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First published online August 24, 2007
doi: 10.1242/10.1242/dev.007674
1 Boston Biomedical Research Institute, Watertown, MA 02472, USA.
2 Department of Medical Biochemistry and Microbiology, Uppsala University
Biomedical Center, PO Box 582, S-75123, Uppsala, Sweden.
3 Department of Biomedicine, Division of Physiology, University of Bergen, Jonas
Lies vei 91, 5009 Bergen, Norway.
4 Center for Stem Cell Biology, Vanderbilt University, Nashville, TN 37232,
USA.
* Authors for correspondence (e-mails: xingbina{at}bbri.org; emersonc{at}bbri.org)
Accepted 14 July 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSupplementary materialREFERENCES |
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Key words: SULF1, SULF2, Heparan sulfate, GDNF, Esophagus, Innervation, Intrinsic neuron, Enteric glial cell, Neural crest progenitor, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSupplementary materialREFERENCES |
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). HS is a linear
polysaccharide chain covalently attached to a protein core of heparan sulfate
proteoglycans (HSPGs) and consists of repeating disaccharide units of uronic
acid linked to glucosamine (Esko and
Lindahl, 2001). During HS biosynthesis in the Golgi, disaccharide
units can be sulfated at the 2-O position of uronic acid and N-, 3-O and 6-O
positions of glucosamine by specific sulfotransferases. Sulfation along the HS
chain is incomplete, thus creating highly sulfated domains separated by
partially sulfated and non-sulfated domains. The highly sulfated domain
interacts with a variety of extracellular signaling ligands and receptors and
is therefore considered the major signaling domain of HS. However, owing to a
lack of biochemical tools and genetic models through which specific sulfate
groups of HS can be modified, it remains controversial whether such
interactions are determined non-selectively by the overall negative charge, or
selectively through establishment of fine sulfated HS sequences - an `HS
code'-within the highly sulfated domain
(Kreuger et al., 2006).
Sulfs are newly discovered extracellular heparan sulfate 6-O-endosulfatases
that have unique structural features, enzymatic activities and signaling
functions (Ai et al., 2005).
Sulfs contain the essential enzymatic sequences conserved among all
sulfatases, as well as distinct hydrophilic sequences that are required both
to dock Sulfs on the cell surface and for their enzymatic activities
(Dhoot et al., 2001;
Ai et al., 2006). SULF1 and
SULF2 in vertebrates have similar substrate specificity towards a selective
subset of 6-O-sulfate groups within the highly sulfated domain of HS chains,
implicating these enzymes as regulators of the `HS code'
(Morimoto-Tomita et al., 2002;
Ai et al., 2003). Sulfs remodel
HS 6-O-sulfation pattern on the cell surface to regulate HS binding to signal
ligands and receptors in a diversity of signaling pathways, including Wnt, FGF
and HGF, BMP and SHH (Ai et al.,
2005). Sulfs are dynamically expressed in the embryonic tissues
(Dhoot et al., 2001;
Danesin et al., 2006;
Lum et al., 2007). Avian SULF1
controls Wnt-dependent myogenic specification and is implicated in
SHH-regulated oligodendroglial specification
(Dhoot et al., 2001;
Danesin et al., 2006).
The functions of Sulfs in mammals are unknown. Recent studies report that
Sulf single- and double-mutant mice appear normal at birth, and
Sulf2-/- and
Sulf1-/-;Sulf2-/- mice have reduced body weight
and double-mutant mice die soon after birth
(Lum et al., 2007;
Lamanna et al., 2006),
although the cellular basis for this growth phenotype was not identified. To
investigate the developmental signaling functions of Sulfs, we established
independent lines of Sulf mutant mice using gene targeting. We identify a
primary neuronal innervation defect of
Sulf1-/-;Sulf2-/- esophagi, providing an
explanation for the severe growth defects in these mice. Furthermore, we show
that this esophageal defect in
Sulf1-/-;Sulf2-/- mice is due to aberrant GDNF
signaling.
The neuronal innervation of muscles along the gastrointestinal tract is
controlled by GDNF, a HS-dependent neurotrophic factor derived from the target
muscles (Baloh, et al., 2000;
Barnett et al., 2002;
Rickard et al., 2003). In the
embryonic esophagi, muscle progenitors express GDNF beginning at embryonic day
(E) 10, peaking between E11 and E16 and diminishing at E18
(Golden et al., 1999). GDNF
not only promotes the proliferation of the enteric neural crest precursors and
support their neuronal and glial differentiation
(Heuckeroth et al., 1998), but
also acts as a target-derived chemoattractant for directed neurite outgrowth
of both `intrinsic' enteric neurons and `extrinsic' neurons whose cell bodies
are located in ganglia outside of the esophagus
(Young et al., 2001;
Yan et al., 2004). Neurons
reach the target muscle during the initiation of GDNF expression in the
embryonic esophagus (Durbec et al.,
1996; Sang and Young,
1997), and the formation of functional innervations and enteric
glial cells initiates in the embryo and proceeds postnatally until completion
around two weeks after birth (Sang and
Young, 1997; Breuer et al.,
2004). The esophageal muscle in the muscularis externa (ME)
matures from smooth muscle to skeletal muscle along with the neuronal
innervation (Rishniw et al.,
2003). In the adult esophagi, the striated muscle in the ME is
innervated by both intrinsic and extrinsic neurons
(Sang and Young, 1998;
Neuhuber et al., 2006),
whereas the smooth muscle in the muscularis mucosae is directly innervated
mostly by intrinsic neurons, which elicit the smooth muscle contractility in
response to extrinsic nerves, as suggested by previous electrophysiological
studies (Kamikawa and Shimo,
1979; Storr et al.,
2001; Worl et al.,
2002). Defects in neural innervation can lead to neonatal death
and a variety of esophageal disorders, such as achalasia and a motility
disorder, congenital idiopathic megaesophagus
(Longstretch and Walker, 1994;
Neuhuber et al., 2006).
GDNF signaling requires 2-O- and 6-O-sulfate groups of HS or heparin, a
highly sulfated HS derivative, for binding to HS and for GDNF signaling
(Barnett et al., 2002;
Rickard et al., 2003). Whether
Sulfs regulate the GDNF signaling pathway has not been investigated. In this
study, we establish that Sulfs are required for GDNF signaling during
esophageal innervation. We show that Sulf mutant HS has a highly selective
increase of the trisulfated disaccharides without affecting other structural
properties, establishing Sulfs as major regulators of cellular HS
6-O-desulfation. Furthermore, SULF1 and SULF2 are dynamically and
differentially expressed by the GDNF-expressing muscle progenitors and
neuronal progenitors to promote GDNF-mediated neurite sprouting. By further
demonstrating that neurite outgrowth of the esophageal explants requires GDNF,
but not neurotrophins or other previously known Sulf-regulated signaling
ligands, we establish functional specificity of Sulfs in the GDNF pathway
during esophageal innervation. Our findings provide the first evidence that
Sulf enzymes are in vivo regulators of an `HS code' that controls HS fine
sulfated structures to coordinate the responses of Sulf-expressing cells to
developmental signals.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSupplementary materialREFERENCES |
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). Genotyping was performed by DNA extraction from
tail followed by PCR. The Sulf single-mutant mice, maintained as heterozygous,
were backcrossed into C57BL6 background (sixth generation). The
Sulf1-/- and Sulf2-/- mice were mated
to generate the double-heterozygous mutants.
Sulf1-/-;Sulf2-/- mice or embryos were
generated by crossing Sulf1+/-;Sulf2+/- mice
with Sulf1-/-;Sulf2+/- mice or by mating
between Sulf1-/-;Sulf2+/- mice. The
Sulf1-/-;Sulf2-/- mice were in a mixed
129SvEv/C57BL/6 genetic background.
Histochemistry
The lower-half thoracic segment of the esophagus was dissected from control
and Sulf1-/-;Sulf2-/- mice, rinsed gently in
ice-cold PBS and fixed in periodate-lysine-paraformaldehyde (PLP) for 2 hours
at 4°C. Tissues were then embedded in OCT and 10 µm serial cryosections
were collected. Sections were stained with Hematoxylin and Eosin (H&E).
Adjacent, unstained sections were used for immunohistochemical studies (see
below).
Single- and multi-label immunohistochemistry
Immunohistochemistry on tissue sections was performed as described
previously (Ai et al., 2003),
except for an additional 1-hour blocking with MOM blocking reagent (Vector)
for mouse monoclonal antibodies. Antigen-antibody complexes were detected
either by fluorescence or by chromogenic substrate. For explant cultures or
whole-mount staining, the washes were extended to 30 minutes each. Primary
antibodies included: (1) rabbit anti-glial fibrillary acidic protein (GFAP)
(Dako; 1:100); (2) rabbit anti-MS1HD (1:100); (3) rabbit anti-MS2HD (1:100);
(4) mouse anti-skeletal fast myosin (clone MY-32) alkaline phosphatase
conjugate (Sigma; 1:300); (5) goat anti-GDNF (R&D Systems; 2 µg/ml);
(6) mouse anti-neuron-specific class III ß-tubulin antibody (clone TuJ1)
(R&D Systems; 1:500); (7) rabbit anti-smooth muscle-specific SM22 (a gift
from Dr Mario Gimona, Austrian Academy of Sciences; 1:1000); (8) rabbit
anti-p75 (Upstate; 1:200); (9) goat anti-GFR
1 (R&D systems; 1
µg/ml); (10) rabbit anti-RET (Santa Cruz Biotechnology; 1:50); (11) rabbit
anti-phosphorylated p44/42 MAPK (Cell Signaling; 1:500).
Esophagus explant cultures
The 
400 µm esophagus was dissected from E11.5 mouse embryos and
cultured in 500 µl growth medium (DMEM plus 10% fetal bovine serum;
Mediatech) on presolidified collagen gel in a 24-well plate. GDNF, NGF, BDNF,
NT3 or NT4 (R&D Systems) were added immediately to the collagen/DMEM
mixture at various concentrations. Esophagus explants were cultured on
collagen gel for 4-5 days before fixation with 4% paraformaldehyde in PBS
followed by immunohistochemistry as described above. After antigen-antibody
complexes were detected by diaminobenzine (DAB) substrate, the explants were
examined using a Nikon TS100 microscope and photographed using a Nikon E4300
camera to show the whole explant with extended neuritis. To quantify the
number of neurons in the explant, immunostained explants were examined using a
Leica DMR microscope and photographed using a Leica DC300F camera.
Tissue culture
Cells were cultured in growth medium plus 1% antibiotics (Gibco). To
activate signaling, cells (1x105 per well of a 24-well plate)
were serum-starved in DMEM for 6 hours before adding GDNF or NGF at various
concentrations. After stimulation, cells were lysed for western blot or for
co-immunoprecipitation.
Immunoblot analysis
Immunoblot analysis was as described previously
(Dhoot et al., 2001). The
intensity of the signal was quantified by Multi-analysis software (Bio-Rad).
The primary antibodies for immunoblots included: rabbit anti-MAPK (1:4000;
Sigma), mouse anti-phosphorylated MAPK (1:1000; Sigma), rabbit
anti-phosphorylated AKT (1:1000; Cell Signaling), rabbit anti-GDNF (0.5
µg/ml); mouse anti-phosphorylated Tyr (1:1000; Calbiochem); rabbit anti-RET
(1:500).
Measurement of contraction forces of the esophagus skeletal muscle and smooth muscle
Esophagus was dissected from P12 or adult mice. To measure contractions of
skeletal muscles, the 5-mm longitudinal esophagus slices were cut out, tied
with silk threads at both ends and hooked up to tungsten needle tips. One of
the needles was connected to a force transducer (AM801, SensoNor, Horten,
Norway) and the other to a micromanipulator to stretch the muscle length to
1.1 times the slack. Platinum electrode wires were placed near the tubes on
both sides. The esophagus slices were stimulated by 1-millisecond duration
square pulses (TSS20, Intracel, UK), one single pulse for twitch and 30 Hz for
1 minute for tetanus. To measure contractions of smooth muscle, the smooth
muscle cross-rings were dissected from 1-mm esophagus tubes and then hooked up
to two needle tips. The rings were stretched to 1.2-1.3 times the slack. The
muscle preparations were then immersed into the normal external solution [150
mM NaCl, 4 mM KCl, 2 mM Ca-methanesulphonate (Ca-Ms), 2 mM Mg-Ms, 5.6 mM
glucose, 5 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH
7.4] in a well on a bubble plate to allow for rapid solution exchange. Force
records were amplified with a bridge-amplifier, which was connected to a
data-acquisition system (PowerLab, ADInstruments, Colorado Spring, CO).
Depolarizing external solution had K-Ms substituted equally for NaCl with
other chemicals in the same concentrations. Compounds used in the recoding
were applied at the following final concentrations: 10 µM atropine, 2
µg/ml alpha-BTx, 100 µM ATP, 124 mM K+, 30 µM histamine
and 30 µM carbachol. All experiments were carried out at 30°C.
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1). To allow GDNF binding to heparin, 20 µl of digested
heparin-conjugated agarose beads were incubated with GDNF in PBS (a total
volume of 100 µl) at room temperature for 1 hour. The amount of GDNF bound
to the heparin-conjugated agarose beads was assayed by immunoblot analysis. To
allow GDNF binding to GFR1, 10 ng GDNF, 1 µg GFR1-Fc and
heparin were mixed in 50 µl PBS for 30 minutes at room temperature. The
GDNF-heparin-GFR1-Fc complex was purified with 10 µl protein
A-agarose beads. The amount of GDNF bound to GFR1 was assayed by
immunoblot analysis.
HS preparation and disaccharide analysis
Mouse embryonic fibroblasts (MEFs) were isolated from the skin of E14.5
mouse embryos after dissociation with dispase II (Boehringer Mannheim; 2
mg/ml). MEFs were cultured in DMEM with 10% fetal bovine serum. The
radiolabeling, preparation and structural analysis of the HS were performed as
described previously (Ai et al.,
2003).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSupplementary materialREFERENCES |
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). Although Sulf mutant embryos express partial Sulf
transcripts and polypeptides recognizable by anti-Sulf antibodies (see Fig.
S1E and Fig. S2D in the supplementary material; data not shown), removal of
exon 2 eliminates the full-length Sulf transcripts (see Fig. S1E and Fig. S2D
in the supplementary material) and disrupts Sulf protein function, as shown by
the disaccharide analyses of 35S-radiolabeled glycosaminoglycans
(GAGs) (Fig. 1A). GAGs were
isolated from cultured primary mouse embryonic fibroblasts (MEFs) of the E14.5
skin that express both SULF1 and SULF2 (see Fig. S1E and Fig. S2D in the
supplementary material). Loss of Sulf1 selectively increased the
abundance of the trisulfated disaccharide IdoA2S-GlcNS6S (ISMS), the known
substrate of Sulf enzymes, by 30% (Ai
et al., 2003), and proportionately decreased the disulfated
IdoA2S-GlcNS (ISM) disaccharide, whereas it had no effect on the other two
disaccharides, GMS and IMS (Fig.
1A, Table 1). By
contrast, Sulf2-/- and
Sulf1+/-;Sulf2+/- MEFs had unchanged levels of
ISMS disaccharide, suggesting redundant Sulf protein activity in MEF cells.
The magnitude of the increase in ISMS in various Sulf mutant MEFs was
generally proportional to the number of mutant Sulf alleles
(Table 1). Most significantly,
HS isolated from Sulf1-/-;Sulf2-/- MEFs had
double the ISMS content (63%), a level comparable to that of heparin, a highly
sulfated HS derivative. Sulf mutant MEFs of all genotypes had normal HS chain
length, anionic properties, N-sulfation and chondroitin sulfate contents (data
not shown). These results establish that Sulf mutants have loss-of-function
mutations, and that Sulf enzymes are the major regulators of HS 6-O-sulfation
in vivo.
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10% less than wild-type
littermates. The Sulf1-/-;Sulf2-/- embryos and
neonates appeared normal at birth (data not shown) and fully viable based on
observed mendelian frequencies at E11.5 (14/107, expected 1/8), at E14.5
(14/69, expected 1/4) and at birth (34/311, expected 1/8). We identified a
primary developmental defect in esophageal innervation of
Sulf-/-;Sulf2-/- embryos that is likely to
cause the severe postnatal growth defect of a majority of
Sulf1-/-;Sulf2-/- pups (41/76) that was evident
as early as postnatal day 3 (P3) (Fig.
1B), with death at P14.
Sulf1-/-;Sulf2-/- pups that survived into
adulthood (35/76) were runted and had greatly reduced fertility
(Fig. 1B,
Table 1). Approximately 60% of
adult Sulf1-/-;Sulf2-/- mice developed
megaesophagus phenotypes as early as 2 months of age, characterized by food
accumulation in the esophagus, coughing, labored breathing and lung infection
(Fig. 1C). The esophagus of
Sulf1-/-;Sulf2-/- mice had an enlarged lumen
with a normal arrangement of the muscle layers and esophageal epithelium by
H&E staining and by immunostaining with specific markers for each cell
type, although these tissue layers appeared much thinner owing to the dilation
of the esophagus (Fig. 1C; data
not shown). We did not observe inflammatory cells in the
Sulf1-/-;Sulf2-/- esophagi at P12 or in
surviving adults (data not shown), ruling out the possibility of immune
cell-mediated tissue injury. In addition, we did not detect compensatory
expression of the intact Sulf gene in single-mutant esophagi (data not
shown).
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1-2%), this suggested that SULF1 and SULF2 have redundant functions that
contribute to the megaesophagus phenotype in
Sulf1-/-;Sulf2-/- mice. To investigate this
possibility, Sulf1 and Sulf2 expression in embryos was
assayed by in situ hybridization and by immunostaining using specific
antibodies generated against the hydrophilic domains of SULF1 (MS1HD) and
SULF2 (MS2HD) (see Fig. S3A,B in the supplementary material). The tissue
distribution of Sulf mRNA completely overlaps with protein expression,
establishing the specificity of the Sulf antibodies
(Fig. 1 and see Fig. S3C in the
supplementary material) and consistent with Sulfs being membrane-docking and
lacking free secretion (Dhoot et al.,
2001). The expression levels of Sulfs were relatively low before
E9.5 (data not shown). At E14.5 and E16.5, SULF1 and SULF2 were expressed at
higher levels in partially overlapping patterns in a variety of embryonic
tissues, including the floor plate of the neural tube, bone, cartilage,
skeletal muscle and lung (see Fig. S3C in the supplementary material)
(Lum et al., 2007).
SULF1 and SULF2 were found to be dynamically and differentially expressed
in the embryonic esophagus (Fig.
2). They were first detectable around E11.5, peaked around E14.5,
decreased dramatically by E18.5 and were undetectable 2 weeks after birth and
in the adult (Fig. 2; data not
shown). SULF1 expression did not co-localize with neuronal ß-tubulin
(TUBB3 - Mouse Genome Informatics), as labeled by the TuJ1 antibody
(Fig. 2F). Instead,
Sulf1 mRNA and protein were detected at the esophageal ME at E14.5,
when the smooth muscle forms prior to maturation into the skeletal muscle
(Fig. 2B-E). To confirm muscle
expression of SULF1, double labeling was performed with antibodies against
SULF1 and the smooth muscle marker SM22 (TAGLN - Mouse Genome Informatics).
Although both antibodies were raised in rabbit, we distinguished
membrane-bound SULF1 from intracellular SM22 by their differential subcellular
localization (Fig. 2C-E). We
found that SULF1 outlined the membrane of SM22-expressing cells at the outer
layer of E14.5 esophagus, and was localized at both outer and inner muscle
layers of the esophagus at E16.5 (Fig.
2H and see Fig. S3D in the supplementary material). Esophageal
muscle progenitors also express GDNF
(Golden et al., 1999). To test
whether SULF1 and GDNF co-localize, we characterized GDNF expression in the
esophagus at E14.5 and E16.5 for comparison with SULF1 expression. GDNF was
found on the cell surface and appeared diffusely across the esophageal ME at
E14.5 (Fig. 2G), including the
SULF1-expressing outer layer, and overlapped with SULF1 at E16.5
(Fig. 2H-J). By contrast, SULF2
was detected within the esophageal muscle layers and was tightly associated
with the neuronal marker TuJ1 (Fig.
2K-N and see Fig. S3E in the supplementary material). As SULF2 and
neuronal ß-tubulin have differential subcellular localization, the
observed close association between these two proteins indicates that SULF2 is
expressed by innervating neuronal progenitors. The differential expression of
SULF1 and SULF2 by muscle progenitors and by innervating neural progenitors
suggests Sulf regulation of neuron-muscle interaction in the embryonic
esophagi.
Esophagi of Sulf1-/-;Sulf2-/- mice have impaired smooth muscle contractility
The esophageal defects in Sulf1-/-;Sulf2-/-
mice may result from impaired muscle contractility owing to defects in muscle
maturation or in neuronal innervation. To distinguish these possibilities, we
characterized the formation of the esophageal muscle
(Fig. 2). The striated muscles
of ME expressed the myogenic genes - including those encoding myosin heavy
chain [MHC (MYHS)], MYF5, MYOD (MYOD1 - Mouse Genome Informatics) and myogenin
- normally at P14, when Sulf1-/-;Sulf2-/- pups
start to die (Fig. 3A; data not
shown). Also, Sulf1-/-;Sulf2-/- esophagi
complete skeletal muscle maturation normally at P15
(Rishniw et al., 2003), as
shown by the expression of fast skeletal myosin in the esophageal abdominal
segment connecting to the stomach (Fig.
3A). In addition, Sulf1-/-;Sulf2-/-
mice that survived into adulthood expressed fast skeletal muscle myosin and
nicotinic acetylcholine receptor clusters normally in the esophageal striated
muscle (Fig. 3A; data not
shown). Furthermore, isolated thoracic longitudinal segments of the adult
Sulf1-/-;Sulf2-/- esophagi produced a
comparable contractile force in response to twitch and tetanus electrical
stimuli with those of controls (Fig.
3B) (Worl et al.,
2002). To distinguish forces generated by nerve stimulation from
forces generated from direct muscle stimulation, we applied alpha-bungarotoxin
(alpha-Btx) to block electrical stimuli-induced nerve input by inhibiting
postsynaptic nicotinic acetylcholine receptors. Alpha-Btx significantly
inhibited both twitch and tetanus forces in both
Sulf1-/-;Sulf2-/- and control esophagi in a
similar manner, demonstrating that the skeletal muscle innervation is normal
in Sulf1-/-;Sulf2-/- esophagi. After the
treatment with alpha-BTx, the remaining forces were resistant to atropine, an
inhibitor of muscarinic receptors on smooth muscle
(Fig. 3B), but were completely
inhibited by further addition of tetrodotoxin in
Sulf1-/-;Sulf2-/- and control esophagi (data
not shown). These results establish that the electrical stimuli-induced forces
are produced largely by skeletal muscle, not by smooth muscle, in the
esophagus. The electrical stimuli elicited relatively low levels of smooth
muscle contractility, as evidenced by the small shoulders after the tetanus
stimuli and by their sensitivity to inhibition by atropine
(Fig. 3B). We observed no
significant difference in skeletal muscle contractility between the
double-mutant and control esophagi. Therefore, the development, maturation and
function of esophageal skeletal muscle is unaffected in
Sulf1-/-;Sulf2-/- mice.
By contrast, smooth muscle contractility was found to be impaired in
Sulf1-/-;Sulf2-/- esophagi. The contractile
forces of the isolated smooth muscle ring at the thoracic segments of the
esophagi were elicited by various stimuli such as electrical, high
K+, carbachol, ATP and histamine
(Storr et al., 2001;
Worl et al., 2002). The smooth
muscle contractility elicited by electrical stimuli (the small shoulders after
tetanus stimuli, Fig. 3B) was
comparable in control and Sulf1-/-;Sulf2-/-
mutant esophagi. In addition, compared with
Sulf1+/-;Sulf2+/- controls, the
Sulf1-/-;Sulf2-/- esophageal smooth muscles
showed a greatly diminished response to carbachol, an agonist of the
muscarinic receptor, but only partially reduced response to other stimuli
(Fig. 3C,D). A similar decrease
in carbachol-induced smooth muscle contractility was observed in
Sulf1-/-;Sulf2-/- esophagi at P15 (data not
shown). No smooth muscle contractility defects were observed in the lower
sphincter muscle of the Sulf1-/-;Sulf2-/-
esophagi (data not shown). The esophageal defects of
Sulf1-/-;Sulf2-/- mice are different from
symptoms of achalasia in which the lower sphincter muscle contractility is
deregulated owing to a loss of inhibitory nitrinergic neurons
(Longstretch and Walker, 1994;
Holland et al., 2002;
Neuhuber et al., 2006), but
are similar to those observed in congenital idiopathic megaesophagus.
Carbachol-induced smooth muscle contractility is likely to correlate with the
level of neuronal innervation in the esophagus, as shown in studies of sprouty
2 mutant mice (Taketomi et al.,
2005). The selective impairment in carbachol-induced smooth muscle
contractility of the Sulf1-/-;Sulf2-/- esophagi
therefore suggests specific neuronal innervation defects rather than a general
disruption of the smooth muscle structure in the muscularis mucosae.
Neuronal innervation and enteric glial cell numbers are reduced in the esophagi of Sulf1-/-;Sulf2-/- mice
To test whether esophageal innervation is defective in
Sulf1-/-;Sulf2-/- mice, we identified
innervating nerve fibers of both intrinsic and extrinsic neurons using the
TuJ1 antibody. At E14.5, before the neurites extend across the ME, the
Sulf1+/-;Sulf2+/- control and
Sulf1-/-;Sulf2-/- esophagi exhibited comparable
staining with TuJ1, GDNF, GDNF receptor 1 (GFR1), RET
(previously known as c-RET), or the low-affinity neurotrophin receptor p75
(NGFR - Mouse Genome Informatics) (Fig.
4A; data not shown). However, at E18.5,
Sulf1-/-;Sulf2-/- esophagi had much reduced
levels of neurite sprouting and innervation density at the smooth muscle of
the muscularis mucosae, although the number of intrinsic neurons and the
circumference of the esophagi were unchanged
(Fig. 4B). In addition, we
found no difference in p75 expression on the cell body or in the number of
p75-expressing intrinsic neurons between control and
Sulf1-/-;Sulf2-/- esophagi
(Fig. 4B). However, p75
expression on neurites innervating the smooth muscle was decreased in
Sulf1-/-;Sulf2-/- esophagi, providing
additional evidence that the innervation of the muscularis mucosae is
defective. Reduced smooth muscle innervation in
Sulf1-/-;Sulf2-/- esophagi persisted
postnatally and in the adult (Fig.
4C and see Fig. S4 in the supplementary material), which directly
affected smooth muscle contractility (Fig.
3C,D) and led to gradual enlargement of the esophageal lumen from
a 20% increase in the circumference of smooth muscle at P15
(Fig. 4C), to almost a doubling
in the circumference in adults (Fig.
1C and see Fig. S4 in the supplementary material).
To test whether enteric glial cells might also be affected in mutant
esophagi, we labeled the esophagus using antibodies against the glial cell
markers, glial fibrillary acidic protein (GFAP) and S100 (S100a1 - Mouse
Genome Informatics) at P15 and in the adult when enteric glial cells form and
mature (Fig. 4C and see Fig. S4
in the supplementary material; data not shown). We found that the number of
GFAP-expressing cells along the thoracic segments of the
Sulf1-/-;Sulf2-/- esophagi was reduced to
one-third and to half of that in the control esophagi at P15 and in adult,
respectively (Fig. 4C and see
Fig. S4 in the supplementary material). Since esophageal innervation is
controlled by GDNF (Yan et al.,
2004) and GDNF supports enteric glial differentiation from neural
crest progenitors (Heuckeroth et al.,
1998), the observed esophageal defects in
Sulf1-/-;Sulf2-/- mice suggest that Sulf
regulation of the GDNF signaling pathway establishes the esophageal
innervation that is required for postnatal feeding and growth.
SULF1 and SULF2 are required for GDNF-dependent neurite outgrowth in embryonic esophageal explants
To investigate whether SULF1 and SULF2 are required for GDNF-induced
neurite outgrowth of the endogenous neurons in the embryonic esophagus,
esophageal explants from E11.5 embryos were cultured on collagen gels
(Yan et al., 2004). After 4
days, the explants adhered to the collagen gel and exhibited a GDNF
dose-dependent neurite sprouting, as shown by TuJ1 immunoreactivity extending
from the explants in GDNF-induced cultures
(Fig. 5A). The sprouting was
not promoted by neurotrophins NGF (NGFß - Mouse Genome Informatics),
BDNF, NT3 (NTF3), NT4 (NTF5) or any other previously known Sulf-regulated
signaling ligand, including FGF2, HGF, VEGF165 (NRP1), Wnts, BMP2 and sonic
hedgehog (Fig. 5A-C and see
Fig. S6 in the supplementary material) (Ai
et al., 2005). Furthermore, heparin, but not dermatan sulfate,
blocked the GDNF-induced neurite outgrowth (see Fig. S6A,B in the
supplementary material), establishing the HS-dependent GDNF activity. The
neurite outgrowth from control esophageal explants could be induced by GDNF at
10 ng/ml, with maximal induction at 50 ng/ml GDNF
(Fig. 5A,C). By contrast,
Sulf1-/-;Sulf2-/- explants failed to extend
neurites at 10 ng/ml GDNF, and the neurite outgrowth was less than half of the
control levels at 20 ng/ml. The defect in the neurite outgrowth of
Sulf1-/-;Sulf2-/- esophageal explants was fully
rescued by 100 ng/ml GDNF (Fig.
5A,C). A lack of neurite outgrowth by
Sulf1-/-;Sulf2-/- esophageal explants at 10
ng/ml GDNF could be due to disrupted GDNF signaling in intrinsic neurons, or
to a reduction in the number of intrinsic neurons. To distinguish between
these two possibilities, we quantified the total number of TuJ1-immunoreactive
intrinsic neurons within the explants after 4 days in culture. We found that
GDNF is essential for neuron number, as control explants cultured without any
neurotrophic factors or in the presence of NGF had only one-third of the
neurons as explants treated with 10 ng/ml GDNF
(Fig. 5E).
Sulf1-/-;Sulf2-/- esophageal explants, although
they showed no significant neurite outgrowth at 10 ng/ml GDNF, had the same
number of intrinsic neurons as the controls
(Fig. 5E), consistent with the
in vivo phenotype. In addition, a few neurons within GDNF-treated control
explants migrated out of the explants (data not shown), whereas neurons of the
Sulf1-/-;Sulf2-/- explants never migrated out
of the explants (Fig. 5D),
suggesting a defect in GDNF-induced neural migration.
|
), suggesting that they have related functions. To test
whether SULF1 regulates GDNF signaling, we first examined whether SULF1
modulates GDNF binding to HS by comparing GDNF binding to heparin-agarose
beads that were predigested with either purified SULF1 or enzymatically
inactive QSULF1(C-A) (Fig. 6A)
(Ai et al., 2003). SULF1
activity significantly decreased GDNF binding to heparin by up to 6-fold at
sub-saturating amounts of GDNF, and by 30% under conditions of excess
GDNF (Fig. 6A). Second, we
tested whether SULF1 activity affects HS-regulated GDNF binding to
GFR1. GDNF and the extracellular domain of GFR1 (GFR1-Fc)
were incubated with various amounts of heparin predigested by SULF1 or
enzymatically inactive QSULF1(C-A) to allow the formation of a
GDNF-heparin-GFR1-Fc ternary complex. As reported previously, heparin
enhanced GDNF binding to GFR1 by 2-fold
(Fig. 6B)
(Rickard et al., 2003).
However, SULF1 had no effect in heparin-regulated GDNF binding to GFR1
(Fig. 6B), indicating that
SULF1-regulated HS 6-O-desulfation does not control GDNF binding to
GFR1. These findings suggest that SULF1 functions in the embryonic
esophagus to control the affinity of GDNF for HS in the extracellular matrix
to facilitate GDNF binding to RET receptor on GDNF-responding innervating
neurons.
|
). These studies of neural cell lines provide evidence that
SULF2 enhances GDNF signaling in neural progenitors during esophageal
innervation. To further demonstrate that Sulfs enhance GDNF signaling in vivo, we assayed the endogenous level of MAPK activation in E14.5 esophagi by immunohistochemistry. Compared with robust MAPK phosphorylation in cell bodies located within the control esophageal muscles, Sulf1-/-;Sulf2-/- enteric neurons showed a much reduced level of MAPK phosphorylation (Fig. 6G). By contrast, Sulf1-/-;Sulf2-/- epithelial cells surrounding the esophageal lumen exhibited normal MAPK phosphorylation (Fig. 6G). Although MAPK phosphorylation can be triggered by several signals, the GDNF pathway is one of the major signaling pathways in esophageal neuronal progenitors at E14.5. Therefore, this observation is consistent with our finding that Sulfs promote GDNF signaling activity, as shown by esophageal explant and cell signaling assays.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSupplementary materialREFERENCES |
|---|
Our studies also show that Sulfs are enhancers of GDNF signaling rather
than obligatory components in the GDNF pathway. Neuroblastoma-glioma cells,
which do not express Sulfs, are capable of transmitting the GDNF signal. In
addition, high concentrations of GDNF rescue the neurite outgrowth defect of
Sulf mutant esophagi in our explant assays, demonstrating that Sulf-deficient
neurons are competent to receive GDNF signal. The regulatory roles of Sulfs in
GDNF signaling contrast with the obligatory roles of GDNF or GDNF receptors,
as demonstrated by the observed differences in the number of enteric neurons
between Sulf mutant mice and mice deficient in GDNF or GDNF receptors
(Baloh et al., 2000). In
addition, we did not observe any significant innervation defects in the Sulf
double-mutant colon by immunolabeling (see Fig. S7 in the supplementary
material). These findings suggest that Sulf activity is not crucial for
enteric neural crest progenitor migration or myenteric plexus formation during
development, consistent with the relatively late onset of Sulf expression.
However, Sulfs are essential for the developmental transmission of GDNF
signaling during esophageal innervation in the embryo. The esophageal smooth
muscle is largely innervated by intrinsic neurons. Although extrinsic
innervation might also be affected, the observed 50% reduction of smooth
muscle innervation in Sulf double-mutant esophagi indicates Sulf function in
intrinsic neurons, which is consistent with the defects in GDNF-dependent
neurite outgrowth of the Sulf mutant esophageal explants.
As Sulf-deficiency leads to specific changes in HS 6-O-sulfation
without disrupting the overall charge or structure of HS chains, our findings
establish that Sulf desulfation generates HS with a distinct 6-O-sulfated fine
structure - an `HS code' - to control the specificity of extracellular
signaling responses to HS-dependent ligands and receptors. HS 6-O-sulfation is
known to be strictly controlled by developmental stage, tissue type and in
tumors (Esko and Lindahl,
2001; Kreuger et al.,
2006). Here, we show that Sulfs have dynamic expression and are
enzymatically active in embryos, indicating their dynamic regulatory roles.
Sulf mutant mice not only provide new tools for studies to decipher the
HS-sulfation code for regulation of ligand-receptor interactions, but also
open new opportunities to investigate the genetic and developmental signaling
mechanisms underlying the motility disorder, congenital idiopathic
megaesophagus. Additionally, Sulf mutant mice provide a new therapeutic model
for developmental investigations of GDNF-mediated neuroprotection in
Parkinson's disease and brain injury
(Airaksinen and Saarma, 2002)
and in other diseases involving Sulf-mediated HS-regulated signaling.
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Supplementary material |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSupplementary materialREFERENCES |
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ACKNOWLEDGMENTS |
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REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSupplementary materialREFERENCES |
|---|
Ai, X., Do, A., Lozynska, O., Kusche-Gullberg, M., Lindahl, U.
and Emerson, C. P., Jr (2003). QSulf1 remodels the 6-O
sulfation states of cell surface heparan sulfate proteoglycans to promote Wnt
signaling. J. Cell Biol.
162,341
-351.
Ai, X., Kusche-Gullberg, M., Lindahl, U. and Emerson, C. P., Jr (2005). Remodeling of heparan sulfate sulfation by extracellular endosulfatases. In Chemistry and Biology of Heparin and Heparan Sulfate (ed. H. G. Garg, R. J. Linhardt and C. A. Hales), pp. 245-258. New York: Elsevier.
Ai, X., Do, A., Kusche-Gullberg, M., Lindahl, U., Lu, K. and
Emerson, C. P., Jr (2006). Substrate specificity and domain
functions of extracellular heparan sulfate 6-O-endosulfatases, QSulf1 and
QSulf2. J. Biol. Chem.
281,4969
-4976.
Airaksinen, M. S. and Saarma, M. (2002). The GDNF family: signaling, biological functions and therapeutic value. Nat. Rev. Neurosci. 3,383 -394.[CrossRef][Medline]
Baloh, R. H., Enomoto, H., Johnson, E. M., Jr and Milbrandt, J. (2000). The GDNF family ligands and receptors - implications for neural development. Curr. Opin. Neurobiol. 10,103 -110.[CrossRef][Medline]
Barnett, M. W., Fisher, C. E., Perona-Wright, G. and Davies, J.
A. (2002). Signalling by glial cell line-derived neurotrophic
factor (GDNF) requires heparan sulphate glycosaminoglycan. J. Cell
Sci. 115,4495
-4503.
Breuer, C., Neuhuber, W. L. and Worl, J. (2004). Development of neuromuscular junctions in the mouse esophagus: morphology suggests a role for enteric coinnervation during maturation of vagal myoneural contacts. J. Comp. Neurol. 475,47 -69.[CrossRef][Medline]
Danesin, C., Agius, E., Escalas, N., Ai, X., Emerson, C. P. and
Soula, C. (2006). Ventral neural progenitors switch toward an
oligodendroglial fate in response to increased sonic hedgehog (Shh) activity:
involvement of sulfatase 1 in modulating Shh signaling in the ventral spinal
cord. J. Neurosci. 26,5037
-5048.
Dhoot, G. K., Gustafsson, M. K., Ai, X., Sun, W., Standiford, D.
M. and Emerson, C. P., Jr (2001). Regulation of Wnt signaling
and embryo patterning by an extracellular sulfatase.
Science 293,1663
-1666.
Durbec, P. L., Larsson-Blomberg, L. B., Schuchardt, A., Costantini, F. and Pachnis, V. (1996). Common origin and developmental dependence on c-ret of subsets of enteric and sympathetic neuroblasts. Development 122,349 -358.[Abstract]
Esko, J. D. and Lindahl, U. (2001). Molecular diversity of heparan sulfate. J. Clin. Invest. 108,169 -173.[CrossRef][Medline]
Golden, J. P., DeMaro, J. A., Osborne, P. A., Milbrandt, J. and Johnson, E. M., Jr (1999). Expression of Neurturin, GDNF, and GDNF family-receptor mRNA in the developing and mature mouse. Exp. Neurol. 158,504 -528.[CrossRef][Medline]
Heuckeroth, R. O., Lampe, P. A., Johnson, E. M. and Milbrandt, J. (1998). Neurturin and GDNF promote proliferation and survival of enteric neuron and glial progenitors in vitro. Dev. Biol. 200,116 -129.[CrossRef][Medline]
Holland, C. T., Satchell, P. M. and Farrow, B. R. (2002). Selective vagal afferent dysfunction in dogs with cogenital idiopathic megaesophagus. Auton. Neurosci. 99, 18-23.[CrossRef][Medline]
Kamikawa, Y. and Shimo, Y. (1979). Cholinergic and adrenergic innervations of the muscularis mucosae in guinea-pig esophagus. Arch. Int. Pharmacodyn. Ther. 238,220 -232.[Medline]
Kreuger, J., Spillmann, D., Li, J. P. and Lindahl, U.
(2006). Interactions between heparan sulfate and proteins: the
concept of specificity. J. Cell Biol.
174,323
-327.
Lamanna, W. C., Baldwin, R. J., Padva, M., Kalus, I., Ten Dam, G., van Kuppevelt, T. H., Gallagher, J. T., von Figura, K., Dierks, T. and Merry, C. L. (2006). Heparan sulfate 6-O-endosulfatases: discrete in vivo activities and functional co-operativity. Biochem. J. 400,63 -73.[CrossRef][Medline]
Lin, X. (2004). Functions of heparan sulfate
proteoglycans in cell signaling during development.
Development 131,6009
-6021.
Longstretch, G. F. and Walker, F. D. (1994). Megaesophagus and hereditary nervous system degeneration. J. Clin. Gastroenterol. 19,125 -127.[Medline]
Lum, D. H., Tan, J., Rosen, S. D. and Werb, Z.
(2007). Gene trap disruption of the mouse heparan sulfate
6-O-endosulfatase gene, Sulf2. Mol. Cell. Biol.
27,678
-688.
Morimoto-Tomita, M., Uchimura, K., Werb, Z., Hemmerich, S. and
Rosen, S. D. (2002). Cloning and characterization of two
extracellular heparin-degrading endosulfatases in mouse and human.
J. Biol. Chem. 277,49175
-49185.
Neuhuber, W. L., Raab, M., Worl, J. and Berthoud, H. R. (2006). Innervation of the mammalian esophagus. Adv. Anat. Embryol. Cell Biol. 185,1 -73.[Medline]
Rickard, S. M., Mummery, R. S., Mulloy, B. and Rider, C. C.
(2003). The binding of human glial cell line-derived neurotrophic
factor to heparin and heparan sulfate: importance of 2-O-sulfate groups and
effect on its interaction with its receptor, GFR1.
Glycobiology 13,419
-426.
Rishniw, M., Xin, H., Deng, K. and Kotlikoff, M. I. (2003). Skeletal myogenesis in the mouse esophagus does not occur through transdifferentiation. Genesis 36, 81-82.[CrossRef][Medline]
Sang, Q. and Young, H. M. (1997). Development of nicotinic receptor clusters and innervation accompanying the change in muscle phenotype in the mouse esophagus. J. Comp. Neurol. 386,119 -136.[CrossRef][Medline]
Sang, Q. and Young, H. M. (1998). The origin and development of the vagal and spinal innervation of the external muscle of the mouse esophagus. Brain Res. 809,253 -268.[CrossRef][Medline]
Storr, M., Geisler, F., Neuhuber, W. L., Schusdziarra, V. and Allescher, H. D. (2001). Characterization of vagal input to the rat esophageal muscle. Auton. Neurosci. 91, 1-9.[CrossRef][Medline]
Taketomi, T., Yoshiga, D., Taniguchi, K., Kobayashi, T., Nonami, A., Kato, R., Sasaki, A., Ishibashi, H., Moriyama, M., Nakamura, K. et al. (2005). Loss of mammalian Sprouty2 leads to enteric neuronal hyperplasia and esophageal achalasia. Nat. Neurosci. 8, 855-857.[Medline]
Tompers, D. M. and Labosky, P. A. (2004).
Electroporation of murine embryonic stem cells: a step-by-step guide.
Stem Cells 22,243
-249.
Worl, J., Dutsch, F. and Neuhuber, W. L. (2002). Development of neuromuscular junctions in the mouse esophagus: focus on establishment and reduction of enteric co-innervation. Anat. Embryol. 205,141 -152.[CrossRef][Medline]
Yan, H., Bergner, A. J., Enomoto, H., Milbrandt, J., Newgreen, D. F. and Young, H. M. (2004). Neural cells in the esophagus respond to glial cell line-derived neurotrophic factor and neuturin, and are RET-dependent. Dev. Biol. 272,118 -133.[CrossRef][Medline]
Young, H. M., Hearn, C. J., Farlie, P. G., Canty, A. J., Thomas, P. Q. and Newgreen, D. F. (2001). GDNF is a chemoattractant for enteric neural cells. Dev. Biol. 229,503 -516.[CrossRef][Medline]
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