Non-canonical nuclear function of glutaminase cooperates with Wnt signaling to drive EMT during neural crest development

Nekooie Marnany, Nioosha; Dady, Alwyn; Duband-Goulet, Isabelle; Relaix, Frédéric; Motterlini, Roberto; Foresti, Roberta; Dufour, Sylvie; Duband, Jean-Loup

doi:10.1038/s41467-025-58573-0

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Published: 01 July 2025

Non-canonical nuclear function of glutaminase cooperates with Wnt signaling to drive EMT during neural crest development

Nature Communications volume 16, Article number: 5554 (2025) Cite this article

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Abstract

Metabolic reprograming has been linked to epithelial-to-mesenchymal transition (EMT) in cancer cells, but how it influences EMT in normal cells remains largely unknown. Here we explored how metabolism impacts delamination and migration of avian trunk neural crest cells, an important progenitor cell population of the vertebrate embryo. We report that delamination exhibits a quiescent metabolic phenotype whereas migration is characterized by OXPHOS-driven metabolism coupled to distinct expression of metabolic, EMT and developmental genes. While glucose and glutamine are required for delamination and migration, we uncover a specific role for glutamine and its catabolizing enzyme glutaminase in the unfolding of NCC delamination. Namely, glutamine is required for nuclear accumulation of glutaminase, which interacts and cooperates with Wnt signaling to regulate EMT gene expression and cell cycle during delamination. Our data indicate that similarly to cancer cells, embryonic cells engage metabolic enzymes for non-canonical signaling functions to connect metabolism with EMT.

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Introduction

Epithelium-to-mesenchyme transition (EMT) refers to the cellular program by which, through diverse scenarios, epithelial cells lose their compact, ordered organization to become individual, migrating cells. This process plays crucial roles during embryogenesis, morphogenesis, and tissue repair, but it can also adversely cause organ fibrosis and promote cancer progression and metastasis¹. EMT is context-dependent and triggered by numerous inducers, whose signaling cascades all converge to the Snail, Zeb, and Twist families of transcription factors (EMT-TFs), which ultimately control the expression of genes for adhesion, polarity, and cytoskeleton organization².

Because it establishes a direct link between the environment and gene networks, cellular metabolism plays a key role in the unfolding of cell fate and function during EMT. Numerous studies uncovered the mutual relationships between EMT and metabolism, and the molecular mechanisms implicated are being unraveled in cancer cells^3,4,5,6. Metabolic reprogramming occurs often as a consequence of EMT during tumorigenesis, enabling cells to adapt to environmental changes. Thus, EMT inducers can alter the cellular metabolic activity⁷ and EMT-TFs can modify the expression of metabolic genes, profoundly affecting the glycolytic flux, oxygen consumption, or rewiring glycolysis to the pentose phosphate (PP) pathway^8,9. Conversely, changes in the metabolic activity of cancer cells favor EMT. A well-described example of this phenomenon is the hypoxia observed in carcinoma that promotes anaerobic glycolysis and activates the hypoxic-inducible factor 1, a key regulator of EMT-TFs^10,11. Another well-documented metabolic process causing EMT in tumor cells is the Warburg effect, in which energy derives mainly from aerobic glycolysis and less from mitochondrial oxidative phosphorylation (OXPHOS)^12,13.

The mechanisms by which metabolic activity induces EMT in cancer cells are extremely diverse. They may rely on the accumulation of metabolites generated in mitochondria through the tricarboxylic acids (TCA) cycle, which serve as cofactors or substrates of histone modifiers involved in epigenetic regulations¹⁴. Alternatively, EMT can result from non-canonical functions of glycolytic enzymes, either through their release into the microenvironment where they act as autocrine factors inducing EMT-TFs or via their translocation into the nucleus where they exert a transcriptional regulatory activity^15,16,17. However, the diverse molecular processes capable of inducing EMT, along with the intrinsic heterogeneity of tumor cells, make it difficult to decipher which metabolic events are decisive in EMT⁶. Moreover, most metabolic alterations found in cancer cells result from extensive genomic alterations and mitochondrial dysfunction. Thus, it is essential to investigate the mechanisms in normal cells that undergo EMT in a predictable stereotyped fashion.

Neural crest cells (NCCs) of the vertebrate embryo represent a powerful model system to tackle this question^18,19. Delamination, an EMT-related process, constitutes the founding event of this progenitor cell population originating from the neural tube (NT), allowing the release of nascent NCCs from their niche into the surroundings where they undergo migration and disperse away until differentiation^20,21,22. Owing to this process, NCCs give rise to a wide array of cell types throughout the body, from cephalic skeletal tissues to melanocytes and peripheral neurons and glia. Recent studies provided evidence that glucose metabolism plays an important role in NCC delamination and migration. In a similar fashion to cancer cells, NCCs at cranial levels rely primarily on aerobic glycolysis to undergo EMT, with Yap/Tead signaling to serve as the intermediate between glycolysis and activation of EMT-TFs²³. Unlike their cranial counterparts, trunk NCCs display mitochondrial-dependent glucose oxidation and mobilize several metabolic pathways downstream glucose uptake to execute their developmental program²⁴. Interestingly, trunk and cranial NCC delamination events are intrinsically different in their kinetics, cellular processes, and in the molecular players recruited^20,25,26, raising the intriguing question that metabolic activity might determine the scenario by which cells undergo EMT.

Here, using in vivo and in vitro strategies, we explored how metabolism drives the transition from delamination to migration in trunk NCCs. We report that delamination and migration differ in their metabolic requirements in relation to the developmental gene networks involved. We show that besides glucose, glutamine is required for NCC EMT and that glutaminase (GLS), the enzyme that catalyzes glutamine transformation into glutamate, plays a major role in this process. Strikingly, GLS function in delamination is independent of its enzymatic activity and is instead related to its nuclear accumulation, suggesting a non-canonical gene regulatory function. In fact, we found that GLS cooperates with β-catenin, a major player in Wnt signaling, to control the expression of EMT-TFs and cell cycle during NCC delamination. Thus, our data indicates that embryonic cells share similar molecular processes with cancer cells, connecting metabolic events with gene regulation during EMT.

Results

To examine the metabolic processes involved in trunk NCC delamination at the transition between their premigratory and migratory states, we used the avian embryo, a model in which the spatiotemporal features of delamination have been precisely mapped and characterized^27,28. Delaminating and migrating NCCs can be discriminated using specific markers. Expressions of the EMT-TFs Snail-2 and Foxd-3 peak at the onset of EMT in synchrony with downregulation of the cell adhesion molecule Cadherin-6B; these markers gradually decline at the initiation of migration, while expression of Sox-10, a marker of migration, increases (Fig. 1a and Supplementary Fig. 1a)^21,29,30,31. In the thoracic region, delamination of the trunk NCC population occurs over a 15-h period from stage 12 of Hamburger and Hamilton (HH12³²;) up to stage 15. At HH12, premigratory NCCs express EMT markers and are about to undergo delamination. At HH13, delamination culminates and, at stages 14 and 15 (HH14-15), while delamination is still underway, the already-delaminated NCCs start migrating (Fig. 1a and Supplementary Fig. 1a). Additionally, avian NCC delamination can be investigated experimentally in vivo³³ and can be mimicked in vitro under defined cell culture conditions using isolated explants of NT fragments³⁴ (see Supplementary Fig. 1b–d). This system can, therefore, be used to investigate the delamination process and its transition to migration, and to decipher the individual and collective behavior of NCCs, thus providing a comprehensive spatiotemporal view of the underlying metabolic events.

NCC transition from delamination to migration is coupled to a metabolic switch

To study the bioenergetics profile of NCCs at the time of delamination, we used a Seahorse XF analyzer to measure on explants of NT the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) as readouts of mitochondrial respiration and glycolysis, respectively. It should be emphasized that delaminating NCCs cannot be collected individually without affecting the delamination process, because they are intimately associated with the premigratory NCCs and the other neuroepithelial cells, forming an integrated developmental unit. As a consequence, metabolic profiling could not be performed exclusively on delaminating NCCs but only on intact NT explants. Therefore, to minimize the influence of this cell population heterogeneity, we performed each measurement on single NT explants from embryos at defined stages encompassing the delamination process from HH12 to HH15 (Supplementary Fig. 1b; see also ref. ²⁴) and, for each of them, we assessed the cellular and molecular features of NCCs in the explants after metabolic profiling using specific markers and appropriate assays. In addition, we analyzed a large number of samples for greater accuracy and to circumvent the intrinsic variability in development progression among embryos.

Intriguingly, we consistently observed two subsets of explants with distinct metabolic states at the onset of culture, a major one (70%) with an “OXPHOS-driven profile” characterized by high OCR, low ECAR, and a high OCR/ECAR ratio, and a minor one (30%) with a “metabolically-quiescent profile” characterized by low OCR, low ECAR, and a low OCR/ECAR ratio (Fig. 1b, c). By combining mitochondrial stress assays with measurements of intracellular ATP, we found that mitochondrial respiration was higher in explants with OXPHOS profile than in quiescent ones (Fig. 1d, e) and largely contributed to energy production (Fig. 1f, g). Interestingly, explants with an OXPHOS profile had a significantly greater capacity to produce large NCC outgrowths after 3 h of culture, exhibiting a higher number of migrating cells and stronger expression of the migration marker Sox-10 compared to the quiescent group (Fig. 1h–j). This suggests that explants with an OXPHOS profile may be in a more advanced development state compared to quiescent cells. We, therefore, performed a bioenergetics survey of individual explants over time in culture during the passage from delamination to migration, which occurs during the first day of culture. We found that explants exhibiting a quiescent profile at 3 h shifted to an OXPHOS profile after 20 h (Fig. 1k), while those with an OXPHOS profile at the beginning maintained the same metabolic activity later on, as reported previously²⁴. This explains the metabolic heterogeneity observed in explants at 3 h of culture. To further establish a correspondence between the stage of NCC development and their metabolic activity, we focused on embryos at stages covering either initiation (HH12-13) or termination (HH14-15) of delamination. We found that a majority of explants at HH12-13 exhibited a quiescent profile, while at HH14-15, most of them displayed an OXPHOS profile (Fig. 1l). In addition, NTs from HH12-13 embryos cultured in vitro for 3 h produced fewer migrating NCCs than those from HH14-15 embryos (Fig. 1m).

Collectively, these data establish that the metabolic activity of NCCs is rewired from quiescence to active mitochondrial respiration at the transition phase between delamination and migration (Fig. 1n). Thus, metabolic profiling is instrumental to discriminate reliably NT explants at delamination and migration and investigate the molecular processes underlying the switch between the two stages.

Glucose utilization changes from delamination to migration

Metabolic switch is often related to changes in metabolite demand and utilization. To explore which metabolic programs are rewired during the transition from delamination to migration, we focused first on glucose metabolism, as it is a main energy supplier. We previously demonstrated that glucose is required for trunk NCC development and that genes of several glycolytic enzymes are expressed in the embryo at the time of migration²⁴. Here, after metabolic profiling of NT explants in vitro, we compared the mRNA levels at delamination and migration of the glucose transporter type-1 (SLC2A1/GLUT-1) and the enzymes phosphofructokinase-P (PFK), lactate dehydrogenase (LDH), and pyruvate dehydrogenase complex (PDH) (Fig. 2a). To demonstrate the relevance of our approach to the in vivo situation, data were compared with those obtained from extracts of the brachial region of embryos at a stage between HH12 and HH13 (HH12/13) and at HH14 (see procedure Supplementary Fig. 1c). Consistent with greater energy production, we found that with the exception of LDH, glycolytic enzymes were up-regulated at migration compared with delamination (Fig. 2b).

Next, we analyzed the effect of pharmacological inhibitors of glycolysis and OXPHOS, namely 2-deoxyglucose (2-DG) and oligomycin (Fig. 2a), on the metabolic activity of NT explants, by recording OCR and ECAR in individual explants alongside the measurement of ATP levels. In explants at delamination, we observed that oligomycin decreased OCR and reduced ATP levels, while 2-DG did not affect OCR but decreased ATP (Fig. 2c–e). In contrast and consistent with our previous data²⁴, explants at migration exhibited a drastic decrease in OCR and ATP with 2-DG and oligomycin (Fig. 2c–e). This indicates that during delamination, rather than fueling glycolysis and OXPHOS, glucose might be utilized by alternative routes, such as the PP pathway, while during migration, NCCs use glucose oxidation and mitochondrial respiration as the main route for energy production. Consistent with this assumption, expression of the gene encoding phosphoribosyl-pyrophosphate synthase (PRPS), a key enzyme of purines and pyrimidines synthesis in the PP pathway, was higher during the delamination phase compared to the migration phase (Fig. 2b). We therefore evaluated the effect of the PP pathway inhibitor 6-amminonicotinamide (6-AN) (Fig. 2a) on NCC metabolic activity. We previously showed that 6-AN exerted a minimal effect on trunk NCC delamination but strongly affected their migration²⁴. Here, we found that, in explants at delamination, 6-AN had no impact on OCR but increased ECAR and ATP (Fig. 2c–f), suggesting that blocking the PP pathway re-diverts glucose towards glycolysis and lactate production. In contrast, in explants at the migration stage, 6-AN treatment changed neither the metabolic profile nor ATP levels (Fig. 2c–f). We next quantified the mRNA expression of enzymes of glucose metabolism in embryos at the time of NCC delamination and migration after inhibition of the PP pathway. We found that in ovo injection of 6-AN in the caudal region of embryos at delamination at HH12/13 caused a significant reduction in PRPS expression and an increase in LDH with no effect on PFK, while no significant changes in these genes were detected when 6-AN was injected at migration at HH14 (Fig. 2g). Altogether, these findings support a change in glucose utilization during NCC transition from delamination to migration.

Glutamine is required for delamination and migration

Because a metabolic switch may also depend on the type of metabolite used, we considered other metabolic pathways that may accompany the NCC transition from delamination to migration. We focused on glutamine, a major contributor to macromolecules and ATP production known to cooperate with glucose in cancer cells³⁵. Using in situ hybridization on whole-mount embryos and on sections, we first explored the expression patterns of key enzymes involved in glutamine metabolism (Fig. 2a) in the trunk of avian embryos. mRNAs for SNAT-2, encoding one of the glutamine transporters in neural cells, glutaminase (GLS) and glutamate dehydrogenase (GDH) were all widely expressed at delamination and migration (Fig. 3a). At the tissue level, mRNAs were distributed throughout the NT and were also detectable in premigratory, delaminating, and early-migrating NCCs (Fig. 3b). We also compared the expression levels of these enzymes in metabolically profiled explants cultured in vitro as well as in embryo extracts at HH12/13 and HH14. We found that SNAT-2, GLS, and glutamine synthase (GLUL) expressions remained constant throughout NCC progression from delamination to migration, while GDH, encoding the enzyme involved in glutamate conversion into α-ketoglutarate, was increased at migration (Fig. 3c). These data are in favor of an active glutamine metabolism during delamination and migration and suggest that, like for glucose, a change in glutamine utilization might occur during transition between the two stages.

To assess the implication of glutamine metabolism in delamination and migration, we used in vivo loss-of-function and in vitro culture approaches (see supplementary Fig. 1b, d). For this purpose, 6-diazo-5-oxo-l-norleucine (DON), a glutamine analog that blocks all metabolic routes downstream of glutamine uptake (Fig. 2a), was injected in the caudal region of HH12-embryos prior to delamination. Analyses by qRT-PCR of the embryos 5 h following injection revealed a robust repression of Foxd-3 and Sox-10 and also a decrease of Snail-2 (Fig. 3d). Accordingly, DON strongly decreased NCC delamination and caused migration defects after 24 h, as assessed by the alteration of Sox-10 pattern in the lower trunk (Fig. 3e). Spatiotemporal analyses of NCC distribution showed that cells were missing at the top of the NT in the caudal trunk and were less numerous and trapped dorsally instead of migrating ventrally in the mid-trunk (Fig. 3f). When applied on NT explants at the onset of in vitro culture, DON provoked a strong decrease in the number of NCCs produced by the NT after 5 h, leading to a reduced population of sparse and less spread NCCs at the NT periphery after 24 h (Fig. 3g, h). Analyses of the kinetics of NCC delamination and migration over time in culture using time-lapse video microscopy revealed that application of DON resulted in a twofold shorter duration of NCC delamination from the NT and an almost entire inhibition of their outward migration (Fig. 3i, j). In agreement with our in vivo observations after injection of DON, these effects were accompanied by a severe diminution of the expression of the delamination and migration markers Snail-2 and Sox-10 in NCCs (Fig. 3k).

Because the DON effect could result merely from alterations in NCC identity or fate, ultimately affecting their delamination and migration capacities, we analyzed the expression of specific markers for the neural epithelium cell populations at the time of NCC emigration as well as for early NCC-derived lineages. We found that in the presence of DON, Sox-2, an early pan NT cells marker excluded from NCCs³⁶, was not detected in cells situated in the NT vicinity, whereas Pax7, an early NCC marker³⁷, was maintained in all of them (Supplementary Fig. 2a), indicating that NCC identity was not modified upon inhibition of glutamine metabolism. Likewise, we found no significant expression of α-smooth muscle actin and βIII-tubulin, two markers of NCC-derived mesenchymal and neuronal cells, respectively (Supplementary Fig. 2b; see also ref. ²⁴), thereby excluding any abnormal premature differentiation of NCCs induced by DON. These results establish that glutamine is necessary for both NCC delamination and migration and for the expression of the associated regulatory genes.

Glutamine and glucose cooperate for delamination and migration

We then investigated whether glutamine and glucose cooperate or function independently during NCC delamination and migration. To this aim, we analyzed the cellular and molecular responses of NT explants confronted in vitro with a medium deprived of glucose, glutamine, or both. In complete absence of both glucose and glutamine at the onset of the culture, NCC delamination was almost abrogated and very few cells were able to migrate away from the NT (Fig. 4a–c). When NT explants were cultured in a medium containing either glutamine or glucose, we observed an initial delamination response of NCCs after 5 h in culture (Fig. 4a), suggesting that each of them can individually support delamination. However, we noted that the number of delaminating cells was much reduced and that the delamination period was shorter than when both glutamine and glucose were added together (Fig. 4b, c). Moreover, NCCs gradually rounded up, and the outward progression of the population became severely compromised (Fig. 4a, d). Glucose supported the progression of the NCC population and the velocity of individual cells with greater efficiency than glutamine (Fig. 4d, e). In agreement with the reduction of the delamination and migration capacities of cells, glucose or glutamine deprivation caused a severe reduction of the number of Snail-2-positive cells (Fig. 4f) and of the expression of Sox-10 (Fig. 4g) and several EMT-TFs (Fig. 4h).

To corroborate the requirement for glutamine and glucose, we evaluated their ability to rescue NCC delamination and migration after a brief period of deprivation of both nutrients at the onset of culture. Using video microscopy, we observed that NCCs readily resumed their delamination process within less than 2 h following the re-introduction of either glutamine or glucose to the culture medium (Supplementary Fig. 3a, b). The morphological aspect of delaminating NCC differed markedly depending on the nutrient provided: with glutamine, cells were packed but poorly spread; with glucose, they appeared more spread but dispersed; and in the presence of both nutrient,s cells were well-spread and dense (Supplementary Fig. 3a). Likewise, sustained front progression was fully restored only when both glutamine and glucose were provided (Supplementary Fig. 3c, compare with Fig. 4d). These observations therefore establish that glutamine and glucose are essential for delamination and migration and suggest that they may play additive and complementary roles.

Metabolic profiling of NT explants under depletion of one or the other nutrient provided additional information on the roles of glucose and glutamine metabolism in delamination and migration. While the explants in the presence of glutamine alone displayed both quiescent and OXPHOS profiles in similar proportions to those with both glutamine and glucose, the explants confronted with glucose alone possessed an OXPHOS profile characteristic of those observed at migration, and none of them displayed a quiescent profile typical of the delamination stage (Fig. 4i, l). Moreover, in the presence of glutamine alone, NCCs were unable to maintain sustained mitochondrial activity and showed a low spare respiratory capacity (Fig. 4j, k). In contrast, in the presence of glucose alone, we found that despite a reduced capacity to undergo delamination and migration, NT explants displayed mitochondrial activity and energy production comparable to those in the presence of both nutrients (Fig. 4m–p; compare with Fig. 1b, c, e–g).

These results indicate that glutamine and glucose cooperate to support effective NCC delamination and migration, with each nutrient playing a specific role at each step. Notably, glutamine appears to contribute to delamination with low energy requirement, suggesting the intriguing possibility that metabolic quiescence during delamination is mainly driven by glutamine metabolism. On the other hand, although essential, glucose oxidation and mitochondrial respiration cannot compensate for the lack of glutamine and are not sufficient for sustained delamination and migration, despite their robust capacity to provide energy.

Glutaminase exhibits a non-canonical function during delamination

To investigate how glutamine metabolism drives NCC delamination and migration, we targeted GLS, the first enzyme of the glutamine pathway. To this aim, we injected in ovo in the caudal region of HH12-embryos prior to delamination of either the CB-839 compound, to block GLS activity (Fig. 2a)³⁸, or siRNAs to GLS to repress its expression. Both strategies resulted in severe alterations of NCC delamination and migration. Specifically, CB-839 significantly decreased Foxd-3 and Sox-10 mRNA levels (Fig. 5a), and strongly reduced the expression domain of Foxd-3 in the dorsal NT at 5 h during delamination and of Sox-10 at 24 h during migration (Fig. 5b, c). In addition, when applied to NT explants in culture, CB-839 strongly diminished the numbers of NCCs and of Snail-2-expressing cells (Fig. 5d). Silencing GLS by siRNAs caused a dose-dependent reduction of its expression with an almost complete deletion at the highest dose (Fig. 5e). Similarly to CB-839 treatment, GLS knockdown repressed Snail-2, Foxd-3 and Sox-10 expression (Fig. 5e), accompanied by a severe reduction of NCC delamination and migration (Fig. 5f, g).

**Fig. 5: Glutaminase is required for delamination and migration.**

To further understand the role of GLS in these events, we compared the metabolic, cellular, and molecular responses of NT explants treated with CB-839 at the delamination and migration phases. We first characterized the metabolic profiles of individual explants before and after the addition of CB-839, and we quantified the expression of NCC markers and glutamine metabolism enzymes, along with ATP levels. GLS inhibition at delamination caused a significant decrease in Snail-2, Foxd-3, and Sox-10 expressions, but had no impact on OCR, ECAR, and ATP levels (Fig. 6a, b, d). Conversely, during migration, CB-839 failed to alter the expression of NCC markers, but it decreased OCR and ATP production (Fig. 6a, b, d). Moreover, CB-839 differentially affected the expression of glutamine enzymes during delamination and migration (Fig. 6c). Notably, GLUL expression was reduced during delamination but not during migration. These data suggest that GLS may play distinct roles during delamination and migration.

**Fig. 6: Glutaminase exhibits a non-canonical function during delamination.**

Because GLS converts glutamine to glutamate, we measured the intracellular concentration of glutamine and glutamate in NT explants at delamination and migration after metabolic profiling. The concentrations of glutamine and glutamate were lower at delamination than at migration (Fig. 6e, f). Moreover, the concentration of glutamate in NCCs was severely decreased by CB-839 during migration but not during delamination (Fig. 6e, f). These data suggest that GLS enzymatic activity is low during delamination, but is enhanced during migration, matching the high levels of ATP. To address whether glutamate derived from GLS activity could affect the responses of NT explants, we performed experiments adding glutamate to the culture medium in place of glutamine to bypass GLS enzymatic activity. We found that the number of NCCs, the proportion of Snail-2-expressing cells, and the levels of Snail-2, Foxd-3, and Sox-10 mRNAs in the presence of glutamate were lower than those measured in the presence of glutamine, indicating that glutamate cannot compensate for the negative impact of glutamine deprivation (Fig. 6g, h; compare with Fig. 4). Similarly, in ovo injection of glutamate along with siRNAs to GLS in embryos failed to restore the normal NCC delamination and migration patterns (Fig. 6i). Intriguingly, albeit unable to rescue NCC delamination, glutamate restored NCC metabolic properties. Indeed, while only an OXPHOS profile was observed in the absence of glutamine (Fig. 4m–q), in the presence of glutamate, the NT explants exhibited the two classical OXPHOS and quiescent profiles that were observed in the presence of glutamine (Fig. 6j–l; compare with Fig. 1b–f). In addition, in the glutamate-containing medium, the expression of enzymes of glutamine metabolism was altered, with a downregulation of GLS and GLUL, and an increase in GDH, indicating that glutamate could activate downstream events fueling the TCA cycle (Fig. 6m).

Altogether, our findings reveal that GLS contributes to the NCC delamination process independently from its enzymatic activity, thereby raising the intriguing possibility that GLS exhibits a non-canonical function. In contrast, at the migration stage, GLS enzymatic activity appears to be important for maintaining ATP in association with active locomotion.

Glutaminase accumulates in the nucleus in delaminating NCCs

To investigate a potential non-canonical function of GLS during delamination, we compared its cellular localization in delaminating and migrating NCCs. We found that GLS was particularly enriched in the nuclei of most of the delaminating NCCs in addition to its normal cytoplasmic localization. In migrating cells, in contrast, GLS was primarily found in the cytoplasm and was less prominent in the nucleus (Fig. 7a, b). Quantification of fluorescence intensity revealed a twofold reduction of nuclear GLS accumulation in migrating cells compared with delaminating cells (Fig. 7c). Treatment of NT explants with CB-839 significantly decreased the proportion of delaminating NCCs exhibiting nuclear GLS but had a minimal effect on GLS localization in migrating cells (Fig. 7a–c). In addition, a similar outcome was observed under glutamine-free conditions in the presence or absence of glutamate (Fig. 7b), suggesting an essential role for glutamine in mediating the nuclear translocation of GLS in delaminating NCCs.

We also investigated the cellular localization of GLS in trunk NCCs in embryos at the delamination stage. We found GLS staining within the nuclei of cells situated along the NT midline in the region of NCC delamination, while migratory NCCs expressing Sox-10 and situated more laterally displayed low GLS nuclear staining (Fig. 7d). Analyses of immunostained cross-sections confirmed the nuclear accumulation of GLS in NCCs at delamination but not at migration (Fig. 7e). Interestingly, at the NT level where delamination occurs, nuclear localization of GLS was not restricted to NCCs but was also detectable in more ventral NT cells. Finally, we analyzed GLS localization in NCCs of embryos injected with siRNAs to silence GLS before delamination. In addition to repressing NCC delamination and expression of EMT-TFs (Fig. 5e–g), GLS ablation caused the near disappearance of GLS staining in the cytoplasm and nuclei of NCCs, contrary to control siRNAs, which did not affect GLS expression and intracellular localization (Fig. 7f). Together, our data establish a strong spatiotemporal and functional correlation between GLS expression in the nucleus and its role in controlling delamination of NCCs.

β-catenin and glutaminase co-distribute in nuclei of delaminating NCCs

Delamination of NCCs in the trunk region is under the control of canonical Wnt-1 signaling³⁹. The most distinctive step of this pathway is the nuclear translocation of β-catenin, a modular protein either associated with cadherins in intercellular contacts or capable of binding transcription factors in the nucleus to regulate gene expression⁴⁰. Previous investigations in delaminating and migratory NCCs revealed a pattern of nuclear β-catenin localization similar to that observed for GLS in the present study⁴¹, prompting us to explore a possible link between Wnt signaling and a non-canonical function of GLS. We, therefore, investigated whether β-catenin and GLS are co-distributed in NCCs in culture during delamination and migration. We found that, in addition to its location at cell-cell contacts, β-catenin was accumulated in the nuclei with GLS in delaminating NCCs (Fig. 8a). In contrast, β-catenin was rarely present in the nuclei of migratory NCCs, while GLS was essentially cytoplasmic (Fig. 8a). Quantification analyses showed that β-catenin and GLS were co-expressed in the nuclei of a majority of cells (Fig. 8b, c). In addition, similarly to GLS, the amount of β-catenin in the nuclei of delaminating NCCs was about twice that of migrating cells (Fig. 8d). In embryos, we confirmed GLS localization in the nuclei of NCCs at delamination, while β-catenin showed diffuse staining in both the cytoplasm and nucleus of cells and was faint in the areas of cell-cell contacts, indicative of a nuclear-shuttling activity (Fig. 8d). Depletion of nuclear GLS by CB-839 in vitro or by siRNA in vivo caused significant disappearance of β-catenin from the nucleus and cytoplasm of NCCs (Fig. 8a–e), raising the possibility that β-catenin and GLS may coordinately accumulate in the nucleus to function in cooperation.

Glutaminase cooperates with Wnt signaling to control delamination

To explore the potential cooperation between GLS and β-catenin during NCC delamination, we investigated whether these molecules may associate in common molecular complexes using cellular and biochemical approaches. First, we utilized the proximity ligation assay (PLA) to assess the interaction frequency between β-catenin and GLS. We found a significantly greater number of puncta in the nuclei of delaminating NCCs than in migratory ones (Fig. 9a, b), and this number was markedly reduced in the presence of CB-839, strongly suggesting that GLS and β-catenin accumulate in common complexes in the nucleus of delaminating NCCs (Fig. 9b). Then, we performed pull-down assays on NT extracts from trunk portions of embryos at HH12-14 with antibodies against GLS followed by Western blotting with anti-β-catenin antibodies. A band corresponding to β-catenin could be visualized in extracts immunoprecipitated for GLS and not in control, clearly indicating that GLS associates with β-catenin to form molecular complexes (Fig. 9c).

In addition to regulating the expression of EMT-TFs, Wnt signaling in NCCs drives G1/S transition during the cell cycle, at which step they segregate from the NT^33,39. We, therefore, investigated the expression of cyclin-E, a member of the cyclin family recruited at the G1/S transition^42,43, in delaminating and migrating NCCs. We found a higher expression of cyclin-E mRNAs in NCCs at delamination than at migration (Fig. 9d). Interestingly, CB-839 decreased cyclin-E levels at both stages, while glutamate decreased cyclin-E expression only at delamination (Fig. 9e). Moreover, glutamine deprivation also strongly decreased cyclin-E expression (Fig. 9e). In agreement with these findings, EdU short-pulse experiments revealed that both CB-839 and glutamine starvation significantly inhibited G1/S transition in NCCs, while glutamate had no effect (Fig. 9f). Altogether, our data indicate that nuclear GLS associates with β-catenin, thus cooperating with Wnt signaling to regulate expression of EMT-TFs and synchronize NCC delamination with the G1/S transition phase of the cell cycle.

Lastly, we investigated whether Wnt signaling may affect GLS nuclear accumulation in NCCs. We previously showed that LiCl treatment, which enhances β-catenin nuclear accumulation and over-activates Wnt signaling, causes NCCs to stop migration and form cell clusters⁴¹. We, therefore, assessed GLS cellular localization in early-migrating NCCs following LiCl treatment. As shown in Fig. 9g–i, LiCl caused a conspicuous accumulation of both β-catenin and GLS in the nuclei of all cells, thereby revealing that over-activation of the Wnt signaling can induce massive GLS translocation to the nucleus. Altogether, these results highlight the intimate connection between Wnt signals and GLS activity in the nucleus of NCCs at the time of their delamination and migration.

Discussion

Here, we report that NCCs engage different metabolic pathways during delamination and migration in relation to specific gene networks, highlighting that glucose and glutamine are both required for these events, each playing a specific role at each step (Fig. 10). Specifically, we show in vitro and in vivo that during delamination, NCCs exhibit a quiescent metabolic phenotype characterized by a limited energy production due to combined low glycolytic and low mitochondrial respiration activities, yet they are metabolically active. Indeed, delaminating NCCs show a preference for anabolic activity via the PP pathway to support the many molecular changes occurring during EMT. During migration, in contrast, NCCs exploit glucose and glutamine oxidation to fuel mitochondrial respiration for efficient energy production. Thus, NCCs adapt their nutrient preference and use to their bioenergetics and biosynthetic demands to their developmental program. Importantly, this metabolic switch is accompanied by changes in the repertoire of the genes for metabolic enzymes deployed, which is characteristic of metabolic reprogramming. Moreover, we found that metabolic activity not only accommodates to NCC development but also actively influences their developmental program, as revealed by functional experiments in which blocking glutamine and glucose metabolism affected expression of EMT-TFs and migration-related genes, causing severe reduction in EMT and migration. Our data, therefore, uncover a poorly investigated facet of EMT that the acquisition of migratory abilities following EMT requires metabolic rewiring in connection with profound cellular and molecular changes. How metabolic pathways are regulated to enable the transition from EMT to migration remains yet to be investigated.

**Fig. 10: NCC cellular metabolism is coupled to a developmental program.**

An original aspect and great strength of our work is that we initially determined the metabolic profile of NT cultures prior to analyzing the contribution of metabolites and gene expression of delamination and migration events. This approach enabled us to clearly assign a quiescent profile to delamination and an OXPHOS-driven metabolism to migrating cells and confidently manipulate our experimental conditions to investigate the metabolic and molecular mechanisms underlying these two main events in NCC development.

The most important finding emerging from our study is the signaling role of glutamine during delamination. This function is not mediated directly by glutamine itself but is executed by GLS, a key enzyme of the glutamine pathway. There is increasing evidence that GLS is involved in EMT in cancers³⁵. For example, in colorectal cancers, GLS overexpression correlates with increased invasiveness and patient mortality⁴⁴. GLS also has a critical role in the development of prostate cancer, while loss of GLS results in cell cycle arrest and apoptosis⁴⁵. However, the mechanism by which GLS contributes to cell invasion remains elusive and has been mostly attributed to its bioenergetics role. Our data in NCCs advance the understanding of this process, showing that GLS function in EMT is mainly dependent on its nuclear localization and consequent modulation of EMT-TFs expression, while its enzymatic activity leading to glutamate production is of minor importance during this event. This raises the intriguing possibility that GLS exhibits a non-canonical gene regulatory function during NCC EMT. Several examples of nuclear translocation of metabolic enzymes, such as pyruvate kinase M2, an enzyme of the glycolytic pathway¹⁷, have been reported in cancer cells in relation to EMT, but so far this has not been reported for GLS.

A nuclear role for GLS is corroborated by our results demonstrating a direct interaction between GLS and β-catenin and their concomitant presence in the nucleus of delaminating NCCs. Furthermore, we found that blocking GLS with either an inhibitor (CB-839) or by siRNA abolished the nuclear accumulation of both GLS and β-catenin. Conversely, activating the Wnt-β-catenin signaling pathway causes massive nuclear accumulation of GLS, thereby revealing an intimate connection between Wnt signals and GLS activity in the nucleus of NCCs at the time of their delamination and migration. These findings, together with the observation that cyclin-E-dependent G1/S transition is dependent on GLS activity, emphasize a critical role for GLS in NCC development where canonical Wnt signaling controls multiple events, from induction and EMT to lineage differentiation. Interestingly, GLS inhibition in prostate cancer has been found to suppress Wnt/β-catenin signaling and inhibit proliferation via repression of cyclin-D1⁴⁵.

Our data suggest a potential molecular function of GLS in NCCs at the time of delamination. In relation to Wnt signaling, a likely hypothesis is that through its association (either directly or indirectly) with β-catenin, this complex contributes to the regulation of the genes involved in the delamination process. However, it cannot be excluded that GLS may perform additional activities not directly related to β-catenin. Indeed, we observed that in vivo nuclear accumulation of GLS is not restricted to NCCs but expands to most NT cells. Although NCCs exhibit specific molecular and cellular features, they do not evolve independently from the rest of the NT but are part of the general patterning program during neurulation. Notably, the cell-cell and cell-substratum adhesion properties of NT cells are gradually restricted over time so that at the time of NCC delamination, they organize as a compact structure unable to disperse away when explanted in in vitro culture⁴⁶. Interestingly, the timing of GLS nuclear accumulation in NT cells coincides with the period during which they retain their ability to disperse. This suggests that GLS may play a key role in coordinating the regulation of NT cell adhesion properties at the time of NCC development.

The recent demonstration that avian cranial NCCs display a Warburg effect during delamination and that this is coupled with genetic programs controlling cell identity and behavior highlights the parallels between NCCs and metastasis^3,23. However, this view was challenged by the fact that trunk NCCs do not share this feature with cranial NCCs but instead elect a different metabolic process based on glucose oxidation combined with mobilization of multiple metabolic pathways during migration²⁴. Our present observation that trunk NCCs utilize nuclear shuttling of GLS as an important intermediate to regulate the gene regulatory network involved in EMT is reminiscent once more of the situation observed in cancer cells. This suggests that despite presenting distinct metabolic features, trunk NCCs share with cancer cells similar processes to connect metabolic events with gene regulation during EMT, establishing that the parallel made between cranial NCCs and cancer cells also applies to trunk NCCs. Finally, these findings illustrate the great diversity of processes used by cells to accomplish their developmental program and raise the possibility that metabolic activity might determine the scenario by which cells undergo EMT.

Methods

Reagents

Bovine plasma fibronectin, 1 mg/ml stock, was from Sigma (cat. no. F1141). Dispase II at 5 U/ml stock solution in Hanks’ balanced saline was from Stemcell Technologies (cat. no. 07913). The following chemical compounds were prepared and used according to the manufacturers’ guidelines: 2-deoxy-d-glucose (2-DG, Sigma, cat. no. D8375); oligomycin-A (Sigma, cat. no. 75351); rotenone (Sigma, cat. no. R8875); antimycin-A (Sigma, cat. no. A8674); carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP, Sigma, cat. no. C2920); 6-aminonicotinamide (6-AN, Cayman Chemicals, cat. no. 10009315); 6-diazo-5-oxo-l-norleucine (DON, Sigma, cat no. D2141); CB-839 (Sigma, cat. no. 5337170001); LiCl (Sigma, cat no. L9650). All chemicals were used in vivo and in vitro at concentrations below those, provoking long-term cellular toxicity or overt morphological malformations. 2-DG was solubilized as a 1 M stock solution in DMEM medium (without glucose, pyruvate and glutamine, Gibco, cat. no. A14430-01) and used at 10 mM; oligomycin, rotenone, antimycin, and FCCP were prepared in DMSO as 10 mM stock solutions, diluted as stock solution in culture medium at 100 µM, and used at 1 µM or 0.75 µM (for FCCP); 6-AN was prepared as a 100 mM solution in DMSO and used at 250–500 µM; DON was prepared as a 30 mM stock solution in H₂0 and used at 30–60 µM; and CB-839 was prepared as a 34 mM stock solution in DMSO and used at 20–50 µM. LiCl was prepared as a 1 M stock solution and used at 30 mM. As 1/1000 dilution of DMSO had no effect on NCC behavior, in experiments using this dilution, the corresponding controls were performed without the addition of DMSO. When DMSO dilution was below 1/1000, the same dilution of DMSO but without the drug was used for controls. Four siRNAs corresponding to different regions of the Gallus gallus GLS mRNA were designed using the sidirect2.rnai.jp website and purchased from Eurogentec: siRNA.1 GLS: 5′-GAAAGGUUGCUGACUAUAUUC-3′; siRNA.2 GLS: 5′-CCAAAGUUCCUUUUUGUCUUC-3′; siRNA.3 GLS: 5′-GGAUUAAGAUUCAACAAAUUG-3′; siRNA.4 GLS: 5′-AUUUUUCUGCAUUAUUUGCUC-3′. The universal siRNA negative control was purchased from Eurogentec.

Embryos

Quail embryos were used throughout the study. Fertilized eggs purchased from a local farm (La Caille de Chanteloup, Corps Nuds, France) were incubated at 37–38 °C until embryos reached the desired developmental stages. Embryos were staged using the Hamburger and Hamilton (HH) chart³² and based on the number of somite pairs.

In ovo injection of drugs and siRNAs

In vivo loss-of-function experiments were performed on embryos at stage HH12 (15–16 somite pairs at 48 h of incubation), i.e., before the onset of delamination. After removing 1 ml of albumin from the egg and opening the shell with curved dissecting scissors to expose the embryo to the experimenter, a drop of Fast Green solution was added on top of the vitelline membrane for easy visualization of the embryonic tissues. Prior to injection, drugs were diluted in PBS at the desired concentration, and siRNAs (mix of 4 siRNAs) were prepared in a solution containing 0.75 µl Lipofectamine 3000 (Thermo Fisher, cat. no. L3000001) and 25 µl Opti-MEM (Thermo Fisher, cat. no. 31985062) and further diluted in medium to achieve final concentrations of 28 and 10 ng/µl. Drugs and siRNAs were injected using glass pipets at the tail bud end of the embryo into the lumen of the NT and under the vitelline membrane in the vicinity of the NT over the unsegmented region and the last 3–5 somites (10 μl delivered per injection). Controls embryos were injected with the vehicle only. The eggs were sealed with tape and were further incubated at 37–38 °C in a moist atmosphere for 5–24 h. Monitoring of NCC development was performed by following expression of marker genes by qRT-PCR at 5 h and whole mount in situ hybridization at 5 and 24 h followed by sectioning (see below).

Generation of NCC primary cultures

NCC cultures were produced from trunk NT obtained from quail embryos at different stages covering the delamination process: stage HH12-13 (17–18 somite pairs), stage 13 (19–21 somite pairs), and HH14 (23–25-somite stage) as described^24,34. After opening the eggshell, the yolk was transferred into phosphate-buffered saline (PBS). The embryo was cut off from the yolk and transferred into PBS in an elastomer-containing dish. An embryo portion of about 750-µm long was excised at the level of the last five somites with a scalpel under a stereomicroscope and subjected to mild enzymatic digestion by treatment with dispase II at 2.5 U/ml for 5–10 min at room temperature. The NT was dissected out manually using fine dissection pins under a stereomicroscope, freed from the surrounding tissues, and transferred for 30–60 min in DMEM medium without glucose, pyruvate, and glutamine (Gibco, cat. no. A14430-01) supplemented with 0.5% fetal bovine serum, for recovery from enzyme treatment. NTs were explanted onto a variety of substrates depending on the aim of the culture (plastic culture dishes, Seahorse plates, chambered glass coverslips or glass coverslips), all coated with fibronectin at 10 µg/ml in PBS (i.e., about 5 µg/cm²) for a minimum of 1 h at 37 °C. To ensure rapid initiation of NCC migration, NTs were positioned with their dorsal side oriented down toward the substratum. Explants were cultured at 37 °C under normoxic conditions in a 5%-CO₂ incubator in DMEM containing 1% serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and supplemented or not with 5 mM glucose, 2 mM glutamine, or both, depending on the aim of the experiment. In some experiments, glutamine was replaced by glutamate (Sigma, cat. no. 49621) used at 10 µM. The NT explants were followed and analyzed routinely during the first 5 h of culture and up to 24 h in some experiments. Throughout each experiment, the morphology of the NT explant, area and progression of the NCC outgrowth, as well as individual cell shape were evaluated, imaged, and assessed regularly under an inverted phase contrast Nikon microscope equipped with 6.3X, 10X, and 20X objectives.

Quantifications of NCC numbers and delamination

To quantify the number of NCCs in outgrowths, phase contrast images of explants encompassing the whole NCC outgrowth were taken with the 10x objective at defined time points of the culture. Cells were counted in a square of 100-µm-side of the outgrowth using ImageJ, and data were reported to the entire outgrowth area measured using ImageJ. To analyze the delaminating and migrating NCC populations in NT explants in culture at the end of the experiments, the NT was removed manually from the dish by gentle flushing of culture medium with a pipet tip, thereby uncovering delaminated cells situated underneath the NT²⁴. Delaminating cells were discriminated from migratory cells using several criteria: their strong Snail-2 content, their central position in the outgrowth often separated from the migration zone by a cell-free gap, their reduced size, and their compact shape. The numbers of delaminated and migrating cells were counted as above in a square of 100-µm side covering the areas of delaminated cells or of migrating cells, in the mid-part of the explant along its long axis and reported to the entire outgrowth area. In some experiments delaminating cells were visualized directly along the border of the NT explant under a phase contrast microscope and followed using time-lapse video microscopy (see below). The duration of NCC delamination was defined as the period of time during which NCCs were continuously seen separating from the NT and undergoing migration.

Seahorse analyses

Bioenergetics profiles of NCC primary cultures were determined using a Seahorse Bioscience XF24 Analyzer as described²⁴. A single NT explanted from embryos at HH13 was deposited precisely at the center of each well of a 24-well Seahorse plate previously coated with fibronectin and containing 100 µl of culture medium, and NCCs were allowed to undergo migration for 2 h (Supplementary Fig. 1B). Before preparation of the plate for Seahorse analysis, phase contrast pictures of the NT explants were taken, and their areas were measured for normalization. Cultures were then rinsed in Seahorse XF medium (Agilent, cat. no 103575-100) without serum and incubated for 30 min at 37 °C in a normal atmosphere in 500 µl assay medium supplemented with the same metabolites as prior to the rinsing step, except serum. Then, the Seahorse assay was run according to the manufacturer’s instructions. After Seahorse metabolic profiling, NCC primary cultures were processed for cellular assays of delamination and migration, immunolabeling, qPCR analyses, or intracellular ATP, glutamine, or glutamate measurements (Fig. S1B). All experiments were normalized with respect to both the NT length and the stage of embryos at the onset of experiments.

Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) values, as readouts of basal mitochondrial respiration and lactate production, were assessed through four cycles of measurement during 30 min, and in some experiments, this was followed by drug injections and three additional cycles of measurement. All drug solutions were prepared from stock solutions in an assay medium supplemented with glucose and glutamine. OCR and ECAR values were used to establish the energetic maps of cells, comprising four quadrants (quiescent, aerobic (OXPHOS), glycolytic, and energetic). Briefly, we assessed OCR and ECAR in NT explants under starvation medium and in conditions with 5 mM glucose and 2 mM glutamine. Basal respiration measured was between 120–200 pmole/min. under the starvation condition and 300–400 pmole/min. in the presence of nutrients, respectively. This difference between the levels of OCR in these two conditions allowed us to fix the OCR threshold between the quiescent and OXPHOS quadrant at 200 pmole/min. The ECAR threshold between the quiescent and glycolytic quadrants was set at 30 mpH/min. to achieve an OCR/ECAR ratio of 4. Data shown in our energetic maps correspond to the mean OCR and ECAR values ± s.d. of the fourth measurement of basal respiration and of the third measurement after inhibitor treatment. The key parameters of mitochondrial respiration (ATP-linked respiration, maximal respiratory capacity, and proton leak) were measured by means of a MitoStress test after sequential additions of oligomycin at 1 µM, FCCP at 0.75 µM, and rotenone + antimycin at 1 µM through three cycles of measurements in 30 min. Coupling efficiency, which represents the proportion of respiratory activity that is used to make ATP, was calculated as the difference between the basal OCR and the minimal, ATP-linked OCR. Spare respiratory capacity, which corresponds to the extramitochondrial capacity available in a cell to produce energy under conditions of increased demand, was calculated as the difference between the maximal OCR and the basal OCR^47,48,49.

Temporal metabolic changes of NTs were investigated using two rounds of Seahorse assays on the same explants. After identifying the initial metabolic profile of NT explants at the first 3 h of culture, the plate was removed from the analyzer, the assay medium was changed for 100 µl of fresh culture medium and the plate was further incubated for overnight at 37 °C in a 5%-CO₂ incubator and then proceeded for the second run of Seahorse assay under the same conditions as for the first one.

ATP and metabolite measurements

Intracellular ATP and glutamine/glutamate levels were measured for each NCC primary cultures after Seahorse analysis using the ATPlite Bioluminescence assay kit from PerkinElmer (cat. no. 6016943), and the Glutamine/Glutamate-Go assay (J8021) assay kit from Promega (cat. no. J8021), respectively, following the manufacturers’ instructions. Measures were normalized to NCC numbers.

Cryosectioning

To obtain embryo sections for immunolabeling or following in situ hybridization, embryos were washed in PBS for 1 h at room temperature and in 15% sucrose solution overnight at 4 °C. Next, they were incubated in 7.5% porcine gelatin (dissolved in 15% sucrose solution) for 2–3 h at 40 °C, embedded in gelatin-sucrose in cup, snap-frozen in chilled isopentane at −70 °C and stored at −80 °C. About 20-µm sections were obtained using the Leica cryostat and collected on Superfrost/Plus slides (Thermo Fisher Scientific). For imaging, the slides were immersed in PBS at 40 °C for 2 h for gelatin removal, washed in PBS, and mounted in Aquatex mounting medium (Merck). Sections of embryos with in situ hybridization were observed and imaged with a Hamamatsu NanoZoomer Digital Slide Scanner and treated with NDP.View2 software. Immunolabeled sections were observed with confocal microscope 20X fluorescence objectives.

Immunofluorescence labeling of NCC cultures and embryo sections

For immunolabeling, the following primary antibodies were used: Rabbit monoclonal antibody (mAb) to Snail-2 (clone C19G7, lots no. 5-7, Cell Signaling, 1/300), mouse mAb to Sox-10 (Clone A2, lot no. B0617, Santa Cruz, cat. no. SC-365692, 1/200), mouse mAb to β-catenin (clone 14, lot 15825, BD-Transduction Laboratories, cat. no. 610153, 1/200), rabbit polyclonal antibodies to GLS (lot no. WG3329173A, Life Technologies, cat. no. PA535365, 1/50), rabbit polyclonal antibodies to Sox-2 (lot no. GR79451-1, Abcam, cat. no. 97959, 1/200), mouse mAb to Pax7 (lot no. 1ea11/14/13, Developmental Studies Hybridoma Bank, University of Iowa, undiluted culture supernatant), mouse mAb to α-SMA conjugated to Cy3 (Clone 1A4, lot no. 086M4829V, Sigma, cat no. C6198, 1/300), and mouse mAb to βIII-tubulin conjugated to Alexa488 (Clone Tuj-1, lot no. B245871, R&D Systems, cat. no. 801203, 1/500). NCC primary cultures were performed on glass coverslips deposited in four-well plates (Nunc, cat. no. 144444) or in eight-well Lab-Tek Chambered Coverglass (Nunc, cat. no. 154511) coated with fibronectin. After removing the NT to uncover delaminating NCC, the cultures were fixed in 4% paraformaldehyde (PFA) in PBS for 15 min at room temperature for detection of all antigens, except β-catenin (45 min in 1.5% PFA). After permeabilization with 0.5% Triton X-100 in PBS for 5 min and washes in PBS, cultures were blocked in PBS-3% BSA and subjected to immunofluorescence labeling using primary antibodies followed by incubation with appropriate secondary antibodies conjugated to Alexa-fluor 488 or Cy3 (Jackson Immunoresearch Laboratories), and processed for DAPI or Hoechst staining to visualize cells’ nuclei before mounting in ImmuMount medium (Shandon). For immunostaining of embryo sections, essentially the same procedure was used. Preparations were observed with a Zeiss AxioImager M2 epifluorescence microscope equipped with 20X-63X fluorescence objectives (Acroplan 10X/0.25, 20X/0.45, Plan-Neofluar 40X/0.75 and 63X/1.25 oil) or with a Zeiss LSM 900 confocal microscope equipped with 10X-40X fluorescence objectives (Plan-Apochrome 40X/1.30 oil). Data were collected using the Zen or Airyscan2 software and processed using ImageJ software. Quantification of fluorescence intensity for GLS and β-catenin staining in single NCC nuclei was performed using ImageJ software, and data were reported as the total fluorescence within the nucleus related to its area.

Whole mount immunolabeling

Trunk regions of embryos were fixed with 4% PFA for 2 h at 4 C°, washed two times with PBS + 0.1% Triton X-100 for 5 min, and incubated with blocking solution (PBS + 0.1% Triton X-100 + 1% BSA, and 0.15 % (w/v) glycine) for 2 days at 4 °C under stirring. They were then incubated for one night at 4 °C with gentle shaking with primary antibodies to GLS (1:50) and Sox-10 (1:200) in a blocking solution. On the following day, the embryos were extensively washed with blocking solution at room temperature and then incubated for another night with secondary antibodies conjugated to Cy3 and Alexa-fluor 488 (1:300) in blocking solution as for the primary antibodies. Lastly, embryos were extensively washed with blocking solution and transferred into a Coverwell imaging chamber (Grace Bio Lab, cat. no. 635041), with their dorsal side oriented toward the bottom. After removing the bulk of liquid to keep them flat, a few droplets of mounting medium were added before mounting with a coverslip, and the preparations were kept for 24 h at room temperature before imaging with confocal microscope 20X fluorescence objectives.

Proximity ligation assay

After 5 h in culture and removing the NT to uncover delaminating NCC, explants were fixed with 4% PFA for 15 min, followed by permeabilization with 0.5% Triton X-100 in PBS for 5 min, rinsing in PBS and blocking in PBS-3% BSA. Cultures were then subjected to immunofluorescence labeling using pairs of primary mouse and rabbit antibodies to β-catenin and GLS diluted in a blocking solution as described above. Non-immune mouse antibodies (Jackson ImmunoResearch, cat. no 015-000-002) were used instead of mouse mAb to β-catenin for controls. Following primary antibodies incubation, the cultures were washed three times in PBS for 5 min and incubated with Duolink Plus and Minus probes (diluted 1:5) followed by Duolink In Situ Orange Starter Kit Mouse/Rabbit (Sigma, cat. no. DUO92102), according to the manufacturer’s guidelines. Confocal microscopy image acquisition was performed as above and quantitative analysis of dots was done on Z-projection images through the entire cell using ImageJ.

Co-immunoprecipitations

Immunoprecipitations were performed with rabbit polyclonal antibodies to GLS (Life Technologies, cat. no. PA535365) and using the Pierce Crosslink Magnetic IP/co-IP kit (Pierce-Thermo Scientific, cat. no 88805). NTs from the trunk region of embryos at HH12-14 were dissected out manually with a scalpel under a stereomicroscope (see supplementary Fig. 1c). The pellet of about 30 neural tubes was lysed with 400 µl IP Lysis Buffer (Pierce-Thermo Scientific, cat. no 87787) supplemented with 1X protease and phosphatase inhibitors (Roche, Complete EDTA-free, cat. no. 05056489001 and PhosSTOP, cat. no. 04906837001) on ice for 30 min. The lysate was centrifuged for 15 min at 15,000×g at 4 °C, and the supernatant was collected and precleared with 25 µl of protein A/G magnetic beads (MCE, cat. no HY-K0202) 30 min at 4 °C. The precleared lysate was collected and an aliquot was kept apart to serve as the control input. Half of the precleared lysate was incubated for 90 min at room temperature with 10 µg of anti-GLS antibody non-covalently coupled to 25 µl of protein A/G magnetic beads according to manufacturer’s instructions. Beads without antibodies incubated with the second half of the precleared lysate served as negative controls. After incubation, beads were collected and washed twice with IP Lysis Buffer and once with ultrapure water (Invitrogen, cat. no 10977-035). Elution was performed by mixing beads with 2X Laemmli sample buffer. The supernatant containing immune complexes was denatured at 95 °C for 5 min prior to electrophoresis.

Immune complexes were separated on 8% SDS-PAGE and transferred onto nitrocellulose membranes (Trans-blot Turbo RTA transfer kit, Bio-Rad, cat. no 1704270), and stained with Ponceau red (Sigma, cat. no78376) for 15 min at room temperature. Membranes were washed twice for 5 min in TBS-T (10 mM Tris pH 8.0, 150 mM NaCl, 0.05% Tween-20) then blocked with 5% non-fat milk for 1 h and probed with the rabbit polyclonal antibodies anti-GLS (Life Technologies, cat. no. PA535365,) and mouse mAb anti-β catenin (Clone 14, BD-Transduction Laboratories, cat. no. 610154), diluted in 1% non-fat milk overnight at 4 °C. After washes in TBS-T membranes were incubated with horseradish peroxidase-conjugated secondary antibodies diluted in 1% non-fat milk for 45 min at room temperature. After washes in TBS-T, protein bands were detected by enhanced chemiluminescence using the Clarity Western ECL (Bio-Rad, cat. no 1705061). Quantification of Western blots was carried out using ImageJ software. Full scan blots are presented in the Source data files.

In situ hybridizations for mRNA detection on whole-mount embryos

In situ hybridization mRNA probes were generated either by PCR (for GLS and GDH) or from plasmid vectors (for Sox-10 and Foxd-3). For PCR probes, primers were designed to generate a 400–500 bp fragment of the mRNA coding sequence based on information available from NCBI databases. The following forward and reverse sequences were selected for GLS, fwd 5′-GAAGGACAAGAGAAAATACCAGTG-3′, rev 5′-TGCTCCAGCATTCACCATAGG-3′; GDH, fwd 5′-TGGGACAATCATGGGCTTCC-3′, rev 5′-CAGTACGCATGATTTGTCGAGC-3′; and SNAT-2, fwd 5′-TACGCTGTCCCAATCCTGAC-3′, rev 5′-TGCTGCAGATGCACCAATGAAT-3′ and the PCR primers were designed as follows. The linker, 5′-GAG-3′, and the sequence of the RNA polymerase T7 binding site, 5′-TAATACGACTCACTATAGGG-3′, were added to the reverse sequences. The synthetized PCR primers (Eurogentec) were used to generate SNAT-2, GLS, and GDH probes by PCR reaction performed with chick embryo cDNA template using Platinum Taq DNA polymerase (Invitrogen, cat. no. 10966018). PCR products were assessed on a gel and purified using the Qiaquick PCR Cleanup kit (Qiagen, cat. no. 28104). Plasmids for Foxd-3 and Sox10 mRNA probe synthesis were from C. Erickson and P. Scotting, respectively^50,51. Linearized plasmid DNA and PCR products were used to synthesize digoxigenin-UTP (Roche) labeled antisense probes with RNA polymerases from Promega and RNA probes were purified with Illustra ProbeQuant G-50 microcolumns (GE Healthcare, cat. no. 28903408).

In situ hybridizations were performed on whole-mount embryos collected at the appropriate developmental stages, and fixed in 4% PFA in PBS for 2 h at room temperature or overnight at 4 °C. Embryos were hybridized overnight at 65 °C with the digoxygenin-UTP-labeled RNA probes in 50% formamide, 10% dextran sulfate and Denhart’s buffer (0.5 µg probe/ml hybridization buffer) and washed twice in 50% formamide, 1x SSC and 0.1% Tween-20 at 65 °C, then four times at room temperature in 100 mM maleic acid, 150 mM NaCl pH 7.5, and 0.1% Tween-20 (MABT buffer). After a 1-h pre-incubation in MABT buffer containing 10% blocking reagent (Roche) and 10% heat-inactivated lamb serum, embryos were incubated overnight at room temperature with the anti-digoxygenin antibody (Roche). After extensive rinsing with MABT buffer, they were preincubated in 100 mM NaCl, 50 mM MgCl₂, 1% Tween-20, and 25 mM Tris-HCl, pH 9.5, and stained with NBT-BCIP (Roche) following manufacturer’s guidelines. Preparations were observed and imaged with a Nikon stereomicroscope.

mRNA quantification by quantitative RT-PCR analyses

For NT explants cultured in vitro, after characterizing the metabolic profiles of individual explants by Seahorse analysis, the total RNA of explants sharing the same metabolic profile were isolated using Trizol (Invitrogen), following the manufacturer’s instructions. For normal, untreated embryos and for embryos injected with drugs or siRNAs, the NT situated in the caudal part encompassing the last five somites up to the tail bud was dissected manually with a scalpel under a stereomicroscope (see supplementary Fig. 1c), and the total RNA was extracted using Trizol (Fig. S1C, D). About 500 ng RNA were used for cDNA synthesis using SuperScript IV reverse transcriptase (Invitrogen, cat. no. 18090050). Quantitative real-time PCR were performed using the Power Syber Green Master Mix (Applied Biosystems, cat. no. 4368708) in a StepOne Plus RT-PCR apparatus (Applied Biosystems). Gene expression was assessed by the comparative CT (ΔΔ_Ct) method with β-actin as the reference gene. The primers used for the quantitative RT-PCR analyses are shown in Supplementary Table 1.

Cell cycle assay

Cell cycling in culture was monitored using the EdU Click-iT Cell Proliferation Kit for Imaging from Life Technologies (cat. no. C10638). Briefly, NCC primary cultures were incubated with EdU at 20 µM in a culture medium for 1 h. Immediately after EdU incorporation, cultures were fixed in 4% PFA in PBS for 15 min at room temperature, permeabilized in 0.5 Triton X-100 for 15 min, and treated for EdU detection using Alexa-555 Fluor azide in accordance with the manufacturer’s guidelines. After DNA staining with Hoechst, cultures were examined as described for immunolabeling.

Time-lapse video microscopy

NCC primary cultures were performed in four-well-chambered coverglass (Nunc, cat. no. 155383) coated with fibronectin. Up to five NTs were positioned with their dorsal side oriented down toward the substratum and distributed separately into each well filled with culture medium, and were maintained at 37 °C in a humidified 5%-CO₂ incubator for about 2 h until the NT adhered firmly to the dish, and NCC initiated migration on the substratum; then the cultures were transferred into a heated chamber (Ibidi) with a humid atmosphere containing 5% CO₂/95% air placed on the motorized stage of a Leica DMIRE2 microscope equipped with a CoolSNAP HQ camera (Roper Scientific). Time-lapse video microscopy was performed with a 10X objective and phase contrast images were captured every 5 min during 16–24 h using the Micromanager software. In some experiments, the acquisition was paused briefly after 2 h for injection of drugs or nutrients in the medium. Trajectories and positions of individual NCC in several NT explants recorded in parallel were tracked using Metamorph 7 software, and the progression of the migration front of the NCC population every hour was measured using a custom GUI (available upon request) written in MATLAB (MathWorks, Natick, MA, USA). The velocity of each NCC was calculated as the ratio between the total length of its trajectory and the duration of the acquisition time.

Statistical methods

Statistical analyses were performed using Prism 7 (GraphPad). For statistical analysis of data, we used a one-way ANOVA parametric test after the validation of normality and equality of variances using Shapiro–Wilk and Brown–Forsythe methods, respectively. For comparison between two conditions we used unpaired t-test two-tailed test after the validation of normality and equality of using Shapiro–Wilk and equality of variances. Otherwise, a non-parametric Mann–Whitney test was used. Unless specified, at least three independent experiments were carried out for each procedure. Each NT explant or embryo was considered an individual sample. The data obtained for each drug or nutrient condition were compared to that of the medium without drug or with both nutrients, respectively. The number of samples n (explants or embryos) analyzed is indicated in each graph in brackets. For statistical analysis of qRT-PCR results of in vitro experiments, mRNA expression from a pool of 1–8 NT explants are presented as mean ± s.e.m. of triplicates per experiment and gene. In in vivo experiments, mRNA expression was measured per embryo after analyzing three replicate measurements acquired per embryo and genes. The data obtained from embryos injected with drugs were analyzed using two-way ANOVA relative to the data of embryos injected with the vehicle. Data were expressed as mean values ± s.e.m. Results are considered statistically significantly different when p < 0.05.

Reporting summary

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

Data availability

Most data were available within the Article and Supplementary Files. Source data are provided via Figshare [https://doi.org/10.6084/m9.figshare.28280384] or can be obtained from the corresponding authors upon reasonable request.

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Acknowledgements

We are grateful to Chantal Thibert and Sakina Torch for their advice on cellular metabolism, Ana Ferrero for her support and for providing the facilities to develop the co-IP analyses, Joana Esteves de Lima for prospective chromatin-IP experiments, and Redouane Fodil for designing the custom GUI to track the progression of the migratory front of NCC populations. Thanks to Xavier Decrouy from the IMRB imaging platform and Xavier Laffray from the histology and imaging platform of the Laboratoire Gly-CRRET for advice. This work was supported by funding from Institut National de la Santé et de la Recherche Médicale, Université Paris-Est Créteil, AAP IMRB cross-teams project to S.D. and R.M., Fondation ARC pour la Recherche sur le Cancer (No. PJA 20181207844) for S.D., the Association Française contre les Myopathies (AFM) via TRANSLAMUSCLE II programs 22946 for F.R., the Fondation pour la Recherche Médicale (FRM, grant EQU20200301021) for F.R., and Labex REVIVE (ANR-10-LABX-73) for F.R. N.N.M. was funded by doctoral fellowship of Université Paris-Est Créteil and by the Labex REVIVE to F.R.

Author information

Nioosha Nekooie Marnany
Present address: Diabetes and Metabolism Research Center, University of Utah, Salt Lake City, UT, 84112, USA
These authors jointly supervised this work: Sylvie Dufour, Jean-Loup Duband.

Authors and Affiliations

Institut Mondor de Recherches Biomédicales, Faculté de Santé, INSERM, Université Paris-Est Créteil, 94000, Créteil, France
Nioosha Nekooie Marnany, Frédéric Relaix, Roberto Motterlini, Roberta Foresti, Sylvie Dufour & Jean-Loup Duband
Laboratoire Gly-CRRET, Université Paris-Est Créteil, 94000, Créteil, France
Alwyn Dady
Laboratoire de Myologie Fondamentale et Translationnelle, CNRS, Université Paris Cité, 75013, Paris, France
Isabelle Duband-Goulet
Ecole Nationale Vétérinaire d’Alfort, 94700, Maisons-Alfort, France
Frédéric Relaix

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Nioosha Nekooie Marnany
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Alwyn Dady
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Isabelle Duband-Goulet
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Roberto Motterlini
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Contributions

N.N.M. conceived the project and designed the experimental approach, performed experiments, analyzed data, and contributed to the manuscript. A.D. provided expertise for in ovo studies and facilities for cryosectioning methods and analyses. I.D.-G. performed the co-IP experiments. F.R. provided financial and logistics support. R.F. and R.M. provided expertise in cellular metabolism and Seahorse technology and revised the manuscript. S.D. designed the experimental approach, performed experiments, analyzed the data, and contributed to the manuscript. J.-L.D. designed the experimental approach, performed experiments, analyzed the data, and wrote the manuscript.

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Correspondence to Nioosha Nekooie Marnany, Sylvie Dufour or Jean-Loup Duband.

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Nekooie Marnany, N., Dady, A., Duband-Goulet, I. et al. Non-canonical nuclear function of glutaminase cooperates with Wnt signaling to drive EMT during neural crest development. Nat Commun 16, 5554 (2025). https://doi.org/10.1038/s41467-025-58573-0

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Received: 08 February 2024
Accepted: 21 March 2025
Published: 01 July 2025
DOI: https://doi.org/10.1038/s41467-025-58573-0