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First published online 9 April 2008
doi: 10.1242/dev.020891
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Research Report |
1 Stockholm University, Wenner-Gren Institute, Developmental Biology, Svante
Arrhenius Väg 16-18, SE-106 91 Stockholm, Sweden.
2 Institute of Microbiology, Department of Biology, Swiss Federal Institute of
Technology, ETH Zurich, CH-8093 Zurich, Switzerland.
3 Max-Planck Institut für Entwicklungsbiologie, Abteilung Genetik,
Spemannstraße 35, D-72076 Tübingen, Germany.
Author for correspondence (e-mail:
mannervik{at}devbio.su.se)
Accepted 25 March 2008
SUMMARY
N-linked glycosylation is a prevalent protein modification in eukaryotic
cells. Although glycosylation plays an important role in cell signaling during
development, a role for N-linked glycosylation in embryonic patterning has not
previously been described. In a screen for maternal factors involved in embryo
patterning, we isolated mutations in Drosophila ALG5, a
UDP-glucose:dolichyl-phosphate glucosyltransferase. Based on the embryonic
cuticle phenotype, we designated the ALG5 locus wollknäuel
(wol). Mutations in wol result in posterior segmentation
phenotypes, reduced Dpp signaling, as well as impaired mesoderm invagination
and germband elongation at gastrulation. The segmentation phenotype can be
attributed to a post-transcriptional effect on expression of the transcription
factor Caudal, whereas wol acts upstream of Dpp signalin by
regulating dpp expression. The wol/ALG5 cDNA was
able to partially complement the hypoglycosylation phenotype of alg5
mutant S. cerevisiae, whereas the two wol mutant alleles
failed to complement. We show that reduced glycosylation in wol
mutant embryos triggers endoplasmic reticulum stress and the unfolded protein
response (UPR). As a result, phosphorylation of the translation factor
eIF2
is increased. We propose a model in which translation of a few
maternal mRNAs, including caudal, are particularly sensitive to
increased eIF2 phosphorylation. According to this view, inappropriate
UPR activation can cause specific patterning defects during embryo
development.
Key words: Drosophila, Glycosylation, Patterning, Unfolded protein response
INTRODUCTION
A common protein modification in eukaryotic cells is N-linked
glycosylation, which occurs on the majority of proteins synthesized in the
endoplasmic reticulum (ER), where a pre-assembled oligosaccharide chain is
transferred to the nascent polypeptide
(Helenius and Aebi, 2004
).
Protein glycosylation has several purposes: it is needed for proper folding
and quality control in the ER, it targets some proteins to different cellular
compartments, and it can affect protein function. Accumulation of unfolded
proteins within the ER triggers the unfolded protein response (UPR)
(Zhang and Kaufman, 2004).
This response increases the folding capacity of the ER and decreases the
folding demand via three ER transmembrane proteins, IRE1 (inositol-requiring
1), ATF6 (activating transcription factor 6) and PERK (PKR-like endoplasmic
reticulum kinase). Activation of IRE1 and ATF6 causes transcriptional
activation of genes needed for folding in the ER, whereas PERK activation
results in a general decrease in translation initiation and selective
translation of specific mRNAs through phosphorylation of eIF2.
Transfer of Met-tRNAi to the 40S ribosomal subunit is
accomplished by GTP-bound eIF2 (Proud,
2005). Following recognition of the AUG start codon, GTP is
hydrolyzed and the eIF2-GDP complex released from the ribosome. Exchange of
GDP for GTP is mediated by eIF2B, and is regulated by phosphorylation of the
alpha subunit of eIF2 at a conserved serine residue, which generates a
competitive inhibitor of eIF2B.
In this work, we show that mutations in wollknäuel, a
UDP-glucose:dolichyl-phosphate glucosyltransferase involved in N-linked
protein glycosylation, disrupts Drosophila embryo development by
affecting the expression of a few key gene regulators. Reduced glycosylation
efficiency in wol mutant embryos triggers the UPR. As a result,
phosphorylation of eIF2 is increased. We propose that some mRNAs are
more sensitive to eIF2 phosphorylation than others, and that this
causes specific patterning defects.
MATERIALS AND METHODS
Fly stocks, P-element transformation and germline clones
Oregon-R or w1118 were used as wild-type
controls. The wol alleles 2L-284 (wol1) and
2L-267 (wol2), as well as the cad allele 2L-264,
were generated on an FRT2L-40A-containing chromosome in a germline
clone EMS screen performed in Tübingen
(Luschnig et al., 2004).
A P-element plasmid containing a 2.8 kb wol/ALG5 genomic region
was constructed by PCR amplification from genomic DNA, cloned into pCaSpeR4
(Thummel and Pirrotta, 1992),
and injected into w1118 embryos according to standard
procedures. Two insertions on the X-chromosome were used in rescue
experiments.
Germline clones were produced as described
(Qi et al., 2008). Males
containing a transgene misexpressing dpp in the Krüppel
(Kr) domain (gift from Hilary Ashe, University of Manchester, UK)
were crossed to wol1 germline clone females. Expression of
dpp was activated by FLPing out transcriptional stop signals
downstream of the Kr promoter
(Struhl et al., 1993).
Cuticle preparation, in situ hybridization and immunofluorescence
Cuticles were prepared as described by Wieschaus and Nüsslein-Volhard
(Wieschaus and Nüsslein-Volhard,
1998) and examined using dark-field microscopy. Whole-mount RNA in
situ hybridization using digoxigenin-labeled probes was performed as described
previously (Jiang et al., 1991;
Tautz and Pfeifle, 1989).
Double labeling was performed as described
(Kosman and Small, 1997).
|
). Rabbit anti-Eve (1:1000 dilution) was provided by
Mike Levine (University of California, Berkeley, CA), guinea pig anti-Cad
(1:800) and rat anti-Hb (1:400) antibodies are described by Kosman et al.
(Kosman et al., 1998) and were
provided by John Reinitz and Steve Small.
Positional cloning
Complementation tests with deficiencies were used to map the wol
alleles, which failed to complement the deficiency Df(2L)TE29Aa-11.
We developed SNP markers from the intergenic regions in the 260 kb interval of
this deficiency. PCR products that could be distinguished by restriction
fragment length polymorphisms were identified, and used in high-resolution
recombination mapping with P-element strains flanking the deficiency. The
wol1 allele in a white- background was
crossed to the white+ P-elements l(2)k16919, located
proximal to the deficiency, or l(2)k14902, positioned distal to the
deficiency. We recovered 25 white-eyed recombinants out of 
20,000 flies
from the cross with the proximal P-element, and four recombinants out of
5000 flies from the distal P-element. Genomic DNA was prepared from the
recombinants, PCR amplified with SNP marker primers, and analyzed after
restriction digest. One recombination event with the l(2)k16919 chromosome
occurred distal to a SNP located 162 kb into the deficiency. Two out of the
four recombinants with l(2)k14902 had recombined proximal to a SNP located 210
kb into the deficiency.
Genomic DNA was isolated from wol1 and wol2 homozygous mutant larvae. Exonic sequences from the genes in this interval were amplified by PCR, sequenced and compared with an FRT2L-40A chromosome from another mutant. We found mutations in the CG7870/ALG5 gene, and confirmed that lethality maps to the ALG5 locus by complementation tests with a PiggyBac insertion (PBac RBe04276, Fig. 3) that became available during the course of this work.
Yeast assays
Saccharomyces cerevisiae strains used were derivates of YG91
(Mat ade2-101 ura3-52 his3-200
alg5::HIS3) and YG355 (Mat ade2-101 ura3-52 his3-200
alg5::HIS3 wbp1-2) (Burda
et al., 1996). Standard yeast media and genetic techniques were
used (Guthrie and Fink,
1991).
The Drosophila ALG5 cDNA was RT-PCR amplified from embryo mRNA,
TA-cloned and sequenced. The wol1 and
wol2 mutations were introduced by site-directed
mutagenesis (Quick-change Kit, Stratagene). Blunt-ended cDNAs were cloned into
the yeast expression vector pCFZ41-GPD
(Mumberg et al., 1995).
Analysis of carboxypeptidase Y (CPY) in ALG5-deficient cells
(YG91) was detected by western blot analysis as described
(Burda et al., 1996).
Western blot and RT-PCR
Protein extracts from 2x106 untreated, 10 mM DTT-treated
or eIF2 RNAitreated S2 cells, or from 30 µl 2- to 4-hour
w1118 or wol1 germline clone embryos
were prepared as described (Lilja et al.,
2007). Proteins (7 µg) were separated by SDS-PAGE, transferred
to PVDF membrane (GE Healthcare), and incubated with a rabbit
phospho-eIF2 (Ser51) antibody (1:1000, Cell Signaling Technology). The
membrane was re-probed with a rabbit anti-human eIF2 (residues 50-150)
antibody (1:200, Abcam). HRP-coupled secondary antibodies were visualized by
ECL (GE Healthcare).
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was performed as described
(Qi et al., 2008). RESULTS AND DISCUSSION
Segmentation defects in wollknäuel mutant embryos
From a screen for maternal genes that are required for Drosophila
embryo development (Luschnig et al.,
2004), we selected mutants that affect segmentation gene
expression. We identified two mutants with defects in posterior embryo
patterning, 2L-284 and 2L-267, that failed to complement each other and are
thus allelic. Cuticle preparations of embryos derived from mothers harboring
2L-284 or 2L-267 germline clones revealed segmentation defects in the
posterior half of the embryo, and a curled-up phenotype resulting from defects
in germband elongation and retraction (Fig.
1B,C). The cuticle phenotype resembles a ball of wool,
Wollknäuel in German, and we therefore named our alleles
wollknäuel 1 and 2 (wol1 and
wol2).
We stained embryos derived from wol germline clones (hereafter called wol embryos) with an engrailed (en) RNA probe (Fig. 1D-F). In the posterior, every second en stripe was missing. Double staining for Even skipped (Eve) protein and en RNA showed that the missing stripes correspond to odd-numbered stripes (Fig. 1G,H). Expression of en regulators was altered in wol embryos (see Fig. S1 in the supplementary material). We therefore examined gap gene expression, as gap proteins control expression of the en regulators. The posterior giant (gt) expression domain was severely reduced (Fig. 1K), although it recovered at later embryo stages (not shown), and the posterior knirps (kni) stripe was shifted posteriorly in wol embryos (Fig. 1, compare M with N).
A major activator of gt and kni expression is Caudal
(Cad), which forms a posterior-anterior protein gradient, being
translationally repressed by Bicoid in the anterior part of the embryo
(Rivera-Pomar and Jackle,
1996). Whereas the maternal cad RNA was present in normal
amounts in wol embryos (Fig. 1,
compare O with P), we found much less Cad protein in wol
embryos than in the wild type (Fig.
1Q,R). This result suggests that either Cad protein stability or
the efficiency of Cad translation is affected by the wol mutations.
Segmentation gene expression in wol embryos was very similar to that
found in embryos derived from cad germline clones
(Fig. 1I,L). There was no
failure in either the terminal system or in the posterior system, both of
which control gap gene expression (see Fig. S2 in the supplementary material).
We therefore favor the notion that reduced Caudal levels cause the
segmentation phenotype in wol embryos.
wol is required for dorsal-ventral patterning and gastrulation movements
We investigated dorsal-ventral patterning in wol embryos by
examining rhomboid (rho) expression. The rho gene
is expressed in two ventrolateral bands in response to the protein Dorsal
(Stathopoulos and Levine,
2002). In addition, rho is activated in dorsal cells by
signaling from the TGF-β protein Decapentaplegic (Dpp)
(Fig. 2A, arrow). In
wol mutant embryos, the Dpp-dependent rho expression pattern
was selectively affected (Fig.
2B). We overexpressed Dpp from a Kr enhancer in a central
domain of transgenic embryos and monitored Dpp activity through another
downstream target gene, Race (also known as Ance)
(Fig. 2C)
(Rusch and Levine, 1997). As
expected, Race mRNA was absent from wol mutant embryos
(Fig. 2D). However, in
wol embryos expressing ectopic Dpp (purple in
Fig. 2E), Race
expression was restored (brown in Fig.
2E). From this result, we conclude that wol activity is
not needed for transduction of the Dpp signal, but is acting upstream of Dpp
signaling. We found that expression of dpp itself is reduced in
wol embryos as compared with wild type
(Fig. 2F,G), which probably
explains the failure to activate Dpp target genes. It appears that an unknown
maternal regulator of dpp expression or, alternatively, mRNA
stability is dependent on wild-type wol activity.
We also noted problems with cell movements during gastrulation in wol mutant embryos. As a result, germband elongation does not proceed normally (Fig. 1 and data not shown), and mesoderm invagination is disturbed (see Fig. S3 in the supplementary material).
wol encodes a UDP-glucose:dolichyl-phosphate glucosyltransferase
Using SNP markers, we mapped the wol locus to a 48 kb interval on
chromosome 2. We sequenced the exons from the nine genes in this interval from
wol homozygous larvae. In the wol1 allele we
found an A-to-T transversion, and in the wol2 allele a
G-to-A transition in the gene annotated as CG7870
(Fig. 3A). CG7870 is predicted
to encode a 326 amino acid protein - the UDP-glucose:dolichyl-phosphate
glycosyltransferase ALG5 (Heesen et al.,
1994). In wol1, there is a R209W substitution
within the glycosyltransferase domain, whereas W316 is changed to a stop in
wol2. The lethality of wol mutants could be
rescued by a transgene containing the ALG5 genomic locus. Whole-mount
in situ hybridization showed that wol RNA is maternally contributed
and expressed zygotically in the salivary glands and proventriculus
(Fig. 3B-D), tissues where a
lot of protein secretion takes place.
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). This glucose is added to the oligosaccharide chain that is
assembled in the ER prior to transfer to the nascent polypeptide by
oligosaccharyl transferase (Helenius and
Aebi, 2004). Substrate recognition by oligosaccharyl transferase
is greatly diminished in the absence of terminal glucoses. Mutations that
disrupt ALG5 function are therefore expected to cause an accumulation of
hypoglycosylated proteins.
To investigate whether the wol1 and
wol2 mutations affect the function of ALG5, we performed a
complementation assay in Saccharomyces cerevisiae. As previously
shown, mutations in yeast alg5 lead to hypoglycosylation of secreted
proteins (Heesen et al., 1994).
This can be assayed by processing of the carboxypeptidase Y (CPY) protein, a
vacuolar protein with four N-linked oligosaccharides. In a
alg5 yeast strain, CPY glycoforms lacking one or two
oligosaccharide chains accumulate (Fig.
3E). Introduction of yeast or Drosophila ALG5 cDNA into
the alg5 strain restored the glycosylation phenotype of CPY,
whereas cDNAs with the wol1 or wol2
mutations failed to do so (Fig.
3E). In a yeast growth assay, ALG5 cDNAs with the wol
mutations only weakly rescued, or failed to rescue, the growth phenotype (see
Fig. S4 in the supplementary material). These results suggest that the
wol mutations either impair the catalytic activity of the ALG5
protein or lead to protein destabilization.
The unfolded protein response is triggered in wol mutant embryos
An important function of N-linked glycosylation is to aid the folding of
proteins in the ER (Helenius and Aebi,
2004). Accumulation of unfolded proteins in the ER induces the UPR
that activates the IRE1 endoribonuclease. In Drosophila, this leads
to the removal of a 23 bp intron from xbp1 mRNA in the cytoplasm
(Plongthongkum et al., 2007;
Ryoo et al., 2007;
Souid et al., 2007), which
generates a translational frameshift that gives rise to transcriptionally
active Xbp1 protein (Plongthongkum et al.,
2007).
We examined xbp1 splicing in wol mutant embryos by RT-PCR. Fig. 4A shows that one band with the size expected from unspliced xbp1 mRNA is obtained from wild-type embryos, whereas both spliced and unspliced products were detected in wol embryos. To confirm that this band corresponds to ER-stress-induced xbp1 splicing, we isolated mRNA from untreated, tunicamycin- or DTT-treated S2 cells. Tunicamycin inhibits N-linked glycosylation, whereas DTT prevents thioester bond formation, and both treatments generate unfolded proteins in the ER. As shown in Fig. 4A, these drug treatments resulted in more of the smaller, spliced xbp1 mRNA than was found in untreated S2 cells. We conclude that the UPR is triggered by tunicamycin and DTT, as well as by mutations in wol.
Another branch of the UPR is activation of the kinase PERK that results in
eIF2 phosphorylation and attenuation of translational initiation
(Kaufman, 2004).
Drosophila PERK (also known as PEK) is maternally contributed to the
embryo (Pomar et al., 2003).
As shown by the western blot in Fig.
4B, a 1.5-fold increase in eIF2 phosphorylation was
detected in wol embryos as compared with wild type, whereas the total
amount of eIF2 remained unchanged. As a control, we prepared protein
extracts from S2 cells treated with DTT or with eIF2
double-stranded RNA. eIF2 phosphorylation was increased by DTT, whereas
both the eIF2 and the phospho-eIF2 bands disappeared in extracts
from eIF2 RNAi-treated cells (Fig.
4B).
Taken together, these results confirm that the UPR is activated in
wol mutants, and indicate that translation might be attenuated in
wol embryos. We propose that this causes the observed patterning
defects. According to this model, reduced maternal wol activity would
lead to accumulation of unfolded proteins in the ER in early embryos, with a
consequent transient increase in eIF2 phosphorylation. Translation of
selected maternal mRNAs, including cad and the activator of
dpp expression, would be particularly sensitive to increased
eIF2 phosphorylation. Reduced amounts of these transcription factors
result in disruption of posterior segmentation and of dorsal-ventral
patterning. Although the UPR plays important developmental and physiological
roles in C. elegans, Drosophila and mammals
(Ryoo et al., 2007;
Shen et al., 2001;
Shen et al., 2005;
Souid et al., 2007;
Wu and Kaufman, 2006), this is
the first report to indicate that inappropriate UPR activation may disrupt
embryonic patterning.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/10/1745
ACKNOWLEDGMENTS
We thank Hilary Ashe, John Reinitz and Steve Small for providing reagents, and Monika Björk at the WCN fly facility for embryo injections. A.H. was supported by a Wenner-Gren fellowship, and grants from the Swedish Research Council to M.M. supported this work.
Footnotes
* Present address: University of Zurich, Institute of Zoology, Developmental
Biology, CH-8057 Zurich, Switzerland 
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