REVIEW

Glycosphingolipid metabolism in cell fate specificationDomenico Russo1,*, Laura Capolupo1,2, Jaipreet Singh Loomba1,2, Lucia Sticco1 and Giovanni D’Angelo1,2,*

ABSTRACTGlycosphingolipids (GSLs) are ubiquitous components of eukaryoticplasma membranes that consist of a ceramide backbone linked to aglycan moiety. Both the ceramide and the glycan parts of GSLs displaystructural variations that result in a remarkable repertoire of diversecompounds. This diversity of GSLs is exploited during embryogenesis,whendifferentGSLsareproducedat specificdevelopmental stagesandalongseveral differentiation trajectories. Importantly, plasmamembranereceptors interactwithGSLstomodify their activities.Consequently, twootherwise identical cells can respond differently to the same stimulusowing to their differentGSLcomposition. Themetabolic reprogramingofGSLs is in fact a necessary part of developmental programs, as itsimpairment results in developmental failure or tissue-specific defects.Moreover, single-cell variability is emerging as a fundamental player indevelopment: GSL composition displays cell-to-cell variability insyngeneic cell populations owing to the regulatory gene expressioncircuits involved in microenvironment adaptation and in differentiation.Here, we discuss how GSLs are synthesized and classified and reviewthe role of GSLs in the establishment and maintenance of cell identity.We further highlight the existence of the regulatory circuits thatmodify GSL pathways and speculate how GSL heterogeneity mightcontribute to developmental patterning.

KEY WORDS: Differentiation, Glycosphingolipid, Golgi complex

IntroductionCellular membranes serve as both barriers and interfaces betweentopologically distinct biological spaces. The lipid composition ofthese membranes varies at different cellular locations. For example,the plasma membrane (PM) is rich in sphingolipids compared tointracellular membranes, which results in the PM having distinctbiophysical properties (Holthuis and Menon, 2014). Sphingolipidscontain a hydrophobic ceramide (Cer) backbone that is composed ofa saturated fatty acid and sphingoid base. This allows sphingolipidsto establish lateral interactions (both homotypic and with sterols)to yield a tightly packed and thick membrane structure (Hannunand Obeid, 2018; Holthuis et al., 2001). Owing to this lipidcomposition, the PM is less permeable to ions and peptidescompared to intracellular membranes, which matches with its‘barrier’ function towards the extracellular environment (Holthuisand Menon, 2014). Sphingolipids also show incomplete miscibilitywith phospholipids, which results in lateral phase partitioning of themembrane and thus in the formation of membrane microdomains(Simons and Ikonen, 1997). Such microdomains have differentaffinities for proteins depending on the length and composition oftheir transmembrane domains, or on their lipid-based membrane

anchoring. Specifically at the PM, sphingolipids participate insignaling events by recruiting signaling molecules to, orsequestering them at, membrane microdomains for the modulationof their activities and for their processing into the endocytic cycle(Holthuis and Menon, 2014; Holthuis et al., 2001; Simons andIkonen, 1997). Given these properties, sphingolipids are proposed tofunction as fundamental membrane organizers and to make upthe fabric of eukaryotic PMs in order to influence the interactionwith the extracellular environment (Hannun and Obeid, 2018;Holthuis et al., 2001).

Interestingly, different cell types exhibit a specific sphingolipidarray at their PMs (Hakomori, 2003; Ngamukote et al., 2007)(Table S1). Indeed, sphingolipids are subjected to remarkablestructural variations that lead to the production of hundreds ofdifferent species (Hannun and Obeid, 2008, 2018). A substantialpart of this variability derives from the heterogeneous elongationof glycan chains that are covalently linked to the sphingolipidbackbone in the synthesis of the class of compounds known asglycosphingolipids (GSLs). GSL-associated glycans range havebetween one and more than 20 sugar residues, with 11 differentmonosaccharide types being used in vertebrates (D’Angelo et al.,2013a). Importantly, the elongation of glycans in GSLs is not drivenby a template; instead, it entirely depends on the relative expressionand organization of their specific synthetic enzymes (Bieberichet al., 2002; Giraudo and Maccioni, 2003). Still, GSL productionis tightly controlled during differentiation programs; as a result,specific GSLs are used as differentiation stage or cell-type-specificmarkers (D’Angelo et al., 2013a). In addition, GSL composition cansubstantially vary among single cells in syngeneic cell populations(Majoul et al., 2002; Russo et al., 2018; Snijder et al., 2009).Furthermore, specific GSL glycans appear to organize interactionswith receptors that are located at the PM in order to modulate theiractivity (Bremer and Hakomori, 1982; Bremer et al., 1984; Coskunet al., 2011; Farooqui et al., 1999; Liu et al., 2008; Mirkin et al.,2002; Mutoh et al., 1995; Park et al., 2012; Toledo et al., 2004).This occurs, for instance, in the case of the GM3-dependentinhibition of epidermal growth factor receptor (EGFR) signaling,which maintains EGFR in an inactive state in the absence of itsligand (Coskun et al., 2011). By contrast, GD1a and GM1 enhanceEGFR activation (Li et al., 2001, 2000; Liu et al., 2004). Thus, twootherwise identical cells can react differently to the same stimulusowing to their different composition in GSLs.

Whereas the role of cell-to-cell variability in GSL compositionin differentiated cells remains to be understood, non-geneticheterogeneity has been proposed to contribute to cell-typediversification in developmental processes (Huang, 2009).Specifically, non-genetic heterogeneity provides cells with transitory‘states’ to potentially orient their fates towards diverging directions(Huang, 2009). Given the role ofGSLs inmodulating cell responses toenvironmental cues, along with their extensive structural variation,cell-to-cell heterogeneity in GSL composition might therefore help ingenerating identity patterns during tissue morphogenesis. In thisReview,wediscuss the role ofGSLs as cell-fate determinants, focusing

1Institute of Protein Biochemistry, National Research Council, Via P. Castellino 111,Napoli, Italy. 2Institute of Bioengineering, Laboratory of Lipid Cell Biology, Écolepolytechnique federale de Lausanne (EPFL) CH-1015 Lausanne, Switzerland.

*Authors for correspondence (giovanni.dangelo@epfl.ch; d.russo@ibp.cnr.it)

D.R., 0000-0003-2171-657X; G.D., 0000-0002-0734-4127

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on (1) how GSL diversity is generated, (2) what GSL changes occurwhen cells differentiate toward alternative fates, and (3) how theGSL metabolism is controlled by differentiation programs. Finally,we will speculate on how GSLs can contribute to tissue patterningand morphogenesis.

GSL synthesisGSL synthesis is initiated at the cytosolic membrane leaflet of theendoplasmic reticulum (ER), where Cer is produced from itsprecursor sphinganine by the consecutive action of enzymes thatcatalyze its acylation and desaturation (Mullen et al., 2012). Cer canthen be converted into several compounds that include sphingosine,Cer-1-phosphate, acyl-Cers, sphingomyelin (SM) and GSLs(Hannun and Obeid, 2018; Holthuis et al., 2001; Merrill et al.,2005). SM and GSLs are synthesized at the interface between theER and Golgi complex and constitute the major sphingolipids at

the PM. For its metabolic conversions, Cer can be galactosylated inthe ER to produce galactosylceramide (GalCer), extracted from ERmembranes by the lipid-transfer protein ceramide transfer protein(CERT) and delivered to the trans-Golgi where SM is synthesized(Hanada et al., 2003), or transported in vesicles to the cis-Golgiwhere it is glucosylated to produce glucosylceramide (GlcCer)(Funakoshi et al., 2000) (Fig. 1). Whereas SM cannot be furtherprocessed in an anabolic direction, GalCer is the precursor ofGSLs from the gala-series, also known as sulfatides, which includesulfo-GalCer, (α2-3)-sialylated GalCer (GM4), di-GalCer (i.e.Gal-GalCer) and di-sulfo-GalCer, which are produced at theGolgi complex where the enzymes for GalCer processing reside(Merrill, 2011) (Fig. 1).

Apart from the gala-series GSLs, all other GSLs have GlcCeras a precursor (D’Angelo et al., 2013a; Merrill, 2011). GlcCer isconverted into lactosylceramide (LacCer; Gal-GlcCer) (Kumagai

Gb3

Gb5 (SSEA-3)

SSEA-4

Globo-Hfucosyl-Gb5

Gb4 Forssmanantigen

ER

Golgi

TGN

Cer SphGalCer

GlcCer

(sulfatide)

GM4di-GalCer

di-sulfo-GalCer

SO3−

sulfo-GalCer

GSL precursors

LacCer

GM3 GM2 GM1a GD1a

GD3 GD2 GD1b GT1b

GT3 GT2 GT1c GQ1c

GT1a

GA2 GA1 GM1b

GD1c

GD1a

LC3

LC4

nLC4 nLC5

Lacto H antigen Lewisy

SSEA-1(Lewis)x

FAPP2

Asialo

Ganglio

Globo

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Cer-1-P

P

acyl-Cer

Key

Sia Glu

GlcNac

Gal Fuc

GalNac

Cer

Sulfate

Phosphate P

Fatty acidSphSO3−

SO3−

GBGT1B3GALNT1

B3GALT

5ST

3GAL2

FUT1/2

GalCerS CerS

GCS

GAL3ST1

GAL3ST1

B4GALT5

ST8SIA1

ST8SIA5

B4GALNT1

B4GALNT1

B4GALNT1

B3GALT4

B3GALT4

B3GALT4

ST3GAL1

ST3GAL1

B3GALT4 ST3GAL1 ST6GALNAC4

ST8SIA

5

B3GAL

T5

B3GALT1

A4GALT

ST3GAL5

B4GALNT1

B3GNT5

CERT

Fig. 1. GSL synthesis and classification and schematic representation of the GSL synthetic pathways. Ceramide (Cer) can be acylated (acyl-Cer),phosphorylated (Cer-1-phosphate) or conveyed to the TGN for the synthesis of SM. Alternatively, Cer is glycosylated for the synthesis of the GSL precursors,glucosylceramide (GlcCer) and of galactosylceramide (GalCer) along the secretory pathway (left panel). GalCer is then processed for the production of sulfatides.GlcCer is galactosylated to lactosylceramide (LacCer), which serves as a common precursor for the different GSL series: globo (red), ganglio (green), asialo(blue) and lacto (purple). Glycosphingolipid-synthetizing enzymes (GSEs) catalyzing the major synthetic reactions are shown in dark orange.

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et al., 2010; Nishie et al., 2010), which is the metabolic branchingpoint for the formation of all remaining GSLs. They arecategorized into four classes (i.e. the globo, lacto, ganglio andasialo series) and their cumulative number exceeds 400 GSLs(D’Angelo et al., 2013a; Merrill, 2011; van Meer et al., 2008).Thus, LacCer is the substrate of (1) GA2 synthase (GA2S) for thesynthesis of GA2 (GalNAc-LacCer) and of asialo-series GSLs(Nagata et al., 1992), (2) of GM3 synthase (GM3S) for thesynthesis of GM3 (NeuAc-LacCer) and of ganglio-series GSLs(Ishii et al., 1998), (3) Gb3 synthase (Gb3S) for the synthesis ofGb3 (Gal-LacCer) and of globo-series GSLs (Kojima et al.,2000), (4) Lc3 synthase (Lc3S) for the synthesis of Lc3 (GlcNAc-LacCer) and of lacto-series GSLs (Biellmann et al., 2008)(Fig. 1).After being conveyed to one of these four major metabolic

directions, GSLs are processed in glycosylation pathways.There, GSLs are often substrates of multiple possible reactionsthat lead to further diverging metabolic directions or to theformation of branched glycan structures (Fig. 1) (D’Angelo et al.,2013a; Merrill, 2011). The elongation of glycan residues in GSLs isindeed the result of the ordered action of glycosyltransferases; theirrelative levels, topological organization within the Golgi stack andpresence in multi-enzymatic complexes are key factors in thedetermination of the metabolic outcome (Maccioni et al., 2011).Along with these parameters, another factor that influences glycanelongation in GSLs is substrate availability. GlcCer, the commonprecursor of most GSLs, can be delivered to specific sub-Golgiregions by different transport mechanisms [i.e. vesicular, or non-vesicular through the action of the lipid transfer protein 4-phosphateadapter protein 2 (FAPP2, also known as PLEKHA8); D’Angeloet al., 2007], where each of these transport routes feeds a distinctglycosylation pathway (D’Angelo et al., 2013b). However, in spiteof the non-deterministic nature of the GSL synthetic system, whenthe database of GSL structures was analyzed (Sud et al., 2007), theywere found to be assembled according to regular patterns;this suggests that structural heterogeneity in GSL structures isnot the result of a random process and points to them having abiological function.A major limitation in our understanding of the structural and

functional features of GSLs derives from technical difficulties:determining the GSL composition of a biological sample remains ananalytical challenge. GSL composition is specific to the species,cell type and condition (Hakomori, 2008). Moreover, GSLs largelydiffer in their abundance, chemical stability and biophysicalproperties, which makes their uniform extraction from biologicalsamples difficult. In addition, the monosaccharide units in GSLchains have very similar chemical structures, which, together withheterogeneous positioning and the anomery of the sugar–sugarbonds and glycan chain branching, complicate GSL analysis(Merrill, 2011). However, the accuracy in resolving GSLcomposition has improved as technologies have improved. Thus,whereas orcinol-sulfuric acid staining and radioactive labeling with3H- or 14C-labeled monosaccharides coupled to chromatographicseparation are still valuable procedures for a rapid and inexpensiveassessment of GSL composition (Schnaar and Kinoshita, 2015),detection with specific lectins or antibodies and mass spectrometry-based methods now represent the golden standards for GSLprofiling (Wuhrer, 2013). Thanks to these advancements, it isnow possible to evaluate GSL changes in biological samples withgood accuracy, although an absolute quantification is often notpossible owing to lack of complete reference standard samples(Farwanah and Kolter, 2012).

In the following sections, we will discuss the changes GSLsundergo during cellular differentiation in developmental processes,as well as during oncogenic transformation of tissues.

GSL reprograming in development, cell differentiation andcancerGSL changes during embryonic development and cellulardifferentiationNumerous studies have reported that the composition of GSLs in themembrane is remodeled during embryonic development (Cochranet al., 1982; Handa and Hakomori, 2017; Kannagi et al., 1983;Ngamukote et al., 2007; Yamashita et al., 1999). Thesecompositional changes have been evaluated during the threemajor developmental stages in mice {i.e. preimplantation[embryonic day (E) 0.5–6.5], gastrulation (E6.5–E10.5) andorganogenesis (E10.5–E17.5)} (Handa and Hakomori, 2017)(Fig. 2). The preimplantation phase is dominated by GSLs of thelacto series [i.e. stage-specific embryonic antigen 1 (SSEA-1) andLey] and globo series (i.e. Forssman antigen, Gb4, SSEA-3 andSSEA-4) (Handa and Hakomori, 2017; Sato et al., 2007). Duringgastrulation, production of the ganglio-series GSLs is induced inboth neuronal and glial cell precursors (Goldman et al., 1984),whereas SSEA-3, Forssman antigen and Gb4 globosides arerestricted to visceral mesoderm cells and to the inner cell mass ofthe growing blastocysts (Handa and Hakomori, 2017). Finally,during the organogenesis phase, the GSLs that are most prominentlysynthetized are gangliosides; their relative amounts change in thenervous system from post gastrulation (E8) to adult ages. Thus,GM3, GD3 and GD2 are expressed at day E8, whereas GM1, GD1a,GD1b, GT1b and GQ1b are induced starting from E14 (Ngamukoteet al., 2007) (Fig. 2).

Changes in GSL expression have also been measured duringin vitro differentiation of pluripotent cells into the three germ layers(i.e. ectoderm, mesoderm and endoderm) (Liang et al., 2010, 2011;Russo et al., 2018) (Table S1). Pluripotent stem cells express GSLsof the globo and lacto series (Breimer et al., 2017; Liang et al., 2010,2011; Russo et al., 2018), including Gb3, Gb4, Gb5 (SSEA-3), α1-2fucosylated-Gb5 (Globo H), sialyl-Gb5 (SSEA-4) and disialyl-Gb5,globo-A, Lc3, Lc4, SSEA-1 and fucosyl-Lc4 (Breimer et al., 2017)(Fig. 3). The levels of globo- and lacto-series GSLs decrease upondifferentiation of pluripotent stem cells to neuronal progenitors,which is followed by the increase in the synthesis of GD3, GM3,GM1 and GD1 (Kwak et al., 2006; Liang et al., 2011;Marconi et al.,2005; Russo et al., 2018). In contrast, when embryonic stem cellsdifferentiate into definitive endoderm, the major GSL that isexpressed is Gb4 (Liang et al., 2011) (Fig. 3). GSL compositiondynamically changes during the differentiation of mesenchymalstem cells (MSCs) from adult bone marrow into multiple celllineages. Indeed, MSCs express SSEA-4 (Bergante et al., 2014;Gang et al., 2007) along with GD1a and GD2 gangliosides(Bergante et al., 2014), whereas in MSC-derived adipocytes, themajor GSLs are GM3 and GD1a (Kojima et al., 2015), and GM3and GD3 are expressed in MSC-derived chondrocytes (David et al.,1993). Moreover, lacto-series GSLs and GM3 are expressed in pre-B-cells, whereas mature and activated B cells express GM3 and theglobo-series GSLs Gb3 and Gb4 (Taga et al., 1995; Wiels et al.,1991; Wipfler et al., 2011) (Fig. 3).

These data indicate that developmental programs areaccompanied by the reprograming of GSL metabolism.

Importantly, active GSL synthesis is required for embryonicdevelopment: both GlcCer synthase (GLS, encoded by UGCG) andLacCer synthase (B4GALT5) (Fig. 1) knockout (KO) mice, which are

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unable to synthesize GSLs through the GlcCer precursor, die byE10.5 (Allende and Proia, 2014; Nishie et al., 2010; Yamashita et al.,2002, 1999). In both cases, the embryo is able to progress through pre-implantation phase, but not beyond formation of the three germ layers(Allende and Proia, 2014; D’Angelo et al., 2013a; Yamashita et al.,1999). Further analyses of mice that harbor defects in the pathwaysof GSL synthesis support the idea that there is a tissue-specific rolefor the GSL subclasses (Allende and Proia, 2014; D’Angelo et al.,2013a). Knockout of B3GNT5 – the gene encoding the firstenzyme involved in the synthesis of lacto-series GSLs (i.e. Lc3synthase) (Fig. 1) – results in either preimplantation lethality ormultiple postnatal defects (Biellmann et al., 2008). Conversely,the genetic disruption of globo or ganglio series GSL productionyields a wide range of immune and neurological phenotypes,respectively (Allende and Proia, 2014). In addition, loss-of-function mutations in three genes that encode enzymes involved inthe synthesis of ganglio-series GSLs cause neuronal disease inhumans (Boccuto et al., 2014; Boukhris et al., 2013; Fragaki et al.,2013; Harlalka et al., 2013; Simpson et al., 2004).Altogether, this evidence highlights that (1) GSL cell

composition is remodeled when cells differentiate, and (2) thatGSL synthesis has a role in differentiation and development. Alongthese lines, aberrant changes in GSL metabolism are coupled toaltered cell differentiation and malignant cell transformation, asdiscussed in the following section.

GSL and cancerAberrations in GSL metabolism have also been linked to cancer(Gouaze-Andersson and Cabot, 2006; Morad and Cabot, 2013;Ogretmen, 2018). In fact, similar to the events during normalembryonic development and tissue lineage differentiation, cellsrearrange their GSL composition during oncogenic transformation(Hakomori, 1998; Hakomori and Zhang, 1997). This rearrangementhas been suggested to contribute to cellular transformation,

metastasization and the emergence of multi-drug resistance(Gouaze-Andersson and Cabot, 2006; Hakomori and Zhang,1997; Jacob et al., 2014; Kovbasnjuk et al., 2005). A recent studyon mammalian target of rapamycin (mTOR)-induced liver cancershowed that hyperactive mTOR signaling results in increased GSLsynthesis (Guri et al., 2017), and that GSL production is strictlyrequired for mTOR-dependent cancer development (Guri et al.,2017), but how exactly do GSLs contribute to the different aspectsof oncogenesis?

Signal transducers, adhesion molecules and growth factorreceptors that participate in malignant transformation anddevelopment of drug resistance are often GSL targets. Forinstance, in breast cancer, increased GD3 and GD2 synthesisfavors stem cell proliferation by fostering the activation of growthfactor receptors on the PM (Liang et al., 2013) and promotingresistance to treatment with Gefitinib, a tyrosine kinase inhibitorthat targets EGFR (Liang et al., 2017). Cisplatin is achemotherapeutic agent that is used for the treatment of anumber of cancers, such as non-small cell lung cancer (NSCLC)and malignant pleural mesothelioma (MPM). It induces Cerproduction, leading to cell cycle arrest and apoptosis (Dasari andTchounwou, 2014; Nowak, 2012). Drug-resistant cancer cellsescape apoptosis by increasing GSL synthesis at the expense of anaccumulation of Cer, which also leads to increased expression ofthe multidrug resistance-associated protein 1 (MRP1), whichstimulates drug efflux (Tyler et al., 2015).

GSL reprograming has a role in the epithelial-to-mesenchymaltransition (EMT), which is the process that enables metastaticcellular invasion in the context of cancer progression. The inductionof EMT in vitro by transforming growth factor β (TGFβ) treatment isaccompanied by a reduction in the levels of asialo-GSLs GM1and GM2, whereas complex gangliosides are, in turn, inducedduring this process (Guan et al., 2009; Mathow et al., 2015).Interestingly, a subpopulation of cells that express low levels of

E0.5 E1.5 E2 E3 E3.5 E4.5 E6.5 E7.5 E8.5 E10.5 E14.5

Preimplantation development Gastrulation and early organogenesis Organogenesis and fetal growth

E17.5

Ganglio-series GSL

GM3, GD2, GD3

GM1, GD1a, GD1bGT1b, GQ1b

Sulfatides SM4

Globo-series GSL

Gb3, Gb4

SSEA-3, SSEA-4

Forssman antigen

Lacto-series GSL

LC3, LC4

SSEA-1, Le y

Fig. 2. Changes of GSL profile in mouse embryonic development. Stage-specific changes of GSL expression during mouse embryogenesis (Cochran et al.,1982; Handa and Hakomori, 2017; Kannagi et al., 1983; Ngamukote et al., 2007; Yamashita et al., 1999). Globo- and lacto-series GSLs are expressedpredominantly during the preimplantation phase and gastrulation (Handa and Hakomori, 2017; Heinrich, 1993). Ganglio-series GSLs, together with sulfatides,start to be synthetized during late gastrulation until the prenatal phase (Goldman et al., 1984; Ngamukote et al., 2007), and concomitantly to embryonic braindevelopment. The three phases of embryonic development are indicated by three different shades of gray. The color-coded rectangles represent lipids thatare expressed at the specific stage; colored lines mean that the lipid is not expressed at that specific stage; the absence of a rectangle or line means thatthe lipid has not been measured at the reported developmental stage.

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epithelial markers has been identified in prostate tumors. Thissubpopulation expresses high levels of SSEA-4 and spontaneouslyescapes from adhesive colonies and forms invadopodia-likemigratory structures. This supports the idea that SSEA-4 is amarker for metastasizing cells that have acquired a mesenchymalnature (Sivasubramaniyan et al., 2015).Moreover, for a number of tumors, the overproduction of a

specific GSL has been reported. These GSLs can be used astumor-associated antigens (TAAs) for the definition of the tumortype and stage (Table 1 and references therein). Importantly,GSLs that serve as TAAs have been exploited to develop vaccinestrategies to elicit a specific cytotoxic and/or humoral immuneresponse against tumor cells (Dobrenkov and Cheung, 2014).GD2-targeted immunotherapy of neuroblastoma has become thefirst GSL-targeting immunotherapy to obtain food and drugadministration (FDA) approval for medical care (Dobrenkov andCheung, 2014). Moreover, innovative strategies to target GSL-TAAs also imply that toxins that use these GSLs as the naturalreceptors in their target cells could be used for cancer treatment;

this is the case for a Shigella toxin, which recognizes Gb3 that isoverexpressed in gastric adenocarcinomas (Geyer et al., 2016).

Thus, metabolic alterations of GSLs are inherent componentsof cancerogenesis as they (1) originate from the malignanttransformation process, (2) contribute to cancer-relevantphenotypes and (3) define cancer-specific cell states.

The regulatory circuits of GSL expressionThe aforementioned metabolic changes in GSLs, both incancerogenesis and developmental contexts, are often theconsequence of a reprograming in the expression of genes thatencode the enzymes that synthesize GSLs. During neuraldifferentiation, for instance, the expression of genes encodingenzymes for the synthesis of globo- and lacto-series GSLs (i.e.A4GALT, encoding Gb3 synthase, and B3GNT5, encoding Lc3synthase) decreases; at the same time, expression of genes encodingenzymes of the ganglio series synthesis pathway (i.e. ST3GAL5,encoding GM3 synthase, and B4GALNT1, encoding GA2/GM2synthase) increases (Liang et al., 2010, 2011; Russo et al., 2018).

Pluripotent stem cells

Endoderm

Mesoderm

Ectoderm

Primordial germ cell

Sperm

Oocyte

Skin and Hair

Neural crest stem cell

Neuronal restricted progenitor

Glial restricted progenitor

Astrocyte

Oligodendrocyte

Neuron

Schwann cell

Neural stem cell

Muscle cell

Cardiocyte

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HSC

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Proerythroblast

Myeloblast

Limphoid progenitor

Erythrocyte

Myelocyte

Hepatocyte

Pancreatic cell

Intestine epithelial cells

NK cell

T cell

B cell

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Myocyte

Adipocyte

Osteocyte

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Gb3 Gb4 SSEA-3 fucosyl-Gb5 (Globo H)

sialyl-Gb5 (SSEA-4)disialyl-Gb5 Globo-ASSEA-1 LC3 LC4

fucosyl-LC4

GM3 GM1 GD3 GD2 GM3 GM1a GD1a GD1b GD2 GD3

GM3 GM1 GD2 GD3

GD3 GalCer

SM4 GalCer SMSSEA-1 SSEA-4

GM1 GD1a GT1b

GM1 sialyl-Le x

GD1a GD1b GT1b GQ1b

Axon

Neuron

YYYYYY

YY YY

GalCer SM4 SM

GT1b GQ1b

GM3 GM1a GD3Le x Gb3 Gb4

SSEA-4 GM1 GD1a GD2

GM3 GD1a

GM3 GD3

asialo-GM1 GM1

GM3 Gb3 Gb4

Globo H

GM3 GD1a GM1 asialo-GM2 Gb3

Gb3 Gb4 LC3 nLC4 LC4 Le x sialyl-Le x asialo-GM1 asialo-GM2

Le a Le b Gb4 Gb3Forssman antigen GD1a

iGb3

GM3 GM1 Gb3

CeramideGM3 GD3 Gb3 Gb4

fucosyl-Gb5 (Globo H)Gb4 LC4 fucosyl-LC4

GM3

GM1

Fig. 3. Change of GSL expression profile during differentiation of pluripotent stem cells. Schematic representation of the production of GSLs during thedifferentiation of pluripotent stem cells into definitive endoderm, mesoderm, ectodermal lineages or primordial germ cells (each lineage is represented by adifferent shade of gray, going from light to dark gray respectively). Detailed information on the changes in the GSL composition during differentiation that arerepresented in the figure are reported in Table S1 and references therein.

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Similarly, during EMT, the production of GSLs is switched from theasialo to ganglio series (Fig. 4A) owing to the induction ofST3GAL5 and ST8SIA1 (encoding GD3 synthase) and to therepression of B3GALT4 (encoding GA1/GM1 synthase) (Mathowet al., 2015). These data suggest that dedicated regulatory circuitsexist to reorient the GSL pathways.During TGFβ-induced EMT, the mothers against

decapentaplegic homolog 3 and 4 (Smad3–Smad4) complexrepresses B3GALT4 by binding to its promoter (Guo et al., 2015),whereas zinc finger E-box-binding homeobox 1 (Zeb1) (atranscriptional target of Smad3–Smad4) binds to and activates thepromoters of both ST3GAL5 (Mathow et al., 2015) and ST8SIA1(Dae et al., 2009). Importantly, exogenous provision of GA1 (one ofthe products of B3GALT4/GA1 synthase) inhibits TGFβ-inducedEMT, which suggests that GSLs are both targets and regulators ofthe same signaling pathway (Guan et al., 2009; Guo et al., 2015)(Fig. 4A). Moreover, we recently demonstrated that, during

differentiation of stem cells into neural cells, the decrease inglobo-series GSLs (owing to A4GALT/Gb3 synthase repression)triggers the expression of the chromatin-remodeling factor autismsusceptibility gene 2 protein (AUTS2), which, in turn, binds to theST3GAL5 promoter where it stimulates local histone acetylation andtranscriptional activation of ganglio-series GSLs (Russo et al.,2018). Similar to what is seen during EMT, the addition ofGSLs that are repressed in differentiated cells (globo-series GSLs)counteracts both the differentiation process and metabolicreprograming (Russo et al., 2018) (Fig. 4B). Interestingly,ganglio-series GSLs (i.e. GM1) have been found to stimulateneuronal differentiation and to sustain the expression of enzymesthat synthesize ganglio-series GSLs (i.e. GM2S) by promotinghistone acetylation at their promoters (Tsai et al., 2016; Tsai and Yu,2014), which ultimately leads to maturation of the neuronalpopulation (Fig. 4C). An increase in histone acetylation at theGM2S (B4GALNT1) promoter was indeed observed in developing

Table 1. Glycosphingolipids as TAAs

Tumor-associated GSL antigens

GSL antigen Cancer type Reference

Globo-series GSLs Gb3 Burkitt lymphomaOvarian cancerColon cancerGastric adenocarcinoma

Farkas-Himsley et al., 1995Arab et al., 1997Kovbasnjuk et al., 2005Geyer et al., 2016

SSEA-3 Breast cancer Chang et al., 2008

Globo H Breast cancerOvarian cancerThyroid carcinoma

Chang et al., 2008Hakomori, 1989Cheng et al., 2016

DSGG Renal cell carcinomaHepatocellular carcinoma

Satoh et al., 1996Wu et al., 2012

SSEA-4 Breast cancerGlioblastoma

Aloia et al., 2015Lou et al., 2014

Ganglio-series GSLs GM3 Acute myeloid leukemia (AML)MelanomaMultiple cancer types

Wang et al., 2012Guthmann et al., 2004Zheng et al., 2018

GM1 Lung cancer Fuentes et al., 1997

Fucosyl-GM1 Hepatocellular carcinomaSmall cell lung carcinoma

Wu et al., 2012Nilsson et al., 1986

GD3 MelanomaNeuroblastomaLung cancer

Cheresh et al., 1985Cheresh et al., 1986Fuentes et al., 1997

GD2 MelanomaNeuroblastoma

Navid et al., 2010Yang and Sondel, 2010

Lacto-series GSLs Lex Breast cancerColon cancerGastric cancer

Hakomori, 1989Hakomori, 1989Hakomori and Zhang, 1997

Lex- Lex Breast cancerColon cancerGastric cancer

Hakomori, 1989Hakomori, 1989Hakomori and Zhang, 1997

Lea- Lea Colon cancerGastric cancer

Hakomori, 1989Hakomori and Zhang, 1997

Ley- Lex Pancreatic cancerMultiple cancer types

Kim et al., 1988Zheng et al., 2018

SSEA-1 Renal cancer Liebert et al., 1987

Lc3 Acute myeloid leukemia (AML)Ovarian cancer

Wang et al., 2012Alam et al., 2017

nLC4 Acute myeloid leukemia (AML) Wang et al., 2012

Sialylated-Lewisa (sLea) Colon cancerPancreatic cancer

Hakomori, 1989Hakomori and Zhang, 1997

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mouse brains, where it correlates with GM2S mRNA expression(Suzuki et al., 2011). This evidence reveals the existence of a two-way relationship between GSL metabolism and transcriptionalprograms that affect cell fate determination.How exactly do GSLs influence gene expression? One

fundamental feature of GSLs is to organize protein–carbohydrateor carbohydrate–carbohydrate interactions with structural proteinsand receptors at the PM (Chakrabandhu et al., 2008; Coskun et al.,2011; Kawashima et al., 2009; Liang et al., 2017; Liu et al., 2004;Mutoh et al., 1995; Park et al., 2012; Russo et al., 2016). The GSL-glycan moiety can, indeed, interact directly with a specific aminoacid residue (Coskun et al., 2011) within a protein domain, or witha glycan portion (Heuss et al., 2013) of PM proteins. In doing so,GSLs regulate PM proteins through (1) their conformation,

(2) their accessibility to ligands, (3) their oligomerisation stateand/or (4) their partitioning into membrane microdomains (Russoet al., 2016).

Thus, GSLs can modify cell signaling in response to specificstimuli. Examples for this are the interactions of GSLs withEGFR (Hofman et al., 2008; Park et al., 2017; Coskun et al., 2011)and the notch ligand delta-like 1 (Dll1) (Heuss et al., 2013). Here,their signaling – and as a consequence, the downstreamtranscriptional responses – are influenced by their interaction withGSLs at the PM. GSLs also affect signal transduction and geneexpression by regulating endocytosis (Lakshminarayan et al., 2014).GSLs interact with the secreted carbohydrate-binding proteingalectin-3, which in turn triggers GSL-dependent biogenesis ofspecific cargo-laden endocytic carriers (Lakshminarayan et al.,

A

B

C

Gb3 AUTS2

AUTS2 promoter

Repressive histone modifications

GM3S promoter

Day 0 Day 13

Gb3

AUTS2

AUTS2 promoterActive histone modifications

GM3S promoter

Neuronal differentiation

Globo-seriesGSLs

Neuronal maturationImmature neurons Mature neurons

Active histone modifications

GM3GM2 GM1a

GM1SGM3

GM2 GM1a

GM2SmRNA

GM1S

GM2S promoter

GM2SmRNA

GM2S promoter

Complex ganglio-series

GSLs

Simpleganglio-series

GSLs

EMTEpithelial phenotype Mesenchymal phenotype

LacCer GA2

GM2S

GM3S promoter GM3

GM3SmRNA

Asialo-series GSLs

LacCer GA2

GM2S

GM3S promoterGM3

TGFb1

GM3SmRNA

Ganglio-series GSLs

Sia Glu GlcNac Gal GalNac Cer

Ganglio-series GSLs

Asialo-series GSLs

GM3Senzyme

GM3Senzyme

ZEB1

GM3SmRNA

GM3Senzyme

GM3

Ganglio-series GSLs

GM2Senzyme

GM2Senzyme

GA1

GA1S

GA1

GA1S

Key

Fig. 4. Regulatory circuits for GSL expression andmetabolism. (A) The asialo- to ganglio-GSL switch during EMT. During the acquisition of themesenchymalphenotype, which is induced by TGFβ1, the transcription factor Zeb1 binds theGM3S (ST3GAL5) promoter and activates its transcription. Increased expression ofGM3S and the concomitant suppression of GA1S (B3GALT4) tip GSL metabolism toward the synthesis of ganglio-series GSLs. (B) The globo- to ganglio-GSLswitch during neuronal differentiation. In parallel with the decrease in the globo-series GSL, the epigenetic regulator AUTS2 is induced. AUTS2, in turn, bindsand activates the promoters of neuronal genes – and of GM3S – inducing GM3S gene expression and favoring neuronal differentiation. (C) Establishmentof complex ganglio-series GSL synthesis during neuronal maturation. GM1 fosters the synthesis of complex gangliosides and neuronal maturation bypromoting the expression of the upstream ganglio-series GSL-synthesizing enzymes (GM2S).

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2014). Through this mechanism, GSLs regulate the exposureof specific receptors and proteins at the PM. Another level of actionfor GSLs on gene expression is directly within the nucleus, whereGSLs have been found at the nuclear lamina where they directlycontact chromatin and influence the activity of promoters (Tsaiet al., 2016).Thus, the impact of GSLs on signal transduction pathways and on

transcriptional programs upon their activation (Regina Todeschiniand Hakomori, 2008). As outlined above, the transcriptionalresponses to changes in cellular GSL content often involveenzymes of the GSL synthetic pathway itself, thus resulting inself-contained regulatory loops that lead to metabolic switches. Thefinal output of these transcriptional metabolic interplays influencescell fate decisions and differentiation programs.

Conclusions and future directionsGlycans of GSLs protrude out of the PM towards the extracellularspace. This peculiar position makes GSLs specifically suited tointeract with glycans and proteins that are present either on the samePM or on the PM of adjacent cells. Through these interactions,GSLs influence signaling, receptor trafficking, cell–cell contactsand adhesion, and, thus, ultimately gene expression and cell fatedetermination. The structural diversity in GSLs and their tissue-specific production suggest that distinct GSLs influence cell fatedecisions towards differentiation trajectories.During development and tissue pattering, individual progenitor

cells are subjected to specific differentiation programs in order toachieve the formation of functional anatomical structures throughmorphogenesis (Basson, 2012). Whereas specific hormonegradients sustain morphogenetic processes, the events that initiatemorphogenesis usually happen in a uniform context, that is, amongundifferentiated and genetically identical cells that are exposed to ahomogeneous environment (Tabata and Takei, 2004).

Cell-to-cell variability in gene expression (either stochastic ordependent on the cellular microenvironment) has been proposed todrive these early morphogenetic events by changing celldifferentiation potential (Huang, 2009). This provides otherwiseidentical cells with the capability to break symmetry within thepopulation (Huang, 2009) (Fig. 5). According to this concept,progenitor cells can follow alternative differentiation trajectories toachieve one of multiple stable states; this eventually leads to cellfate decisions that depend on the oscillating expression of a keyfactor (Huang, 2009). Besides proteins and nucleic acids, smallmolecules, which include lipids and, specifically, GSLs, are ableto influence these cell differentiation programs. Moreover, GSLcomposition varies among cells in a syngeneic cell populationowing to cell cycle phase (Majoul et al., 2002), the localmicroenvironment (Snijder et al., 2009) or to metabolic circuits(Russo et al., 2018). Whether this variability is involved insymmetry-breaking events in morphogenesis remains to beaddressed (Fig. 5).

Research devoted to the dissection of the role for GSLsin regulatory gene expression circuits at single-cell resolution inadvanced models of development and morphogenesis (i.e. inorganoids) is probably the missing and required step to attain asufficient body of knowledge on the role of GSLs in development,morphogenesis and tissue patterning.

Competing interestsThe authors declare no competing or financial interests.

FundingG.D.A. acknowledges the financial support of Associazione Italiana per laRicerca sul Cancro (AIRC) (MFAG 10585), of the Italian Ministry of Health(GR-2011-02352256) and of Ministero dell’Istruzione, dell’Universita e della Ricerca(MIUR) (PON_00862). J.S.L. is supported by the H2020 Marie Skłodowska-CurieActions INCIPIT PhD program.

Key

Pluripotent stem cells

Stable equilibrium

Multiple attractor states

Symmetry breaking

event

Symmetrical cell division

Pluripotent daughterstem cells

Differentiated cells

Pluripotent stem cells

Asymmetrical cell divisionPluripotent daughter

stem cells

Committed cell progenitors

GSL profile of Pluripotent stem cells

GSL profile of Progenitorsand differentiated cells

Fig. 5. Symmetry breaking in cell differentiation. During stem cell differentiation, symmetrical or asymmetrical cell divisions occur. Symmetrical cell divisionssustain the maintenance of pluripotent stem state whereby a stable transcriptional and epigenetic profile is inherited by pluripotent daughter stem cells in a‘stable equilibrium’. Asymmetrical cell divisions lead to the generation of cell progenitors diverging toward multiple differentiation trajectories due to unevenpartitioning of key differentiation factors between the two daughter cells. Here, destabilized daughter cells transition towards new stable transcriptional andepigenetic arrangements (i.e. attractor states). GSLs influence differentiation programs, and their asymmetrical segregation might sustain bifurcation eventsin cell fate decisions.

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Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.219204.supplemental

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