups. Scale bar represents 40 µm. (e) LEF1 (magenta) and EOMES (cyan) expression in mesoderm explants are consistently expressed across treatment groups (Azaserine, 2-DG-BrPA, YZ9, PD) after 16 h. 3 independent experiments for all groups. Scale bar represents 40 µm. (f) GO biological process enrichment of downregulated genes in mesoderm explants after PD or YZ9 treatments. P-values from Fisher’s exact tests on the X-axis.

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This file contains Supplementary Fig. 1 and Supplementary Tables 1 and 2. Supplementary Fig. 1: Uncropped images of western blots shown in this study. Raw data used in Fig. 3d. Graph show mean ± SEM. n = 2 independent experiments. Supplementary Table 1: List of antibodies used in this study. Table show antibodies and dyes used. Supplementary Table 2: List of qPCR primers used in this study. Table show qPCR primers used.

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Cao, D., Bergmann, J., Zhong, L. et al. Selective utilization of glucose metabolism guides mammalian gastrulation. Nature 634, 919–928 (2024). https://doi.org/10.1038/s41586-024-08044-1

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Received: 19 July 2023
Accepted: 12 September 2024
Published: 16 October 2024
Version of record: 16 October 2024
Issue date: 24 October 2024
DOI: https://doi.org/10.1038/s41586-024-08044-1

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Abstract

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Main

Regional glucose waves in embryos

HBP guides fate transition in epiblast

Embryo models link HBP to fate change

HBP links to EMT in posterior epiblast

HBP mediates ERK in mesoderm transition

Committed mesoderm does not rely on HBP

Late glycolysis in lateral mesoderm

Glycolysis supports mesoderm migration

Discussion

Methods

Data reporting

Mouse embryo recovery

Embryo culture at gastrulation

Tetraploid complementation

Metabolism and signal modulation experiments

Nutrient-sparse and rescue experiments

Cell culture, gastruloids and in vitro mesoderm-directed-differentiation assay

In situ mesoderm explant assay

Glucose-uptake assay

DQ gelatin assay

Invadopodia assay

Labelling active mitochondria

Immunofluorescence staining

Image data acquisition and processing

Time-lapse live imaging

Multiphoton live imaging for NAD(P)H autofluorescence

Image analysis

Angle of GLUT expression in the epiblast

Primitive streak distal elongation percentage

Basement membrane breakdown

FITC–fibronectin invadopodia assay

ERK-KTR nuclear:cytoplasmic ratio quantification

Proliferation quantification

AIVIA-based image analysis

Directed-differentiated mesoderm cell counting

Western blot

qPCR with reverse transcription

RNA-sequencing sample collection and data analysis

Reanalysis of single-cell sequencing data

Statistics and reproducibility

Reporting summary

Data availability

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Extended data figures and tables

Supplementary information

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