Notch signalling in the paraxial mesoderm is most sensitive to reduced Pofut1levels during early mouse development
© Schuster-Gossler et al; licensee BioMed Central Ltd. 2009
Received: 04 July 2008
Accepted: 22 January 2009
Published: 22 January 2009
The evolutionarily conserved Notch signalling pathway regulates multiple developmental processes in a wide variety of organisms. One critical posttranslational modification of Notch for its function in vivo is the addition of O-linked fucose residues by protein O-fucosyltransferase 1 (POFUT1). In addition, POFUT1 acts as a chaperone and is required for Notch trafficking. Mouse embryos lacking POFUT1 function die with a phenotype indicative of global inactivation of Notch signalling. O-linked fucose residues on Notch can serve as substrates for further sugar modification by Fringe (FNG) proteins. Notch modification by Fringe differently affects the ability of ligands to activate Notch receptors in a context-dependent manner indicating a complex modulation of Notch activity by differential glycosylation. Whether the context-dependent effects of Notch receptor glycosylation by FNG reflect different requirements of distinct developmental processes for O-fucosylation by POFUT1 is unclear.
We have identified and characterized a spontaneous mutation in the mouse Pofut1 gene, referred to as "compact axial skeleton" (cax). Cax carries an insertion of an intracisternal A particle retrotransposon into the fourth intron of the Pofut1 gene and represents a hypomorphic Pofut1 allele that reduces transcription and leads to reduced Notch signalling. Cax mutant embryos have somites of variable size, showed partly abnormal Lfng expression and, consistently defective anterior-posterior somite patterning and axial skeleton development but had virtually no defects in several other Notch-regulated early developmental processes outside the paraxial mesoderm that we analyzed.
Notch-dependent processes apparently differ with respect to their requirement for levels of POFUT1. Normal Lfng expression and anterior-posterior somite patterning is highly sensitive to reduced POFUT1 levels in early mammalian embryos, whereas other early Notch-dependent processes such as establishment of left-right asymmetry or neurogenesis are not. Thus, it appears that in the presomitic mesoderm (PSM) Notch signalling is particularly sensitive to POFUT1 levels. Reduced POFUT1 levels might affect Notch trafficking or overall O-fucosylation. Alternatively, reduced O-fucosylation might preferentially affect sites that are substrates for LFNG and thus important for somite formation and patterning.
The evolutionarily conserved Notch signalling pathway mediates direct cell-to-cell communication in a wide variety of developmental contexts in different species and regulates cell-fate decisions, proliferation and apoptosis [1–6]. Notch genes encode large transmembrane proteins that act at the surface of a cell as receptors for proteins encoded by the Delta and Serrate (Jagged in mammals) genes. Like Notch, the transmembrane ligands Delta and Serrate have a variable number of EGF-like repeats in their extracellular domains [7–9]. Upon ligand binding, the intracellular portion of Notch is proteolytically released, translocates to the nucleus, and by complexing with a transcriptional regulator of the CSL family, RBP-jκ in mouse, activates transcription of target genes [10–16].
Notch modification by O-linked fucose residues is essential for Notch signalling in vivo both in Drosophila and mammals [17–19]. O-linked fucose residues are attached to specific Ser or Thr residues in epidermal growth factor-like sequence repeats of Notch [20, 21]. The transfer of fucose to these residues is catalyzed by protein O-fucosyltransferase 1 (POFUT1), which is encoded by Ofut1 in Drosophila and Pofut1 in mammals . In addition, in certain cell types POFUT1 acts independently from its fucosyltransferase activity as a chaperone and is required for Notch folding and presentation on the cell surface [23, 24]. Pofut1 null (Pofut1tm1Pst/tm1Pst) mutant mouse embryos are severely growth retarded on E9.5 and die around E10 with a phenotype resembling embryos lacking the common downstream effector RBP-jκ or presenilins, which are required for the release of the intracellular domains of Notch receptors, suggesting that Notch signalling is globally inactivated through all four mammalian Notch receptors .
O-linked fucose residues on EGF repeats serve as substrates for further modification by Fringe (FNG) proteins, fucose-specific beta1, 3 N-acetylglucosaminyltransferases that modify Notch in the trans-Golgi and modulate the interactions of Notch receptors with their ligands [20, 21]. Notch modification by Fringe differentially affects the ability of ligands to activate Notch receptors in a context-dependent manner [25–27]. For example, in the Drosophila wing disc Fringe potentiates a cell's ability to respond to Delta and inhibits its ability to respond to Serrate , whereas in the presomitic mesoderm of mouse embryos Lunatic fringe (LFNG) appears to attenuate Delta1-like (DLL1)-mediated activation of Notch1 . In vitro LFNG may enhance DLL1-mediated signalling and inhibits Jagged1-mediated signalling through Notch1, or potentiate both Jagged1- and DLL1-mediated signalling via Notch2  indicating a complex modulation of Notch activity by differential glycosylation.
The context-dependent effects of Notch receptor glycosylation by LFNG suggest that different developmental processes might have different requirements for O-fucosylation by POFUT1. However, this cannot be assessed due to the early embryonic lethality of Pofut1 null mutants. Here, we identify a spontaneous mutation in the Pofut1 gene that leads to a hypomorphic allele. Mice homozygous for this allele display defects in the axial skeleton consistent with the known patterning functions of Notch in somitogenesis, but have no apparent defects in other early Notch-dependent processes such as left-right determination, vascular remodelling or neuronal differentiation. Our results suggest that aspects of somite formation and patterning that depend on Notch function are processes that are most sensitive to the level of POFUT1 in early mammalian embryos.
Cax, a novel spontaneous mouse mutation affecting axial skeleton development
Identification of Pofut1 as the gene affected by the caxmutation
To address whether the Pofut1 cax mutation affects the Pofut1 coding sequence, we amplified Pofut1 cDNA from mRNA purified from homozygous Pofut1 cax kidney and C3H wild type mice, and sequenced at least two independently generated cDNA clones. No mutation in the coding sequence was detected (data not shown), suggesting that Pofut1 cax affects Pofut1 transcription. Consistent with this idea, a probe from the coding region revealed by in situ hybridization overall reduced levels of Pofut1 transcripts in a considerable portion of homozygous Pofut1 cax mutant embryos (Figure 2D, b and 2d, and data not shown) that are also apparent in the PSM and developing somites (compare regions indicated by red lines Figure 2D, c and 2d).
Identification of the Pofut1 cax mutation
The primers used to amplify fragment 4/11 are located in fragments 4/10 and fragment E5, and should therefore be present in mutant DNA (Figure 3A, lanes c, d, and k, l). Thus, we reasoned that an insertion into fragment 4/11 might prevent amplification of this fragment from mutant DNA by conventional PCR. Therefore, we employed long range PCR with fragment 4/11 primers, and amplified an approximately 6 kb fragment from mutant DNA (arrowhead in Figure 3C). Sequencing of this fragment identified an intracisternal A particle (IAP) insertion into Pofut1 cax intron 4 (Figure 3D). The presence of the insertion in the Pofut1 cax genomic DNA was independently verified by PCR reactions that specifically amplify the junction fragments (lanes c, d and g, h in Figure 3D). In E10.5 cax mutant embryos (n = 12) Pofut1 mRNA levels were reduced to approximately 25% of wild type (n = 6) levels (Figure 3E) as determined by TaqMan real time PCR. Western blot analyses with anti-POFUT1 antibodies  showed also reduced POFUT1 protein levels in cell lysates from Pofut1 cax embryos (Figure 3F). The protein reduction could not precisely be determined due to the normally low levels of endogenous POFUT1 in embryos and additional background bands. Thus, the Pofut1 cax allele carries an insertion of a retrotransposon that most likely underlies variably reduced Pofut1 mRNA and POFUT1 protein levels. This variable reduction might also explain the variable phenotype of cax mutant embryos and mice.
Effects of the Pofut1 cax mutation on Notch dependent processes
Consistent with obvious alterations of Hes1, Hey1, and HeyL expression in the paraxial mesoderm also the expression patterns of genes that are important or indicative for somite patterning and polarity, and whose normal expression patterns depend on Notch activity, were altered in Pofut1 mutants. Dll1 expression was virtually abolished in the somites but upregulated in the neural tube of Pofut1 null mutant embryos (n = 3; Figure 4B d). Similarly, Uncx4.1, which is normally expressed in a regular pattern delineating posterior somite halves (Figure 4B i), was severely downregulated in somites of Pofut1 null mutants (n = 5; Figure 4B l), and expression of Tbx18, which normally delineates anterior somite halves (Figure 4B e), was expanded throughout somites (n = 3; Figure 4B h) indicating loss of somite polarity.
In cax mutants anterior-posterior somite patterning was also abnormal, as indicated by the reduced and broadened Dll1 expression domains in somites (n = 11; arrows in Figure 4B b), fuzzy expression domains and disorganized stripes of expression of Tbx18 (n = 27) and Uncx4.1 (n = 10; arrows in Figure 4B f, j). In heteroallelic Pofut1cax/tm1Pstembryos anterior-posterior somite patterning was more severely affected than in Pofut1cax/caxembryos: the somitic Dll1 stripes were essentially lost (Figure 4B c), Tbx18 expression domains were fuzzy and expanded (Figure 4B g) and, Uncx4.1 stripes were irregular and scrambled (Figure 4B k). Similarly, expression of Cer1, Mesp2, and Papc was disrupted by the cax mutation further supporting the notion that somite compartmentalization is affected. Cer1, whose expression is normally restricted to the anterior somite compartments of the prospective and most recently formed two somites (Figure 4B m), was expressed in one broad domain (n = 25; red line in Figure 4B n) similar to Pofut1 null mutants (n = 2; Figure 4B p). Mesp2 which is normally expressed in one or two distinct stripes of variable width (Figure 4B q, and data not shown) showed always only one not clearly delineated expression domain in cax mutant embryos (n = 31; red line in Figure 4B r), and the distinct domains of Papc expression (Figure 4B u) appeared as one blurred domain in cax (n = 36; red line in Figure 4B v). In heteroallelic Pofut1cax/tm1Pstembryos (n = 3, respectively), the expression patterns of these genes were similarly disrupted (Figure 4B o, s, w). In Pofut1 null mutants (n = 6) expression of these genes was severely downregulated in addition to their abnormal patterns (Figure 4B t, x).
We have identified a novel allele, Pofut1 cax , of the mouse Pofut1 gene that leads to reduced Pofut1 mRNA and protein levels. Reduction of POFUT1 in embryos homozygous for this allele consistently affects anterior-posterior somite patterning, which at least in part is due to abnormal Lfng expression in the anterior PSM, but apparently has no impact on other early developmental processes outside the paraxial mesoderm known to be dependent on Notch signalling. Our data suggest that Notch signalling in distinct developmental contexts is differentially sensitive to the levels of POFUT1 and/or POFUT1-dependent modifications.
The complementation test in conjunction with the map position of Pofut1 cax and the intermediate phenotype of Pofuttm1Pst/Pofut1 cax heteroallelic embryos demonstrated that cax is an allele of Pofut1 and that the cax mutation leads to reduced Pofut1 function. Since the coding sequence of Pofut1 is not altered in the Pofut1 cax allele, the enzymatic properties of POFUT1 are not affected. However, we observed a significant variable reduction of Pofut1 mRNA and protein, which provides a plausible explanation for reduced POFUT1 activity. Most likely the IAP insertion that occurred close to the 3' end of intron 4 is responsible for reduced Pofut1 mRNA either by interfering with transcription or by destabilizing the message. Insertional mutagenesis by IAPs is not uncommon [42–45], and other insertions into introns that cause mutations have been reported [43, 46]. The C3H/He inbred strain of mice appears to have a particularly high frequency of IAP insertional mutations [43, 44, 46].
Whereas loss of POFUT1-mediated Notch modification appears to block all Notch activity (this paper and ), reduced POFUT1 levels in embryos homozygous for the Pofut1 cax allele affects predominantly and consistently anterior-posterior somite patterning. Disruption of normal cyclic Lfng expression in the PSM likely contributes to these abnormalities since overexpression or interfering with the cyclic expression of Lfng was shown to cause somite compartmentalization defects similar to the loss of Lfng function [30, 33, 47]. One potential explanation for the apparent high sensitivity of Notch signalling during somitogenesis to Pofut1 levels could be that normal Pofut1 mRNA levels are particularly low in the presomitic mesoderm (PSM), where Notch signalling is critical for somite patterning, and Pofut1 levels in mutants fall only in the PSM below a critical threshold. However, substantial differences in expression levels in different tissues were not apparent in wild type embryos at E9.5 (Figure 2D a, c), a stage at which cax mutants showed already substantial defects in their somites. However, we cannot exclude that such differences may exist but were not detected by the limited quantitative resolution of in situ hybridization.
In Drosophila, the POFUT1 protein appears to be required for efficient presentation of Notch at the cell surface  and/or for the constitutive trafficking of the Notch receptor to early endosomes and downregulation of signalling . It has been proposed that POFUT1 also acts in the mouse PSM as a chaperone that is essential for Notch1 presentation at the cell surface , whereas in CHO and ES cells POFUT1 was not required for stable surface expression but for ligand binding and Notch activation . If POFUT1 is required for Notch presentation at the cell surface in the PSM reduced POFUT1 levels might cause reduced Notch levels at the cell surface, which in turn could lead to attenuated Notch activity. If that were indeed the case one would have to assume that Notch trafficking in the PSM is particularly sensitive to POFUT1 protein levels, for which there is no experimental evidence at present.
Alternatively, different fucosylation sites may require different levels of POFUT1 activity for efficient modification, and reduced POFUT1 levels might affect some sites more than others. Since conserved O-fucosylation sites may have distinct functions with respect to Notch activation and/or trafficking , differential O-fucosylation may result in context-dependent effects. Such effects have indeed been observed for the Notch1 receptor in mice, where mutation of the O-fucosylation site in EGF repeat 12, which is essential for ligand binding, results in a hypomorphic allele which is compatible with apparently normal embryonic development but affects post-natal growth and T-cell development . Context dependent effects might also depend on the role of O-linked fucose residues as substrates for further modifications by FNG glycosyltransferases. In mice, there are three fringe proteins, LFNG, RFNG and MFNG, which are expressed in distinct patterns during development [52–54]. Loss of RFNG has no obvious consequences , no in vivo data on the function of MFNG have been reported, and loss of LFNG leads to severe anterior-posterior somite patterning defects [30, 56] suggesting that FNG modification of Notch is of particular importance for somite patterning. Since fringe proteins modify different regions of Notch in vitro , and not all O-fucosylated EGF repeats are substrates for fringe activity , reduced O-fucosylation might preferentially affect sites that are substrates for LFNG and thus important for somitogenesis.
Our findings and conclusions conflict with previous findings suggesting that somite segmentation is less sensitive to reduced Notch activity than neural tissue . In these experiments, a Notch allele was used that gives rise to a processing-defective Notch protein. This mutant Notch protein can be processed to NICD by an unidentified protease more effectively than wild type Notch, and it was suggested that this protease is more active in the paraxial mesoderm . Thus, processing-defective Notch could generate more residual Notch activity in the PSM than in neural tissue providing the basis for the mild somite defects observed by Huppert at al. . In addition, segment border formation was used as the major criterion, but anterior-posterior patterning was in general more affected than segmentation (border formation) in their studies. Since Notch activity is not essential for border formation, but pivotal for anterior-posterior somite patterning , the results of Huppert et al.  could also be interpreted in favour of a high sensitivity of somite compartmentalization to reduced Notch activity and POFUT1 levels that we observed in cax mutant embryos.
Reduction of Pofut1 expression to approximately 25% affects expression of Notch target genes and Notch-dependent processes differently in different tissues. Cyclic Lfng expression and anterior-posterior somite patterning is highly sensitive to the level of POFUT1 in early mammalian embryos whereas other early Notch-dependent processes apparently are not. Reduced POFUT1 levels might affect trafficking and/or O-fucosylation of Notch as well as its further modification by LFNG due to abnormal Lfng expression. Since FNG modification of Notch appears to be of particular importance for somite patterning, and not all O-fucosylated EGF repeats are substrates for Fringe activity, we propose that reduced O-fucosylation might preferentially affect sites that are substrates for LFNG and thus important for somitogenesis. The hypomorphic Pofut1 cax allele should facilitate to further dissect the roles of POFUT1 for Notch signalling in different developmental contexts and at later stages of development.
The recessive cax mutation arose spontaneously in a C3H/HeJ colony of mice at The Jackson Laboratory and has been maintained on this strain background. The inbred mouse strain carrying the mutation is designated C3H/HeJ-Pofut1 cax /J, Jackson Laboratory Stock Number 7782. Phenotypic analysis was performed with cax mutants on a mixed genetic background due to the low breeding performance of the inbred line.
Intersubspecific F1 hybrids were generated by mating C3H/HeJ-cax/cax mutants with CAST/Ei-+/+ mice. An intercross of these F1 hybrids produced 62 F2 mice with mutant phenotypes (cax/cax genotype) that were used to determine the initial map position of the cax mutation. A backcross of (C3H/BL6xCAST/Ei) F1 hybrids to C3H/B6-cax/cax mutant mice produced 1339 N2 mice with an unambiguous mutant tail phenotype that were analyzed for high resolution mapping. Initially, we mapped cax with respect to the flanking simple sequence length polymorphism (SSLP) markers D2Mit22 and D2Mit409 using all 1339 backcross animals. We identified 62 recombinants, which were genotyped for SSLP markers located between D2Mit22 and D2Mit409.
In situ hybridization
Whole mount in situ hybridization was performed according to . The probes used were originally obtained from Dr. Manfred Gessler (Hey1, HeyL), Dr. Martyn Goulding (NeuroD, neurogenin), Dr. Tom Gridley (Lfng), Dr. Ryoichiro Kageyama (Hes1, Hes7), Dr. Andreas Kispert (Uncx4.1, Tbx18), Dr. Janet Rossant (Cer1, nodal), Dr. Yumiko Saga (Mesp2), or isolated in our laboratory (Dll1, Pofut1, Papc).
Embryos were fixed in 4% PFA, dehydrated, embedded in paraffin, sectioned at 10 μm, stained with Nuclear Fast Red, and embedded in Mowiol (Calbiochem).
For immunohistochemical analysis embryos were fixed in 4% paraformaldehyde, and in case of NF165 staining subsequently treated with proteinase K. Primary antibodies used were anti-PECAM (Pharmingen) diluted 1:100, and anti-NF165 (DSHB clone 2H3) diluted 1:50. Secondary antibodies were biotinylated anti-rat and anti-mouse (Vectastain) diluted 1:200. The signal was intensified with the ABC system (Vectastain) and bound antibodies were detected with DAB (Sigma).
PCR Analysis of Pofut1mRNA
To search for mutations in the open reading frame of Pofut1 mRNA from C3H/HeJ-cax/cax or C3H/HeJ embryos was prepared either with magnetic Dynabeads (Dynal Novagen) or with a Direct mRNA kit (Qiagen). cDNA was synthesised with Superscript II Reverse transcriptase (Invitrogen or Promega). cDNA was amplified in two overlapping fragments using the following gene specific primers: for Exon 2-Exon4: CTG CTT CTG CTG CTG TTG CTG C and CAG TGC GAG CAC AGG ATG CTC, and for Exon 5-Exon7: CAA TGG ACC CAG AGA TTT CCT GCA and GGT TGA GGG TGG GAG GTG GG. Exon1 was amplified from genomic DNA with the primers GCC ATT GTG CGG TGC ATT G and AAG CAG AGG GTT CCG GAG GC. PCR fragments from at least two independent PCR reactions were subcloned either into TpGEM easy or pCRII Topo vectors and sequenced.
PCR Analysis of genomic DNA
To identify gross abnormalities of the Pofut1 gene about 20 kb of the promoter region, the introns and the untranslated 3'region were amplified by PCR as overlapping fragments of about 0.5 to 1 kb in length. Primer sequences were selected based upon http://www.ensembl.org. Primer sequences used to amplify the fragments shown in the figures are as follows: fragment 4/10: TCCATTTTGCCCTTTCAAAGGT and ACACAGAATCCTTTCTGCAATCTTTC; fragment 4/11: GCACTGCCACTGGGGCTAGT and CCCAGGCAGTGCGAGCA; fragment E5: AGATTTCCTGCAAAAGAGCATCCT and GAGCTAAAATCCAGACTTGGTGGA; fragment 5/1: CATTCATCTGCGCATTGGCT and AGTGGGACTGCAGATCACTCCC. Sequences of all other primers are available upon request.
A DNA fragment containing the insertion in the cax mutation was amplified with the long range PCR Kit from Qiagen using primers GCACTGCCACTGGGGCTAGT and CCCAGGCAGTGCGAGCA, followed by a nested PCR reaction with primers GAAAGATTGCAGAAAGGATTCTGT and AGGATGCTCTTTTGCAGGAAATCT. The fragment was subcloned in the TopoXL vector (Invitrogen) and sequenced. Primer pairs used to specifically detect the 5' and 3' ends of the IAP insertion in the cax allele were: AGGGCTCTTTTTGCGTCCTGT and TGGCGCTGACATCCTGTGTT, and CCCAGGCAGTGCGAGCA and TCAAGATCAGACTTACCTCGTTCC, respectively.
Southern Blot hybridization
Southern Blot hybridizations were performed according to standard procedures with a Pofut1 cDNA probe containing exons 5 and 6 and the 5' region of exon7.
Quantitative Real-time PCR analysis
RNA was prepared from individual embryos using the RNeasy Minikit (Quiagen), and reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturers' instructions. Pofut1 cDNA was quantified on an ABI 7900HT using two gene specific assays detecting portions of exons 1 and 2, and exons 6 and 7, respectively, (TaqMan gene expression assays Mm 01240157 m1 (for Pofut1 exon 6–7), Mm 00475567 m1 (for Pofut1 exon 1–2)) each measured in quadruplicate using wild type embryo cDNA as biological calibrator, GAPDH and HPRT as endogenous controls (TaqMan gene expression assays Mm 00446968 m1 (for Hprt exon 6–7), Mm 999999159 g1 (Gapdh exon 1–2), and additional no-template, no reverse transcription and blank controls. Data were evaluated using the SDS RQ Manager (V1.2 ABI) and the delta-delta Ct method [60, 61]. Analysis of variance (ANOVA) was carried out using the statistical software package BIAS for Windows Version 8.6.3.
Western Blot analyses
12 wild type and cax mutant embryos, respectively, and four Pofut1tm1Pst/tm1PStE9.5 embryos were pooled, and lysed in 2× Lämmli buffer. The equivalent of one embryo from pooled embryo lysates was loaded per lane. Proteins were separated by SDS-polyacrylamid gel electrophoresis and blotted onto transfer membranes (Immobilon, Millipore). After blotting membranes were incubated with Qentix™ Western Blot Signal Enhancer (Pierce), blocked with 10% milk powder in TBST at 4°C over night, incubated with rabbit anti-POFUT1 antibodies  diluted 1:1000 in TBST with 1% milk powder for 3 hr at 4°C, washed five times in TBST, incubated with HRPOD conjugated donkey anti-rabbit (GE Healthcare, 1:10000 in TBST with 1% milk powder), and washed five times in TBST. Bound antibodies were detected using ECL Western Blotting Detection Reagents (GE Healthcare) using a Fujifilm LAS 3000 gathering signals every 3 min over an 21 minute interval.
We thank Pamela Stanley, Albert Einstein College of Medicine, New York, for the generous gift of the Pofut1tm1Pstmice and critical discussion, Abdou Mafda, University of Limoges, France, for the generous gift of anti-POFUT1 antibodies, Pat Ward-Bailey and Rod Bronson of The Jackson Laboratory for assistance with the initial genetic mapping and pathology, and Anatoli Heiser at the Medical School for assistance with the backcross and fine mapping. This work was supported by NIH grant RR01183 to The Jackson Laboratory and, by DFG funding to AG as part of the Cluster of Excellence REBIRTH (From Regenerative Biology to Reconstructive Therapy).
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