Targeted Disruption of a Murine Glucuronyl C5-epimerase Gene Results in Heparan Sulfate Lacking L-Iduronic Acid and in Neonatal Lethality*
Jin-Ping Li
,
Feng Gong
¶,
Åsa Hagner-McWhirter
,
Erik Forsberg
,
Magnus Åbrink
||,
Robert Kisilevsky **,
Xiao Zhang 
and
Ulf Lindahl
From the
Department of Medical Biochemistry and
Microbiology, University of Uppsala, The Biomedical Center, Box 582, SE-751 23
Uppsala, Sweden, the **Department of Pathology,
Queen's University, Richardson Laboratory, Kingston, Ontario K7L 3N6, Canada,
and the 
Division of Molecular
Neuropharmacology, Neurotec Department, Karolinska Institutet, Huddinge
University Hospital, Novum, 141 86 Huddinge, Sweden
Received for publication, May 23, 2003
, and in revised form, June 4, 2003.
 |
ABSTRACT
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The glycosaminoglycan, heparan sulfate (HS), binds proteins to modulate
signaling events in embryogenesis. All identified protein-binding HS epitopes
contain L-iduronic acid (IdoA). We report that targeted disruption
of the murine D-glucuronyl C5-epimerase gene results in a
structurally altered HS lacking IdoA. The corresponding phenotype is lethal,
with renal agenesis, lung defects, and skeletal malformations. Unexpectedly,
major organ systems, including the brain, liver, gastrointestinal tract, skin,
and heart, appeared normal. We find that IdoA units are essential for normal
kidney, lung, and skeletal development, albeit with different requirement for
2-O-sulfation. By contrast, major early developmental events known to
critically depend on heparan sulfate apparently proceed normally even in the
absence of IdoA.
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INTRODUCTION
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The sulfated polysaccharide, heparan sulfate
(HS),1 is a ubiquitous
component of proteoglycans on cell surfaces and in the extracellular matrix.
HS chains interact with a variety of proteins, including growth
factors/morphogens, cytokines, enzymes, extracellular matrix proteins, and
thus modulate important processes in development and homeostasis
(1,
2). Selective protein binding
is mediated by saccharide domains containing sulfate groups in specific
patterns, along with L-iduronic acid (IdoA) units that promote
ligand apposition through their conformational flexibility
(3). The biosynthesis of HS is
initiated by polymerization of D-glucuronic acid (GlcA) and
N-acetyl-D-glucosamine (GlcNAc) residues in alternating
sequence and is pursued through a series of modification reactions that
include N-deacetylation/N-sulfation of GlcNAc,
C5-epimerization of GlcA to the C5-epimer, IdoA, and, finally
O-sulfation at various positions
(4). Mutational studies in
Drosophila and targeted disruption of murine genes involved in HS
biosynthesis, or encoding HS proteoglycan core proteins, have demonstrated a
critical role for HS proteoglycans in developmental processes
(57).
The observed phenotypes vary dramatically in severity, from failure to
gastrulate (8) to selective
disturbance of mast cell function in otherwise seemingly healthy mice
(9). Some of the biosynthetic
enzymes occur in multiple isoforms with potentially different functions and
thus different deletion phenotypes
(911).
IdoA occurs in all protein-binding HS domains so far identified. Here we show
that mouse embryos lacking GlcA C5-epimerase synthesize an abnormal HS, with
GlcA but no IdoA residues and a highly distorted sulfation pattern. The
resulting phenotype is lethal, with defects that can be differentially
ascribed to HS structural alterations. However, it also shows apparently
normal organ development, believed to depend on selective HS-protein
interactions. These findings raise intriguing questions as to the need for
regulation in HS biosynthesis.
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EXPERIMENTAL PROCEDURES
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Gene Targeting ConstructA 19-kb genomic clone containing
3'-untranslated region and 3 exons of the GlcA C5-epimerase gene
(Hsepi) was isolated from a bacteriophage mouse (strain 129/Sv)
genomic library (Stratagene). Three SacI fragments of this genomic
clone were separately cloned into Bluescript plasmid
(12). A 2-kb fragment
immediately upstream of exon 3 was inserted downstream of a PGK-neo
cassette as a short arm homologous sequence to the endogenous gene, and a 4-kb
fragment downstream of exon 3 was inserted upstream of the PGK-neo
cassette as a long arm of homology to the endogenous gene. The targeting
vector construct had a total size of 11 kb.
Homologous Recombination in ES Cells and Generation of Hsepi-deficient
MiceThe targeting vector was linearized by restriction enzyme
NotI and electroporated into R1 embryonic stem (ES) cells. Clones
expressing the neo-resistant gene were selected by including G418
(350 µg/ml; Invitrogen) in the cell cultures and analyzed for target gene
homologous recombination. The clone showing homologous recombination was
detected by Southern blot analysis using an external 700-bp fragment as probe
(Fig. 1A). The
positive clone was injected into C57BL/6 blastocysts, and chimeric male
founder mice were crossed with C57BL/6 females. The offspring was genotyped
for the mutation by tail biopsies using a PCR method described below. The
heterozygous mice were intercrossed to produce Hsepi mutant mice.
Phenotype studies were performed on mice with mixed genetic background
(129/SvJ/Sv and C57BL/6).

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FIG. 1. Targeted disruption of the Hsepi gene and generation of
Hsepi-deficient mice. A, structure of the Hsepi
gene (top), the targeting vector (middle), and the mutant
Hsepi gene following homologous recombination (bottom). The
expected size of products obtained by digestion with BglII
(B) and SalI (S), and visualized by hybridization
with the indicated probe, is shown for the wild type locus (top) and
for the mutant allele (bottom). The orientation of the neo
cassette is indicated. B, Southern blot of genomic DNA from embryos
of the intercross of Hsepi+/
heterozygous mice, after digestion with BglII and SalI.
Samples were hybridized with the external probe shown in A. C,
epimerase assay. The internal organs of embryos at d.p.c. 18.5 were
homogenized, and supernatants were incubated with C5-3H-labeled
epimerase substrate as described previously
(12). Activities were based on
the amounts of 3H2O generated. Samples 1 and
2, homozygous mutant; samples 3 and 8, wild type;
samples 47 and 910, heterozygous. Each
bar represents three independent assays.
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Genotype AnalysisGenotypes were determined by genomic
Southern blot analysis (Fig.
1A) or by PCR. Genomic DNA from ES cells and tail
biopsies were isolated as described previously
(14) and digested with
BglII and SalI. The resulting fragments were separated in
agarose gel and subsequently blotted onto a nylon membrane, followed by
hybridization with a 700-bp external probe as shown in
Fig. 1A. The PCR
amplification was performed using the following primers:
5'-AGTGTTCAAAGGATAAACTACAA-3' (upstream) for both wild type and
mutant, 5'-ACTCCATGCTGCTCTGAC-3' (downstream) for wild type
(729bp), and 5-GGAAGGATTGGAGCTACGGGGGT-3' (downstream) for mutant (521
bp).
HS Structure AnalysisPregnant mice (17.518.5 d.p.c.,
intercross of heterozygous mice) were injected with 0.5 mCi of
[6-3H]GlcN and were sacrificed after 6 h. Whole embryos were
homogenized in 4 M urea, 1% (v/w) Triton X-100, 50 mM
Tris-HCl, pH 7.4. Proteoglycans in the extracts were purified by
chromatography on DEAE-Sephacel, digested with chondroitinase ABC, and
subjected to alkaline
-elimination. Released HS chains were recovered by
anion-exchange chromatography. The purified HS samples were
N-deacetylated by hydrazinolysis and treated with HNO2 at
pH 1.5 and 3.9 to convert the chains to disaccharides, which were subsequently
reduced with NaB3H4
(15). The metabolically and
chemically 3H-labeled disaccharides were analyzed by anion-exchange
HPLC on a Partisil-10 SAX column. Products obtained on deamination of HS at pH
1.5 only (cleavage of chains at N-sulfated GlcN units
(15)) were analyzed by gel
chromatography on a Bio-Gel P-10 column (1.3 x 185 cm). The column was
eluted with 0.5 M NH4HCO3 at a flow rate of 2
ml/h.
Morphological AnalysisEmbryos from heterozygote matings
were dissected under microscopy. Skeletal preparations of newborn mice were
generated using an alcian blue-alizarin red staining method as described
previously (16). For
histological analysis, newborn mice were immediately fixed in 10% formalin.
The fixed animals were sectioned transversally at seven levels, each level
yielding nine consecutive 4-µm sections. The sections were stained with
hematoxylin and esoin.
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RESULTS AND DISCUSSION
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Targeted Disruption of Hsepi Results in Neonatal Lethality with
Respiratory FailureThe GlcA C5-epimerase is encoded by a single
Hsepi gene with three exons and accounts for the formation of IdoA
units in HS biosynthesis (12,
13). To assess the functional
significance of IdoA residues in HS, we have generated
Hsepi-deficient mice by gene targeting in ES cells. A targeting
vector was constructed to create a functional mutation by deletion of exon 3
(Fig. 1A)
corresponding to the 341 C-terminal amino acid residues of the 618-residue
protein. Following electroporation, homologous recombination in the
Hsepi gene was found in one ES cell clone, identified by Southern
blot analysis, with no additional sites of integration (not shown).
Microinjection of this clone into C57BL/6 blastocysts yielded chimeric animals
and germ line transmission. No overt defects were seen in heterozygous
animals.
Genotype analysis of offspring from intercrosses between heterozygous mice
(Fig. 1B) showed
essentially Mendelian heritance (22% wild type, 57% heterozygous, 21%
homozygous; n = 168), indicating no early embryonic death. The
offspring of heterozygous intercrosses was born on d.p.c. 18.5. Wild type
(Hsepi+/+) and heterozygous
(Hsepi+/) littermates showed
no aberrant phenotype and were fertile, with a lifespan of more than one year.
By contrast, Hsepi/
pups died immediately after birth, apparently from respiratory failure.
Analysis of embryo (d.p.c. 18.5) extracts demonstrated that
Hsepi/ pups lacked
detectable GlcA C5-epimerase activity, whereas heterozygotes showed decreased
activity compared with wild type animals
(Fig. 1C).
HS from
Hsepi/ Mice
Lacks IdoAHS from
Hsepi+/+,
Hsepi+/, and
Hsepi/ embryos (d.p.c.
17.518.5) was analyzed for composition, with particular regard to the
occurrence of IdoA. Samples were cleaved to disaccharides by exhaustive
deamination with nitrous acid, and the products were reduced with
NaB3H4 and analyzed by anion-exchange HPLC
(Fig. 2A). Notably,
all three samples showed the same overall proportions of
non-O-sulfated versus O-sulfated 3H-labeled
disaccharide units (Table I).
The Hsepi+/+ and
Hsepi+/ patterns of
O-sulfated disaccharides were indistinguishable and typical of normal
HS, with a predominance of mono-O- and di-O-sulfated,
IdoA-containing species. By contrast, these disaccharides were completely
absent from the Hsepi/
samples, which instead showed increased proportions of GlcA-containing
species, including a disaccharide with 2-O-sulfated GlcA
(Fig. 2A;
Table I). Gel chromatography of
metabolically [3H]GlcN-labeled HS indicated similar chain length
for the various samples (not shown), whereas the
Hsepi/ polysaccharide
emerged more retarded than the corresponding
Hsepi+/+ and
Hsepi+/ products on
anion-exchange chromatography (Fig.
2B). This difference in charge density was reflected by
changes in N-substitution pattern, as demonstrated by size analysis
of fragments obtained by selective deaminative cleavage of the HS chains at
N-sulfated glucosamine residues
(Fig. 2C). The
proportion of disaccharides, derived from contiguous N-sulfated
domains in mutant HS, was increased relative to wild type HS, whereas that of
intermediate sized (4- to 14-mer) fragments was decreased. Total
N-sulfation was calculated to 51 and 43% of disaccharide units in
Hsepi/ and
Hsepi+/+ HS, respectively. These
findings indicate a change in HS structure, from a largely intermixed
distribution of N-substituents to a pattern of extended
N-sulfated domains, along with a switch from
-IdoA(2-OSO3)-GlcNSO3-(upper structure in
Fig. 2D) to
-GlcA-GlcNSO3(6-OSO3)-(lower structure in
Fig. 2D) as the
predominant sulfated disaccharide unit.

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FIG. 2. Structural analysis of HS from embryonic mice. A, HS
samples were N-deacetylated by hydrazinolysis and treated with
HNO2 at pH 1.5 and 3.9 to convert the chains to disaccharides,
which were subsequently reduced with NaB3H4. The
3H-labeled (metabolically and chemically) disaccharides were
analyzed by anion-exchange HPLC on a Partisil-10 SAX column. Peaks represent:
1, GlcA-aManR and IdoA-aManR (separately
resolved by paper chromatography); 2,
GlcA(2-OSO3)-aManR; 3,
GlcA-aManR(6-OSO3); 4,
IdoA-aManR(6-OSO3); 5,
IdoA(2-OSO3)-aManR; 6,
IdoA(2-OSO3)-aManR(6-OSO3). The
aManR ([1-3H]anhydromannitol) residues are derived from
GlcN units (N-acetylated or N-sulfated) in intact HS.
Upper panel (+/+), HS from wild type embryos; lower
panel (/), HS from mutant embryos. B,
anion-exchange chromatography (DEAE-Sephacel, linear salt gradient) of
metabolically 3H-labeled HS from
Hsepi+/+ (open circles),
Hsepi+/ (closed
circles), and
Hsepi/ (open
triangles) embryos. C, gel chromatography on Bio-Gel P-10 (1.3
x 185 cm column) of products obtained on deamination at pH 1.5
(selective cleavage at N-sulfated GlcN units) of metabolically
3H-labeled HS from
Hsepi+/+ (upper panel) and
Hsepi/ (lower
panel) embryos. The sizes of even-numbered oligomers are indicated
above the peaks in the upper panel.
N-Acetyl/N-sulfate ratios were calculated based on peak areas.
The material in the peaks corresponding to the excluded volume (4245
ml) is resistant to heparitinase I, thus presumably not related to HS.
D, structures of the predominant N- and O-sulfated
disaccharide units found in wild type
(Hsepi+/+)(upper structure)
and in mutant
(Hsepi/)(lower
structure) HS.
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TABLE I Composition of disaccharides obtained upon deaminative cleavage of HS
isolated from embryonic (d.p.c 18.5) animals
Disaccharides were analyzed as described in the legend to
Fig. 2. Results are expressed
to account for the hexuronic acid composition of
-GlcA/IdoA-2R'-GlcNR'', 6R'-disaccharide units, in which
R' is H or , and
R'' is or
COCH3.
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Phenotype of
Hsepi/
MiceAll
Hsepi/ mice lacked
kidneys (Fig. 3A), but
showed no overt abnormalities in any other abdominal organs. The lungs of
mutant animals were poorly inflated and immature with thickened, cell-rich
alveolar walls (Fig.
3B). Furthermore, all
Hsepi/ animals showed
bilateral iris coloboma (Fig.
3C). Abundant skeletal abnormalities included shorter
body length (average 19 mm for
Hsepi/ animals, 26 mm
for wild type littermates) with generally excessive mineralization
(Fig. 3D), lack of
proximal phalanges and tarsal bones (Fig.
3, E and F), "twisted" tail
(Fig. 3G), post-axial
polydactyly in one or both forelimbs (Fig.
3H), malformed ribcage and sternum and often cleft palate
(not shown). However, the brain, heart, liver, gastrointestinal tract,
pancreas, and skin all appeared normal. Given the recognized importance of HS
in early embryonic patterning and morphogenesis
(5,
6), the selective nature of the
IdoA-deficient phenotype, albeit severe, was unexpected. The requirements for
defined HS structure in several functionally important interactions thus would
not seem to include the presence of IdoA units.

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FIG. 3. Phenotype display of
Hsepi/ and
Hsepi+/+ embryos born at d.p.c.
18.5. A, dissected urogenital systems of wild type (+/+) and
homozygous mutant (/) male embryos. a, adrenal gland;
b, bladder; k, kidney; t, testis. Note complete
absence of kidneys in the mutant embryo. B, lungs from
/ mutants are immature and poorly inflated, with thickened,
cell-rich interalveolar septa, compared with wild type +/+ lungs. Tissues were
fixed in 10% formalin, sectioned, and stained with hematoxylin and esoin.
C, iris coloboma of mutant mice. D, skeleton samples were
stained with alcian blue-alizarin red to distinguish ossified tissue
(red) from cartilage (blue). Multiple abnormalities are
seen, as described under "Results and Discussion." E and
F, all examined
Hsepi/ embryos
(n = 4) lacked the two proximal phalanges of the fore paws
(E) and the inner phalanx of hind paws (F; arrow
indicates tarsal bones). All
Hsepi/ embryos showed
twisted tail (G) and polydactyly on one or both forelimbs
(H).
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Comparison of the numerous defects in the
Hsepi/ mice with
phenotypes due to elimination of other enzymes involved in HS biosynthesis
shows similarities as well as differences. HS synthesized by mice deficient in
N-deacetylase/N-sulfotransferase isoform 1 (NDST-1) is low
in N-sulfate, hence in IdoA (sulfated and non-sulfated) and total
O-sulfate compared with wild type HS
(10).
NDST-1/ pups show an
immature lung phenotype similar to that of
Hsepi/ mice and die
mostly immediately after birth (although
30% die E14.5E18.5).
Skeletal defects of
NDST-1/ animals are
generally less pronounced than in
Hsepi/ mice, whereas
severe skull deformities and eye defects are more common
(5). The
NDST-1/ mice
have apparently normal kidneys, contrary to the
Hsepi/ embryos.
However, kidney agenesis is found also in
Hs2st/ mice
(17,
18) deficient in the HS
2-O-sulfotransferase that catalyzes 2-O-sulfation of both
IdoA and GlcA in HS biosynthesis
(4,
19). The HS synthesized by
Hs2st/ cells is devoid
of 2-O-sulfate groups
(20), whereas the overall
content of IdoA is similar to that of wild type HS
(21).2
The Hs2st/ mice
further show several skeletal abnormalities indistinguishable from those seen
in Hsepi/ animals
(17,
18). On the other hand,
contrary to the
NDST-1/ and
Hsepi/ mice, the
Hs2st/ mutants had
apparently normal lungs and remained alive for some hours
(17).
Differential Dependence of Development on HS Fine
StructureThe accumulated information provides some clues to
structure/function relations in HS biology. The multiple functions ascribed to
HS proteoglycans throughout embryonic development involve several signaling
networks, including FGF, Wnt, TGF-
, and Hedgehog pathways
(6,
22). The corresponding
signaling mechanisms appear either unaffected or perturbed by the lack of IdoA
in HS. Indian Hedgehog is expressed during gastrulation in mammals and is an
important regulator of developmental processes. Elimination in mice of the
EXT1 protein, which is essential for HS biosynthesis, resulted in loss of HS,
disruption of gastrulation, and embryonic lethality before E8.5
(23). In
EXT1/ embryos
Indian Hedgehog failed to bind to the appropriate target cells, whereas wild
type embryos showed strong binding. This critical interaction can apparently
be mediated also by grossly perturbed HS lacking either IdoA or
2-O-sulfate residues. The up-regulation in N-sulfation and
6-O-sulfation observed in both
Hsepi/
(Table I) and
Hs2st/ HS
(20) could conceivably
"compensate" for the loss of IdoA and 2-O-sulfate
residues, respectively, in some interactions crucial to development. On the
other hand, we have now identified certain phenotype traits that critically
depend on the presence of IdoA. Some of these features can be differentially
related to the sulfation state of this monosaccharide component, as revealed
by the Hsepi/ and
Hs2st/ phenotypes.
Lung maturation thus requires a minimal proportion of IdoA units (or a
structure associated with such units) that do not need to be sulfated. Other
events, including kidney, iris, and skeletal development, depend on HS with
2-O-sulfated IdoA residues. The distinctive
NDST-1/
phenotype points to signaling mechanisms with yet other requirements for HS
structure (5). The regulation
and temporal aspects of the expression and functional recruitment of HS
epitopes continue to provide challenges for future work.
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FOOTNOTES
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* This work was supported by grants from Swedish Medical Research Consil
(2309), the Swedish Cancer Society (4708-B0201XAA), the European
Commission (QLT-CT-1999.00536), and Polysackaridforskning AB (Sweden). The
costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
¶ Present address: Inst. of Blood Transfusion, Box 130, Beijing, 100850,
Peoples Republic of China. 
|| Present address: Dept. of Veterinary Medical Chemistry, Swedish Agriculture
University, Box 575, 751 23 Uppsala, Sweden. 
To whom correspondence should be addressed: Dept. of Medical Biochemistry and
Microbiology, University of Uppsala, The Biomedical Center, Box 582, SE-751 23
Uppsala, Sweden. Tel.: 46-18-4714241; Fax: 46-18-4714209; E-mail:
jin-ping.li{at}imbim.uu.se.
1 The abbreviations used are: HS, heparan sulfate; IdoA,
L-iduronic acid; GlcA, D-glucuronic acid;
Hsepi, HS D-glucuronyl C5-epimerase gene; NDST-1,
N-deacetylase/N-sulfotransferase isoform 1 gene; Hs2st,
HS 2-O-sulfotransferase gene; ES, embryonic stem; d.p.c., days
postcoitus; HPLC, high performance liquid chromatography. 
2 M. Kusche-Gullberg, personal communication. 
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ACKNOWLEDGMENTS
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We are grateful to Lena Kjellén, Valerie Wilson, Marion
Kusche-Gullberg, Dorothe Spillmann, Per Lindahl, and Alan McWhirter for
critical reading of the manuscript. The technical assistance of Lena Nylund is
acknowledged.
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REFERENCES
|
---|
- Bernfield, M., Gotte, M., Park, P. W., Reizes, O., Fitzgerald, M.
L., Lincecum, J., and Zako, M. (1999) Annu. Rev.
Biochem. 68,
729777[CrossRef][Medline]
[Order article via Infotrieve]
- Perrimon, N., and Bernfield, M. (2000)
Nature 404,
725728[CrossRef][Medline]
[Order article via Infotrieve]
- Casu, B., and Lindahl, U. (2001) Adv.
Carbohydr. Chem. Biochem. 57,
159206[Medline]
[Order article via Infotrieve]
- Esko, J. D., and Lindahl, U. (2001) J.
Clin. Invest. 108,
169173[Free Full Text]
- Grobe, K., Ledin, J., Ringvall, M., Holmborn, K., Forsberg, E.,
Esko, J. D., and Kjellen, L. (2002) Biochim. Biophys.
Acta 1573,
209215[Medline]
[Order article via Infotrieve]
- Nybakken, K., and Perrimon, N. (2002)
Biochim. Biophys. Acta
1573,
280291[Medline]
[Order article via Infotrieve]
- Tsuda, M., Kamimura, K., Nakato, H., Archer, M., Staatz, W., Fox,
B., Humphrey, M., Olson, S., Futch, T., Kaluza, V., Siegfried, E., Stam, L.,
and Selleck, S. B. (1999) Nature
400,
276280[CrossRef][Medline]
[Order article via Infotrieve]
- Lin, X., Buff, E. M., Perrimon, N., and Michelson, A. M.
(1999) Development (Camb.)
126,
37153723[Abstract/Free Full Text]
- Forsberg, E., Pejler, G., Ringvall, M., Lunderius, C.,
Tomasini-Johansson, B., Kusche-Gullberg, M., Eriksson, I., Ledin, J., Hellman,
L., and Kjellen, L. (1999) Nature
400,
773776[CrossRef][Medline]
[Order article via Infotrieve]
- Ringvall, M., Ledin, J., Holmborn, K., van Kuppevelt, T., Ellin,
F., Eriksson, I., Olofsson, A. M., Kjellen, L., and Forsberg, E.
(2000) J. Biol. Chem.
275,
2592625930[Abstract/Free Full Text]
- HajMohammadi, S., Enjyoji, K., Princivalle, M., Christi, P., Lech,
M., Beeler, D., Rayburn, H., Schwartz, J. J., Barzegar, S., De Agostini, A.
I., Post, M. J., Rosenberg, R. D., and Shworak, N. W. (2003)
J. Clin. Invest. 111,
989999[Abstract/Free Full Text]
- Li, J. P., Gong, F., El Darwish, K., Jalkanen, M., and Lindahl, U.
(2001) J. Biol. Chem.
276,
2006920077[Abstract/Free Full Text]
- Crawford, B. E., Olson, S. K., Esko, J. D., and Pinhal, M. A.
(2001) J. Biol. Chem.
276,
2153821543[Abstract/Free Full Text]
- Laird, P. W., Zijderveld, A., Linders, K., Rudnicki, M. A.,
Jaenisch, R., and Berns, A. (1991) Nucleic Acids
Res. 19,
4293[Medline]
[Order article via Infotrieve]
- Maccarana, M., Sakura, Y., Tawada, A., Yoshida, K., and Lindahl, U.
(1996) J. Biol. Chem.
271,
1780417810[Abstract/Free Full Text]
- Kessel, M., and Gruss, P. (1991)
Cell 67,
89104[Medline]
[Order article via Infotrieve]
- Bullock, S. L., Fletcher, J. M., Beddington, R. S., and Wilson, V.
A. (1998) Genes Dev.
12,
18941906[Abstract/Free Full Text]
- Merry, C. L., and Wilson, V. A. (2002)
Biochim. Biophys. Acta
1573,
319327[Medline]
[Order article via Infotrieve]
- Rong, J., Habuchi, H., Kimata, K., Lindahl, U., and
Kusche-Gullberg, M. (2001) Biochemistry
40,
55485555[CrossRef][Medline]
[Order article via Infotrieve]
- Merry, C. L., Bullock, S. L., Swan, D. C., Backen, A. C., Lyon, M.,
Beddington, R. S., Wilson, V. A., and Gallagher, J. T. (2001)
J. Biol. Chem. 276,
3542935434[Abstract/Free Full Text]
- Bai, X., and Esko, J. D. (1996) J. Biol.
Chem. 271,
1771117717[Abstract/Free Full Text]
- Baeg, G. H., Lin, X., Khare, N., Baumgartner, S., and Perrimon, N.
(2001) Development (Camb.)
128,
8794[Abstract/Free Full Text]
- Lin, X., Wei, G., Shi, Z., Dryer, L., Esko, J. D., Wells, D. E.,
and Matzuk, M. M. (2000) Dev. Biol.
224,
299311[CrossRef][Medline]
[Order article via Infotrieve]