The Lc3-synthase gene B3gnt5is essential to pre-implantation development of the murine embryo
© Biellmann et al; licensee BioMed Central Ltd. 2008
Received: 11 June 2008
Accepted: 12 November 2008
Published: 12 November 2008
Glycosphingolipids (GSL) are integral components of mammalian cell membranes that are involved in cell adhesion and cell signaling processes. GSL are subdivided into structural series, like ganglio-, lacto/neolacto-, globo- and isoglo-series, which are defined by distinct trisaccharide cores. The β1,3 N-acetylglucosaminyltransferase-V (B3gnt5) enzyme catalyzes the formation of the Lc3 structure, which is the core of lactoseries derived GSL.
The biological significance of the glycoconjugates produced by the B3gnt5 enzyme was investigated by inactivating the B3gnt5 gene in the mouse germline. The disruption of the B3gnt5 protein-coding region in mouse embryonic stem cells resulted in reduced Lc3-synthase activity, supporting its specific contribution to lactoseries derived GSL synthesis. Breeding of heterozygous mutant mice failed to produce any viable progeny homozygous for the B3gnt5-null allele. The genotypic examination of embryos from heterozygous crosses showed that the disruption of the B3gnt5 gene leads to pre-implantation lethality. This finding was compatible with the expression pattern of the B3gnt5 gene in the pre-implantation embryo as shown by in situ hybridization. The analysis of GSL profiles in embryonic stem cells heterozygous for the B3gnt5-null allele confirmed the reduced levels of lactoseries derived GSL levels and of other GSL species.
The disruption of the B3gnt5 gene in mice affected the expression of lactoseries derived GLS and possibly of protein-bound β3GlcNAc-linked glycans, thereby demonstrating an essential contribution of these glycoconjugates in early embryonic development, and supporting the importance of these glycoconjugates in cell differentiation and adhesion processes.
Glycosphingolipids (GSL) represent a large family of glycoconjugates, which are found abundantly on cellular membranes. GSL are classified into different series defined by their respective core structures. In vertebrates, the main GSL series are called ganglio-, lacto-, globo-, isoglobo-, and muco-series . The functional significance of GSL is diverse since these glycoconjugates have been implicated in processes such as cell adhesion, cell migration, regulation of signaling proteins and binding of pathogens and toxins [2, 3]. The repertoire of GSL expressed by an organism is subject to changes according to cell type and developmental stage. Consequently, several stem cell and differentiation markers of early embryonic development, such as the stage-specific embryonic antigens SSEA-1, -3 and -4, represent carbohydrate epitopes carried by GSL [4–6].
The B3gnt5 enzyme has been shown to be key in the expression of sulfoglucuronylglycolipids (SGGL) in the developing nervous system . SGGL are expressed in a regulated manner during embryonic brain development and again postnatally [10–13]. For example, SGGL carry the HNK-1 epitope, which has been implicated in the regulation of synaptic plasticity [14, 15]. In addition, the developmentally regulated expression of SGGL coincides with certain cell migration and differentiation phases .
The B3gnt5 enzyme also initiates the formation of the SSEA-1 epitope, which is identical to the Lewis X antigen. SSEA-1 corresponds to the trisaccharide Galβ1,4(Fucα1,3)GlcNAc which is first found on 8-cell stage embryos and in mouse embryonic stem (ES) cells . The SSEA-1 epitope, which is mainly found on lactoseries derived GSL, is believed to participate in the regulation of cell adhesion during embryogenesis, cell differentiation, and development of the neuronal system [4, 18]. In order to study the developmental and physiological processes mediated by lactoseries derived GSL, we have inactivated the B3gnt5 gene in mice by homologous recombination in ES cells. This mouse model suggests an essential contribution of the lactoseries derived GSL series in the very early stages of mouse development.
Genotype analysis of B3gnt5 heterozygous crosses at various stages of murine development
By disrupting the B3gnt5 gene, we aimed to improve current appreciation of the functions of lactoseries derived GSL in vivo. Our study suggests an essential role of this GSL series in pre-implantation embryonic development by showing that B3gnt5-null blastocysts could not be retrieved from heterozygous matings. The expression of the B3gnt5 gene from the two-cell stage up to the blastocyst stage would suggest a role for lactoseries derived GSL in cell-cell adhesion, possibly contributing to embryo compaction and implantation. Similarly, the SSEA-1 antigen has been shown to mediate the tight adhesion of blastomeres, since the addition of multivalent SSEA-1 structures promotes embryo decompaction . The formation of the SSEA-1 antigen requires the action of the α1,3 fucosyltransferase FUT9 . The disruption of the Fut9 gene in mice abolished the formation of the SSEA-1 antigen in pre-implantation embryos, but this loss did not affect the compaction and implantation processes . This finding showed that the fucose residue is critical for marking the antigen but that fucose is dispensable for the potential adhesion properties of the lactoseries derived structure.
The early lethality observed in the B3gnt5 gene disruption model contrasts with the phenotype described for mice lacking the ceramide glucosyltransferase Ugcg gene, which initiates GSL biosynthesis (Fig. 1). Ugcg homozygous-null embryos have been reported to die between embryonic days E7.5 and E9.5 . Since Ugcg activity is required for the formation of lactoseries derived GSL, the reverse order would be expected for the onset of lethality in the Ugcg and B3gnt5 gene disruptions. A similar paradoxical situation was observed in the globoside GSL pathway when comparing the phenotypes resulting from the disruptions of the α1,4 galactosyltransferase Gb3-synthase  and β1,3 N-acetylgalactosaminyltransferase Gb4-synthase  genes. Mice lacking the Gb3-synthase gene developed normally and did not show any physiological abnormality . By contrast, the disruption of the Gb4-synthase gene arrested development at the blastocyst stage and prevented the implantation of mouse embryos . This apparent discrepancy may have different causes. It is possible that the loss of Ugcg activity is compensated for by other glycosyltransferase activities expressed during pre-implantation stages. However, no glycosyltransferase has been described with such activity yet and no proteins similar to the Ugcg enzyme could be retrieved from the mouse genome so far. Alternatively, it is possible that the expression of the Ugcg gene in unfertilized oocytes  yields enough of the Ugcg enzyme to sustain the formation of Glc-Cer until gastrulation.
The enzymatic characterization of the B3gnt5 protein has shown a significant N-acetylglucosaminyltransferase activity towards the monosaccharide Gal . Therefore it is conceivable that B3gnt5 may act on Gal-termini found on N- or O-linked glycans. However, it is likely that pre-implantation lethality is in fact mediated by a defect of GSL biosynthesis, since defects in N- or O-linked glycan elongations lead to post-implantation lethal phenotypes at the earliest . Indeed, the inactivation of the B3gnt2 gene, a paralog of B3gnt5 that mediates the formation of poly-N-acetyllactosamine chains on glycoproteins, does not impair embryonic development .
Even if the pre-implantation lethality observed in the B3gnt5-null embryos is caused by a defect of GSL biosynthesis, the question remains whether the loss of B3gnt5 solely affects the lactoseries derived GSL pathway. Our analysis of GSL profiles in ES cells bearing a B3gnt5-null allele showed decreased levels of additional GSL structures, such as the gangliosides GM3, GM1, GD1 and the globosides Gb3, Gb5 (Fig. 5). Earlier work has documented the physical interaction of glycosyltransferases involved in GSL biosynthesis , suggesting that the loss of a single enzyme might destabilize the localization and thereby the activity of other enzymes.
The disruption of the B3gnt5 gene leads to the earliest lethality reported for a Golgi-localized glycosyltransferase, thereby underlining the essential role of lactoseries derived GSL and possibly of protein-bound β3GlcNAc-linked glycans in pre-implantation embryogenesis. Because of this early lethality, the role of these glycoconjugates in subsequent developmental pathways could not be determined in the present model. The expression pattern of the B3gnt5 gene at gastrulation and at later stages would support the involvement of B3gnt5 products in processes such as neurogenesis and brain development. The production of a conditional B3gnt5 gene targeting model will certainly bring new insights on the role of lactoseries derived GSL in organ development and functions.
The targeting vector was assembled by flanking the PGK-Neo cassette of the pPGK-Neo plasmid  with two fragments of 129Sv/J genomic DNA fragments isolated from a λFIX-II bacteriophage library (Stratagene). The left arm, a 1.7-kbp BstXI-NdeI fragment that includes 400 bp of the B3gnt5 exon 4, was inserted blunt-end into the NotI site of pPGK-Neo and the right arm of the targeting vector was subcloned blunt-end as a 3.6-kbp BstXI-XbaI fragment into the EcoRV site of pPGK-Neo. Using this construct, a stretch of 745 bp encoding the catalytic domain of B3gnt5 was replaced by the PGK-Neo cassette (Fig. 2A). The targeting vector was linearized at the unique Sac II site and 10 μg were electroporated into 5 × 106 mouse embryonic stem (ES) cells of line R1  and TC1 . Cells were seeded on gelatin-coated Petri dishes and cultured in KO DMEM (Gibco) containing 15% fetal calf serum (Gibco), 1000 U/ml leukemia inhibitory factor (ESGRO, Gibco) and G418 (200 μg/ml). After 5 days of selection, 300 clones were picked and tested for homologous recombination by PCR and genomic Southern blotting.
Genotyping of ES cell clones
Homologously recombined B3gnt5 alleles were identified by PCR amplification of a 1920-bp fragment comprising the boundary of PGK-Neo up to the genomic DNA proximal to the left arm of the targeting vector. The PCR reaction was carried out using 50 ng of genomic DNA with the primers 5'-TACTACCCTGTCTAGGAGCAGTTG-3' and 5'-CATCGCATTGTCTGAGTAGGTGTC-3' for 35 cycles at 94°C for 45 s, 52°C for 1 min, and 72°C for 2 min. Homologous recombination at the B3gnt5 locus was confirmed by genomic Southern blotting. Genomic DNA (5 μg) samples were digested with EcoRI transferred to Hybond-N membranes (GE Healthcare) and hybridized to a 530-bp EcoRI-BstXI genomic fragment (Fig. 2A) as a probe. The detection of a 2.3-kbp fragment was indicative of a targeted B3gnt5 allele, whereas the wildtype allele was detected as a 3.0-kbp fragment.
Generation and breeding of B3gnt5-targeted mice
The homologously recombined ES cell clones were karyotyped using the standard potassium chloride method . ES cell clones harboring 40 chromosomes were injected into blastocyst-stage embryos. The resulting chimeric males were bred at 8 weeks of age with C57BL/6 females and germ-line transmission was observed with the birth of agouti offspring. Mice harboring the targeted B3gnt5 allele were backcrossed for five generations to the C57BL/6 background.
Genotyping of mouse samples
The B3gnt5-wildtype and -null alleles were detected in DNA isolated from tail biopsies, from embryonic-day-10 (E10) whole embryos and from blastocyst-stage embryos. Blastocysts were harvested from time-mated pregnant females by flushing the uterine horns with M2 buffer (Sigma). The tissue samples were digested with a 25 μg/ml proteinase K solution at 56°C for 16 h and the reaction was stopped by incubation at 95°C for 10 min. The wildtype allele was detected by PCR using the primers 5'-GGCTCAAGATGTCCTCCTCTTA-3' and 5'-ACATGGTCCTGTGGCAAGATTC-3' that yielded a 651-bp fragment. The null allele was detected as a 789-bp PCR fragment using the primers 5'-ACTCGTCAAGAAGGCGATAGAA-3' and 5'-CGGCCATTGAACAAGATGGATT-3'. These PCR reactions were both run for 35 cycles at 94°C for 1 min, 60.5°C for 45 s, and 72°C for 1 min. Another PCR protocol was applied to detect the B3gnt5-null allele in blastocysts. The primers 5'-CATCAGCCGCTACAGTCAAC-3' and 5'-CATCAGAGCAGCCGATTGTC-3' yielded a 326-bp fragment corresponding to a fragment of the PGK-Neo cassette. The corresponding PCR conditions were 35 cycles at 94°C for 45 s, 63.5°C for 40 sec, and 72°C for 40 s.
Glycosyltransferase activity assays
N-acetylglucosaminyltransferase activity was assayed in B3gnt5-targeted ES cells as described previously . ES cells (1 × 107) were released by trypsin digest, washed in PBS twice and lyzed in 200 μl of 2% Triton X-100 in 50 mM cacodylate buffer, pH 7.0, 20 mM MnCl2 for 15 min on ice in presence of a protease-inhibitor cocktail (Complete EDTA-free, Roche). Reactions were run at 37°C for 4 h using 25 μl of post-nuclear supernatant in 50 μl reactions of 50 mM cacodylate buffer, pH 7.0, 20 mM MnCl2, 5% Me2SO, 0.75 mM ATP, 0.5 mM UDP-GlcNAc including 5 × 104 cpm of UDP- [14C]GlcNAc (GE Healthcare). Reactions were stopped by adding 500 μl ice-cold H2O. The reaction products were purified by C18 SepPak cartridges (Waters) and measured by scintillation counting .
Isolation of pre-implantation embryos
Pre-implantation embryos were obtained from superovulated females. Superovulation was carried out by intraperitoneal administration of 50 IU pregnant mare serum gonadotropin (PMSG) (Intervet, Veterinaria AG Zürich, Switzerland) and 25 IU human chorionic gonadotropin (hCG) (Intervet) 48 h later . Embryos were removed at E1.5 (2–4-cell stage) and E3.5 (blastocyst stage). To obtain embryos at the 4-cell up to the morula stage, 2-cell embryos were cultivated in M16 media (Sigma) until the desired stage was reached. E3.5 blastocysts were isolated by flushing of the uterine horn.
Embryos were fixed for 1 h at room temperature in 4% paraformaldehyde in PBS and washed twice in PBS/0.1% Tween (PBT) and dehydrated once in 25%, 50%, 75% and twice in 100% methanol/PBT. The dehydration was followed by rehydration in the reverse order of the MeOH/PBT series 75%, 50%, 25% for 5 min each. The embryos were permeabilized in RIPA buffer and refixed in 4% PFA/0.2% glutaraldehyde. Embryos were washed in a 1:1 mixture of PBT/hybridization solution (50% formamide, 5× SSC, 0.1% Tween-20, 0.1% SDS, 50 μg/ml E. coli tRNA, 60 mM citric acid) for 10 min and then in hybridization solution. In situ hybridization was performed as described previously . As a control for the specificity of the labelings in each hybridization experiment, control embryos were hybridized with an equal concentration of a sense probe transcribed from the same template as the antisense probe. After staining over night at 4°C, embryos were post fixed in 4% paraformaldehyde/0.1% glutaraldehyde in PBT for 1 h and washed twice in PBT, then cleared in glycerol: PBT (1:1) and stored in glycerol: 2 mM EDTA in PBT (4:1). Hybridization results were documented using a Zeiss Axiovert 200 M microscope (Carl Zeiss AG, Feldbach, Switzerland). The B3gnt5 908-bp riboprobes were prepared as described previously . T7 and SP6 riboprobes were made using a DIG RNA labeling kit (Roche, Switzerland) and alkaline hydrolyzed to reduce the size of the riboprobes to about 300 bp.
GSL were extracted from mouse ES cells three times with chloroform:methanol:water (4:8:3, v:v:v) . The extracts were pooled, dried under N2 and re-dissolved in chloroform:methanol:PBS (1:163:160, v:v:v). A 1 ml SepPak tC18 cartridge (Waters) was conditioned with 2 ml methanol and 2 ml water. The dissolved lipid extract, corresponding to 50 mg wet weight of cell pellets were applied to the SepPak tC18 cartridge, followed by washing with 3 ml water. GSL were eluted with 1 ml chloroform:methanol (98:2, v:v), 2 ml chloroform:methanol (1:3, v:v) and 1 ml methanol . Eluates were dried under N2 and subjected to ceramide glycanase digestion, 2-aminobenzamide-labelling and NP-HPLC analysis as described earlier . One minute fractions were collected from the NP-HPLC and aliquots were subjected either directly or after desalting with 100 μl custom packed graphite columns (ENVI-Carb, Supelco) to MALDI-MS.
The MALDI matrix was prepared by suspending 10 mg DHB in 1 ml of 50% acetonitrile, containing 1 mM NaCl. Sample and matrix were mixed on the MALDI plate at a ratio of 1:1 and allowed to dry at room temperature. The dried spots were re-crystallized by applying < 0.1 μl ethanol. MALDI mass spectra were recorded in positive ion mode, using an Applied Biosystems 4800 Proteomics Analyzer (Applied Biosystems). Averages of 2000 to 5000 laser shots were used to obtain MS/MS spectra. The collision energy was set at 1 kV and the air pressure inside the collision cell was set at 2 × 10-6 Torr.
We thank Dr. Birgit Ledermann from the Institute of Laboratory Animal Science, University of Zürich for her technical assistance with the ES cell culture and the Functional Genomic Center Zurich (FGCZ), Switzerland and Dr. P. Gehrig, FGCZ for their support with the MALDI-TOF-TOF-mass spectrometer. This research was supported by the Swiss National Science Foundation Grants PP00A-106756 and 3100A0-116039 to TH.
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