Synergistic regulation of Notch signaling by different O-glycans promotes hematopoiesis

Glycosylation of Notch receptors by O-fucose glycans regulates Notch ligand binding and Notch signaling during hematopoiesis. However, roles in hematopoiesis for other O-glycans that modify Notch receptors have not been determined. Here we show that the EGF domain specific GlcNAc transferase EOGT is required in mice for the optimal production of lymphoid and myeloid cells. The phenotype of Eogt null mice was largely cell-autonomous, and Notch target gene expression was reduced in T cell progenitors. Moreover, EOGT supported residual Notch signaling following conditional deletion of Pofut1 in hematopoietic stem cells (HSC). Eogt : Pofut1 double mutant HSC had more severe defects in bone marrow and in T and B cell development in thymus and spleen, compared to deletion of Pofut1 alone. The combined results show that EOGT and O-GlcNAc glycans are required for optimal hematopoiesis and T and B cell development, and that they act synergistically with POFUT1 and O-fucose glycans to promote Notch signaling in lymphoid and myeloid differentiation.


Introduction
Notch signaling is highly conserved and plays crucial roles in cell fate determination and tissue development (1).There are four Notch receptors (NOTCH1 to NOTCH4) that can be activated by canonical Notch ligands (DLL1, DLL3, DLL4, JAG1 and JAG2) to induce Notch signaling in mammals.Notch ligand binding and Notch signaling are regulated by glycosylation of the extracellular domain (ECD) of Notch receptors (2,3).
Structural studies reveal direct interactions between Notch ligands and O-fucose in specific epidermal growth factor-like (EGF) repeats of NOTCH1 (4,5).Following Notch ligand engagement, an ADAM metalloprotease cleaves NECD, followed by a second cleavage by the g-secretase complex.Notch intracellular domain (NICD) complexes in the nucleus with the transcriptional repressor RBP-Jk, and co-activators including Mastermind-like-1 (MAML1), to induce the expression of Notch target genes, including Hairy enhancer-of-split (Hes) and Hairy-related gene families, which regulate the expression of many other genes (6,7).Within the hematopoietic system, Notch signaling plays important roles in regulating different stages of lymphoid and myeloid development (8,9).DLL4-induced NOTCH1 signaling is indispensable for T cell development in the thymus.Thus, conditional inactivation of Notch1 or Dll4 using Mx1-Cre is sufficient to block T cell development (10,11).However, Notch2 is also required for optimal development of early T cell progenitors (12).DLL1-induced NOTCH2 signaling is essential for the generation of marginal zone B cells (MZ-B) in the spleen (13,14).O-fucose glycans extended by LFNG and MFNG promote the formation of MZ-B cells (15) and all three FNG genes contribute to T and B cell development (16).
Consensus sites within EGF repeats in the NECD of Notch receptors carry O-fucose, O-glucose and O-GlcNAc glycans (17) (Figure 1).EGF repeats with appropriate consensus sites occur in ~50 proteins of the proteome, including Notch receptors and Notch ligands (19,20).O-fucose is transferred by protein Ofucosyltransferase 1 (POFUT1), which is further extended by the Fringe family of glycosyltransferases (LFNG, MFNG and RFNG).Misexpression of Lfng in thymus disrupts T cell development (21)(22)(23).Conditional deletion of Pofut1 in the bone marrow leads to the disruption of hematopoiesis with an increase in granulocytemonocyte progenitors (GMP), and a reduction in common myeloid progenitors (CMP) (24).This causes a block in T cell production in thymus and MZ-B cell production in spleen, accompanied by an increase in granulocytes in spleen.Notch  The addition of O-GlcNAc to EGF repeats was first identified in Drosophila (36), and the EGF domain specific O-GlcNAc transferase EOGT was subsequently revealed (37,38).Eogt null mice exhibit defective perinatal retinal angiogenesis, similar to that observed in mice with disrupted Notch signaling (39).Cell-based experiments showed that EOGT promotes the binding of DLL Notch ligands and Notch signaling (39).Eogt is expressed with Pofut1 in many cells of the immune system (Figure 1).However, their expression levels vary greatly and inversely in some immune cell types, indicating potentially different functional roles (Figure 1).In this paper, we identify roles for Eogt in the regulation of Notch signaling during hematopoiesis, lymphopoiesis and myelopoiesis.In addition, we show that Eogt supports Notch signaling in the absence of Pofut1 and O-fucose glycans.

Mice
Mice with an inactivating mutation in the Eogt gene were generated at Nagoya University and previously described (39).Pofut1[F/F] mice were also previously described (26).Transgenic mice expressing Vav1-iCre (B6.Cg-Commd10 Tg(Vav1-icre)A2Kio /J Strain) were a kind gift from Britta Will and Paul Frenette at the Albert Einstein College of Medicine, NY, USA.Compound mutant mice termed Pof cKO and EPof dKO, with and without Vav1-iCre, were generated by intercrossing.C57Bl/6J male congenic mice expressing CD45.1 (B6.SJL-Ptprc a Pepc b /BoyJ #002014) were obtained from the Jackson Laboratory (Bar Harbor).Genotyping was performed by PCR of genomic DNA using primers that distinguish wild-type and mutant alleles (Supplementary Table 1).Mice were housed in a barrier facility, allowed to eat and drink ad libitum, and used in experiments at 7-8 weeks of age.All experiments were performed with permission from the Albert Einstein Institutional Animal Care and Use Committee under approved protocol numbers 20170709 and 00001311.Euthanized mice were weighed, bone marrow from femurs, tibias, fibulae, & patullae was isolated, thymus and spleen were weighed before making single-cell suspensions.

Antibodies
Supplementary Table 2 has the full description and commercial source of each antibody used in this work.

Flow cytometry of immune cell subsets
Single-cell suspensions were prepared from bone marrow (BM) by crushing femurs, tibias, fibulae, and patullae in a mortar and rinsing vigorously with 20 ml cold FACS binding buffer (FBB: Hank's balanced salt solution (HBSS) with glucose (Corning), 1 mM CaCl 2, 2% bovine serum albumin (BSA, fraction V, Sigma) and 0.05% sodium azide, (pH 7.2-7.4).The BM cell suspension was passed through a 70-mm strainer.Thymus or spleen was weighed and squeezed through a 70 mm strainer in 1 ml FBB.Thymocytes were washed in 10 ml cold FBB twice.Bone marrow and splenocytes were centrifuged and incubated in 1 ml RBC lysis buffer (eBiosciences) for 3-5 min before adding 10 ml cold FBB.After centrifugation and resuspension in 5 ml cold FBB, single cell suspensions were counted in a Coulter counter.Cells were centrifuged at 4°C, resuspended in 2 ml cold PBS with 1 mM CaCl 2 and 1 mM MgCl 2 , pH 7.2, centrifuged and resuspended in 100 ml Zombie NIR dye for live/dead assessment, according to the manufacturer's protocol (Zombie NIR Fixable Viability Kit, BioLegend).After 30 min at 4°C in the dark, 2 ml cold FBB was added.Cells were centrifuged at 4°C, resuspended in 4% paraformaldehyde (PFA, Emsdiasum) in PBS pH 7.2, and incubated 15 min at 4°C in the dark.Cells were washed twice with 2 ml cold FBB, resuspended at 10 6 cells/ml in FBB and stored at 4°C for up to 3 months.For analysis by flow cytometry, ~10 6 cells were washed with 1 ml FBB, resuspended in 90 ml FBB containing 1 ml Fc block (rat-anti-mouse CD16/CD32), and incubated for 15 min on ice.Abs diluted in FBB (10 ml) were added and the reaction mix was incubated for 30 min at 4°C.Cells were washed twice in 1 ml FBB and resuspended in ~500 ml FBB.For all samples, immunofluorescence was analyzed using a Cytek ™ Aurora or BD LSRII flow cytometer and data FCS files were analyzed using FlowJo software (Tree Star).Gating strategies shown in Supplementary Figures 1-3 were based on previous work (16,40,41) and include the Abs used in the analysis of BM, thymus or spleen immune cell subsets.Data on live cells were collected as frequency percent based on gating strategies.Absolute cell numbers in the different immune cell subsets were calculated from the starting number of live cells and the frequency percent of each subset.Significant differences between genotypes in either frequency percent or absolute cell number are highlighted in the main text while complementary data are presented in supplementary figures as described in the text.

Bone marrow transplantation
Cell suspensions from bone marrow of 7-8 week Eogt[+/-] and Eogt[-/-] males were prepared as described above.Approximately 3x10 6 cells were resuspended in 50 ml sterile HBSS (Gibco) and injected using a 28-gauge insulin needle via the retro-orbital plexus into CD45.1+C57BL/6 lethally irradiated recipients.A split dose of 550 rads g-irradiation per recipient male was given twice, with a 16 h interval.After 7 weeks, recipients were euthanized, bone marrow, thymus, and spleen were analyzed for lymphoid and myeloid cell subsets by flow cytometry after gating on donorderived cells positive for anti-CD45.2-PacificBlue.

Isolation of CD4/CD8 double negative T cells
Fresh thymocytes were resuspended in isolation buffer (PBS lacking cations, pH 7.2-7.4,containing 0.1% BSA, 2 mM EDTA and 1 mg/ml glucose) on ice.For T cell depletion, ~3-5x10 7 thymocytes were incubated with 20 mg anti-CD4 (rat IgG2b clone GK1.5, BioXCell) and 37.5 mg anti-CD8a (rat IgG2a clone 53-6.72;BioXCell) in 5 ml buffer for 20 min at 4°C with tilted rotation.After centrifugation, cells were resuspended in 5 ml buffer, and incubated with 250 ml sheep anti-rat IgG Dynabeads (Thermo Fisher Scientific) for 30 min at 4°C with tilted rotation.The tube was placed in a magnet for 2 min, unbound cells were centrifuged and resuspended in 250 ml Dynabeads for a second 30 min incubation at 4°C.After Dynabeads removal, unbound DN T cells were centrifuged, counted and RNA was extracted from the cell pellet with 1 ml TRIZOL (Ambion) as described below.
Quantitative RT-PCR DN T cells from ~3-5x10 7 thymocytes were pipetted vigorously in 1 ml TRIZOL and incubated for 5 min at RT before adding 200 ml chloroform.Tubes were vortexed for 15 sec, incubated at RT for 2-3 min, and centrifuged at 12,000 × g for 15 min at 4°C.The aqueous phase was transferred to a new tube, and 500 ml isopropanol was added.Samples were incubated for 10 min on ice and centrifuged at 12,000 × g for 10 min at 4°C and the supernatant discarded.The RNA pellet was washed once with 1 ml 70% ethanol.Samples were vortexed for 10 sec and centrifuged at 7,500 × g for 5 min at 4°C and the supernatant discarded.The RNA pellet was air-dried for 5-10 min and dissolved in 50 µl RNase-free water.Samples were placed in a 55-60°C water bath for 15-20 min and the RNA concentration determined by Nanodrop.cDNA was prepared from ~250 ng RNA using the ReverTra Ace ® qPCR RT Master Mix with gDNA Remover (Dc.DiagnoCine) following the manufacturer's protocol.Each sample was assayed in triplicate using a 384 well plate.Relative gene expression was calculated in relation to Hprt and Gapdh by the log2 dCCT method.Primer sequences used for qRT-PCR are in Supplementary Table 1.

Histopathology
Spleen was collected, weighed, washed and stored in 10% natural buffered formalin (NBF) at 4°C.The samples were processed for paraffin embedding and longitudinal tissue sections (5 mm) were stained with hematoxylin and eosin (H&E), scanned by the 3D Histech P250 High-Capacity Slide Scanner and analyzed using Case Viewer 2.4 software.

Statistics
Comparisons are presented as mean ± SEM.Significance was determined by both two-tailed and one-tailed (denoted by parentheses) unpaired, parametric, Student t-test analysis (unless otherwise noted) using Prism software version 9.1.

Loss of Eogt affects myelopoiesis and lymphopoiesis
Initial comparisons of Eogt[+/+] and Eogt[+/-] heterozygotes revealed no significant differences in T, B and myeloid subset populations in either absolute cell numbers or in the frequency % of different cell subsets (Supplementary Figure 4 and not shown).Therefore, data from Eogt[+/+] and Eogt[+/-] mice were combined as Control.Thymus and spleen weights, as well as bone marrow (BM) cellularity, were similar in Eogt null and Control mice (Supplementary Figure 5A).In bone marrow, the absolute numbers of CD19+/B220+ B cells, and CD11b+/Gr1+ granulocytes were significantly increased in the Eogt[-/-] population (Figure 2A).In Eogt[-/-] thymus, the frequency % of CD4/CD8 double negative 1 (DN1) T cell progenitors was reduced, although the absolute number Frontiers in Immunology frontiersin.org of DN1 T cells was unchanged (Supplementary Figure 5B).The absolute number of DN2 T cell progenitors was strikingly reduced in Eogt null cells, while the absolute number of DN4 T cells was significantly increased (Figure 2B).By contrast, the numbers of early T cell progenitors (ETP) and DN3 T cells were unchanged (Supplementary Figure 5B).The proportion of double positive (DP) T cells was slightly decreased, while CD4+ and CD8+ single positive (SP) T cells were increased in Eogt[-/-] thymocytes (Figure 2B).These effects were also observed in mice lacking the three Fringe genes (Fng tKO) (16, 42).Consistent with inhibition of Notch signaling in the Eogt null thymus, there was a significant increase in thymic B cells (CD19+/B220+) and the frequency of myeloid cells (CD11b+) (Figure 2B), but no change in natural killer (NK) T cells (Supplementary Figure 5B).In spleen, no histopathological changes were observed in Control versus Eogt[-/-] sections (n=3 for each, not shown), and the absolute numbers of T cells, B cells, marginal zone progenitors (MZ-P) and myeloid cell subsets were similar in Control versus Eogt[-/-] mice (Supplementary Figure 5C).However, significant increases were observed in the absolute number of follicular B (Fo-B), MZ-B, CD19+ and B220+ B cells in Eogt[-/-] mice (Figure 2C).By contrast, a decrease in the frequency % of natural killer T cells (NK1.1+) and dendritic cells (CD11b/c+) was observed (Figure 2C).Thus, EOGT and O-GlcNAc glycans are required for the optimal generation of lymphoid and myeloid cells from HSC.

Notch signaling is reduced in Eogt[-/-] DN T cell progenitors
Notch ligand binding was examined using thymic DN T cell progenitors from 7-8 week Eogt[+/-] and Eogt[-/-] mice.No significant changes were observed in either NOTCH1 cell surface expression, or the binding of soluble ligands DLL1, DLL4, JAG1 or JAG2 between Eogt[+/-] and Eogt[-/-] DN T cell progenitors (Figure 4A).However, there were significant reductions in the expression of two Notch target genes, Hes1 and Il2ra, consistent with reduced Notch signaling (Figure 4B).The expression of Il2ra was also reduced in DN T cell progenitors from mice lacking the three Fringe genes, along with Dtx1, although not Hes1 (16).The reduction in Notch signaling target gene expression in Eogt null T cell progenitors, the changes in T cell subset numbers and frequencies, and the increased numbers of B and myeloid cells observed in Eogt[-/-] thymus, indicate that EOGT and O-GlcNAc glycans are necessary for optimal Notch signaling and T cell development.The altered B cell and myeloid subsets in spleen of Eogt[-/-] mice are also consistent with reduced Notch signaling.Finally, the B cell and myeloid hyperplasia in Eogt[-/-] BM indicate that Notch signaling is required for regulating the differentiation of certain progenitors during hematopoiesis.

Eogt supports lymphoid and myeloid development in HSC lacking Pofut1
Previous work showed that inactivation of Pofut1 using Mx1-Cre causes a reduction in T lymphopoiesis in thymus and myeloid hyperplasia in bone marrow (24).In another study, conditional deletion of Pofut1 in bone marrow was shown to cause a milder T cell phenotype than deletion of RBP-Jk (25).To determine whether Eogt and O-GlcNAc glycans support Notch signaling and hematopoiesis in the absence of Pofut1, we used Vav1-iCre transgenic mice to generate conditional deletion (cKO) of Pofut1 (henceforth referred to as Pof cKO), and deletion of both Eogt and Pofut1 (henceforth referred to as EPof dKO), in hematopoietic stem cells.The absolute number of BM cells was significantly increased in both Pof cKO and EPof dKO mice (Figure 5).Gating strategies used to define HSC, HSPC, myeloid and lymphoid cell subsets in BM are shown in Supplementary Figure 6.Short-term (ST)-HSC, and LSK (Lineage-Sca1+cKit+) cell numbers were increased in both Pof cKO and EPof dKO mutants, but the frequency % of HSPCs was increased only in EPof dKO BM (Figure 5).By contrast, the frequency % of MPP subsets was reduced except for a small increase in the MPP3 population in EPof dKO BM (Figure 5).The frequency % of lymphoid primed multipotent progenitor cells (MPP4/LMPP) and common myeloid precursors (CMP) were reduced in Pof cKO and further reduced in EPof dKO BM, and megakaryocyte erythrocyte progenitors (MEP) were decreased in both single and double mutants (Figure 5).By contrast, the frequency % of granulocyte-monocyte progenitors (GMP) was increased in Pof cKO (as observed previously (24)), and further increased in EPof dKO BM (Figure 5 and Supplementary Figure 7).The proportion of CD11b+/Gr-1+ granulocytes was increased, but the proportion of common lymphoid progenitors (CLP) was reduced in EPof dKO BM (Figure 5).The frequency % of T-and B-cells in BM was also decreased in EPof dKO mice (Figure 5).NOTCH1 was expressed at the surface of Lin-Sca1+ cells which were increased in frequency in Pof cKO and EPof dKO BM (Supplementary Figure 8).Binding of DLL1 and DLL4 Notch ligands was low and similar in control and mutant Sca1+ cells (Supplementary Figure 8).

Highly disrupted development of T cells in EPof dKO thymus
Deletion of Pofut1 in HSC via Vav1-iCre led to a marked decrease in thymus weight and a similar reduction was observed in EPof dKO thymus (Figure 6A).The reduced size was accompanied by a dramatic change in T cell maturation (Figure 6B).Early thymic progenitors (ETP) were greatly decreased in absolute number, and each DN T cell progenitor population (DN1 to DN4) was also reduced in absolute cell number in Pof cKO, and even further reduced in EPof dKO thymocytes (Figure 6C and Supplementary Figure 9).Interestingly, however, the frequency % of DN4 T cells in both single and double mutant thymocytes was increased relative to control (Supplementary Figure 9), as observed in thymus lacking all Fringe activities (16).The DN T cell population, which includes few DN T cell progenitors and all the non-T cell populations, was greatly increased in Pof cKO and EPof dKO, as expected.This reflected loss of Notch signaling leading to the generation of B cell, myeloid and NK cell subsets in thymus (Figure 6D and Supplementary Figure 9).Notably, effects were more severe in EPof dKO than Pof cKO thymus, suggesting that the loss of Notch signaling was greater in EPof dKO thymus.

Defective B, T and myeloid cell development in EPof dKO spleen
Both Pof cKO and EPof dKO mice had an enlarged spleen (Figure 7A).Absolute numbers of splenocytes and spleen weight were increased in Pof cKO, and further increased in EPof dKO mice (Figure 7B).Histological analysis revealed extramedullary hematopoiesis in both Pof cKO and EPof dKO spleens, with larger areas of extramedullary hematopoiesis observed in EPof dKO spleen (Figure 7A).
The absolute numbers of single positive CD4+ and CD8+ T cells were significantly reduced in Pof cKO, and further reduced in EPof dKO spleen (Figure 7C and Supplementary Figure 10).While CD19+ and B220+ B cells were proportionally increased in both Pof cKO and EPof dKO spleen, the frequency % of the CD19+/B220+ B cell population was reduced (Figure 7D).The frequency of Fo-B and the number of MZ-B cells were also reduced (Figure 7D and Supplementary Figure 10).While the absolute number of MZ-P cells did not change in the mutants, the frequency % of MZ-P precursors was increased in EPof dKO spleen (Figure 7 and Supplementary Figure 10).Myeloid cell subsets such as dendritic cells (CD11b/c+) and Gr-1+ granulocytes were increased in greater proportion in EPof dKO compared to Pof cKO spleens.Natural killer T cells were reduced in frequency in both Pof cKO and EPof dKO spleens (Figure 7D and Supplementary Figure 10).

Discussion
Defining specific roles for the glycans that regulate Notch signaling in lymphoid and myeloid development facilitates our understanding of cell fate decisions controlled by Notch signaling, and of potential consequences for people with congenital diseases that perturb Notch signaling (2,43).Several congenital diseases inhibit the synthesis or extension of O-glycans that regulate Notch signaling and may induce immune cell defects.Mutations in EOGT cause Adams Oliver syndrome (44) and autosomal dominant mutations in POFUT1 cause Dowling Degos Disease 2 (DDD2) (45).Here we show that the generation of certain lymphoid and myeloid subsets in bone marrow, thymus and spleen was perturbed in mice lacking Eogt.In bone marrow, loss of EOGT caused increased numbers of B cells and granulocytes.In thymus, the phenotype of Eogt null mice was similar, but not identical to, mice which lack all three Fringe genes (Fng tKO) (16).T cell progenitors DN1 and DN2 were reduced in frequency or absolute number, whereas DN4 T cell progenitors were increased, DP T cells were slightly reduced in frequency, and CD4+ and CD8+ SP T cells were increased in absolute numbers.Inhibition of Notch signaling in thymus is well known to lead to the generation of B cells and myeloid cells in thymus (9,11,46,47).Eogt null thymus contained significantly increased numbers of B cells and granulocytes, and an increased frequency of myeloid cells.In spleen, Eogt null splenocytes included increased numbers of several B cell subsets, although there were no effects on T cells, unlike in Fng tKO mice which had a reduced frequency of T cells in spleen (16).Deletion of RBP-Jk by Vav1-iCre results in an increased frequency of CD19+ B cells (48), also observed in Eogt null spleen.Eogt null mice showed increased absolute numbers of CD19+ B cells, increased Fo-B cells, and a decrease in natural killer T-cells and dendritic cells in the spleen.The overall Eogt null phenotype was largely cell autonomous following bone marrow transplantation.In addition, expression of Notch target genes Hes1 and Il2ra was reduced, similar to Fng tKO DN T cell progenitors that had reduced Il2ra and Dtx1 expression (16).The combined data provide strong evidence that EOGT and O-GlcNAc glycans are required for optimal Notch signaling in the development of lymphoid and myeloid cells from HSC.
Further evidence that EOGT and O-GlcNAc glycans support Notch signaling in lymphoid and myeloid development was obtained in compound mutant mice lacking Eogt and conditionally lacking Pofut1 in HSC.EPof dKO lymphoid and myeloid populations in BM, thymus and spleen were more affected compared to Pof cKO.We conclude that the loss of Eogt and O-GlcNAc glycans in EPof cKO HSC exacerbated the deficits in T, B and myeloid differentiation evident in Pof cKO mice.This result, and our findings that Eogt is required for optimal Notch signaling and the differentiation of HSC, provide an explanation for the observation that Pofut1:Mx1-Cre T cell deficiencies were not as severe as those obtained in RBP-Jk : Mx1-Cre thymus (25).In the absence of POFUT1 and O-fucose glycans, the O-GlcNAc glycans transferred by EOGT to Notch receptors can support a low but significant level of Notch signaling.Thus, as shown here and discussed in a recent review (49), the O-fucose and O-GlcNAc glycans on Notch act synergistically to provide optimal Notch signaling in lymphopoiesis and myelopoiesis.
The conclusions obtained from these experiments are necessarily limited by the difficulty of demonstrating structural changes in the Oglycans on Notch receptors predicted to change in the absence of relevant glycosyltransferase(s).For example, to obtain sufficient NOTCH1 from splenic T cells for analysis by mass spectrometry, it was necessary to activate the T cells in culture thereby increasing cell surface NOTCH1 by ~10-fold (50).Furthermore, the effects of conditional deletion of Eogt in HSC would allow us to define the contribution, if any, of Eogt null stroma to the Eogt null phenotype.It would also be important to determine Notch ligand binding and Notch target gene expression in cells from different HSPC lineages in Pof cKO and EPof dKO mice.Single cell RNA-seq of different mutant HSC and HSPCs would greatly expand our understanding of the pathways affected by altered Notch signaling due to loss of regulation by O-glycans in HSC.Moreover, it would be important to investigate roles for the O-glycans on Notch ligands by deleting Pofut1, Eogt and related glycosyltransferase genes in stromal cells.Another important question for future work is whether changes in immune subset cell numbers reflect apoptosis or proliferation of one or more populations.This question and changes in Notch target gene expression are difficult to determine because changes in Notch signaling lead to changes in cell fates.For example, thymus lacking POFUT1 or EOGT + POFUT1 has few, if any, T cell progenitors and largely comprises B cells, myeloid and NK cells making comparisons with wild type or Eogt null thymocytes meaningless in terms of underlying mechanisms.Thus, the major benefit of the experiments reported here is to pinpoint cell fate decisions regulated differently by the strength of Notch signaling which in turn is regulated by the Oglycans of Notch receptors.In addition, our findings contribute to the growing body of evidence that glycans are integral to the functions of all glycoproteins, glycolipids, glycophosphatidylinositol (GPI)anchored proteins and proteoglycans.Glycans must be included as essential contributors to life, along with proteins, nucleic acids and lipids, to facilitate progress in the personalized medicines of the future (51).participation of their animals in this study because the mice were bred in my colony at Albert Einstein following approval of the IACUC.I am therefore the owner.

FIGURE 1
FIGURE 1 Expression of Eogt and Pofut1 in immune cell subsets.The diagram depicts the ECD of mouse NOTCH1 showing the O-glycans attached at respective consensus sites in the 36 EGF repeats.The sugar that initiates each O-glycan is shown in the key.The potential sugar extensions from Ofucose and O-GlcNAc glycans are also shown.The expression of Eogt and Pofut1 in a number of immune cells is taken from ImmGen Gene Skyline ULI RNASeq data group for Eogt and Pofut1 gene expression (18) (http://rstats.immgen.org/Skyline/skyline.html).The definition of each immune cell type is given at the link.Expression of Eogt and Pofut1 vary inversely in certain immune cell subsets.

4
FIGURE 4 Notch signalling in Eogt null T cell progenitors.Representative flow cytometry profiles and histogram quantification of (A) Cell surface NOTCH1, or binding of DLL1-Fc, DLL4-Fc, JAG1-Fc and JAG2-FC to fixed CD4/CD8 DN T cell progenitors from Eogt[+/-] or Eogt[-/-] mice.Mean fluorescence index (MFI) for anti-Fc Ab was subtracted from MFI for Notch ligand or NOTCH1 Ab (MFI-control).Symbols represent Eogt[+/-] (blue circles) and Eogt[-/-] (red circles) DN T cells.Fixed cells had been stored for up to 3 months at 4°C. (B) Transcripts from DN T cell progenitors of Eogt[+/-] or Eogt[-/-] mice were subjected to qRT-PCR as described in Materials and Methods.Relative expression was determined based on the average delta Ct obtained for Gapdh and Hprt combined.Each symbol represents a mouse of 7-8 weeks.Data are presented as mean ± SEM. *p <0.05, **p<0.01 based on two-tailed Student's t test or (*) p <0.05 based on one-tailed Student's t test.