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First published online August 24, 2007
doi: 10.1242/10.1242/dev.007674

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SULF1 and SULF2 regulate heparan sulfate-mediated GDNF signaling for esophageal innervation

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.

View larger version (56K): [in this window] [in a new window]   Fig. 1. Sulf1-/-;Sulf2-/- mice have cellular HS 6-O-sulfation, postnatal growth and dysfunctional esophageal phenotypes. (A) Disaccharide analysis of HS. Radiolabeled HS was isolated from MEFs of wild-type, Sulf1-/-, Sulf2-/- and Sulf1-/-;Sulf2-/- embryos at E14.5, followed by disaccharide analysis. Individual disaccharides are represented as the percentage of the total radioactivity. Data presented are mean and standard deviation of a minimum of two independent samples of each genotype. In disaccharide abbreviation, M stands for the 2,5-anhydromannitol deamination products of GlcNS residues. **, P<0.01; *, P<0.05. (B) Comparison of the body size and weight between wild-type, Sulf1-/-, Sulf2-/- and Sulf1-/-;Sulf2-/- female mice at P12 and after weaning (n=6 for each group). (C) Histology of the adult esophagus and lung of wild-type control and Sulf1-/-;Sulf2-/- mice. Sulf1-/-;Sulf2-/- mice have enlarged esophagi with food accumulated inside (compare a1,a2 with b1,b2, respectively) and develop lung infections (n=14). Eso, esophagus; ME, muscularis externa; MM, muscularis mucosae. Scale bars: 100 µm.

View larger version (138K): [in this window] [in a new window]   Fig. 2. SULF1 and SULF2 are differentially expressed in the embryonic esophagus. (A-G) SULF1 esophageal expression at E14.5. Sulf1 mRNA (B) and protein (C) were detected at the outer layer of the esophagus and the protein was mostly on the cell surface (insert in C). The outer layer of the esophagus expresses SM22, a smooth muscle marker (D). Immunostaining with the rabbit anti-MS1HD antibody and the rabbit anti-SM22 antibody together identified SULF1 on the membrane of SM22-expressing cells (E); the insert shows a magnified image of the outlined staining. SULF1 did not colocalize with the TuJ1 staining (F). GDNF was detected diffusively across the esophageal muscle layers and partially overlapped with SULF1 in the outer layer (G). (H-J) SULF1 expression and colocalization with GDNF in the esophageal muscle layers at E16.5 by double staining. (K-N) SULF2 esophageal expression at E14.5. Cross-sections were double stained with SULF2 and TuJ1 antibodies. SULF2 tightly associated with TuJ1 staining. Scale bars: 100 µm.

View larger version (26K): [in this window] [in a new window]   Fig. 3. The esophagi of Sulf1-/-;Sulf2-/- mice have normal skeletal muscle function, but impaired smooth muscle contractility. (A) Postnatal development of the esophageal skeletal muscles in control and Sulf1-/-;Sulf2-/- pups. The cross-sections of Sulf1+/-;Sulf2+/- control and Sulf1-/-;Sulf2-/- mice were immunostained with antibodies against skeletal muscle markers including myosin heavy chain (MHC) and MYF5 or incubated with Cy3-conjugated alpha-bungarotoxin (alpha-BTx) to identify acetylcholine (ACh) receptors on muscle. The completion of the esophageal skeletal muscle formation at P15 was assayed by immunostaining of the abdominal segments with an alkaline phosphatase-conjugated mouse antibody against fast skeletal myosin (sk-Myosin). The antigen-antibody complex was visualized using the substrate BM purple. Arrowheads mark the junction between the esophagus and the stomach. The Sulf1-/-;Sulf2-/- esophagi have completed skeletal muscle formation in the esophagus at P15. Scale bars: 100 µm. (B) Physiological measurements of the esophageal skeletal muscles. The lower-half thoracic segments of the esophagi of the adult Sulf1+/-;Sulf2+/- control and Sulf1-/-;Sulf2-/- mice were subject to twitch and tetanus stimuli. Muscle contractility was measured and compared with those in the presence of selective ion-channel blockers (n=3). The Sulf1-/-;Sulf2-/- esophagi exhibited comparable skeletal muscle contractility in response to the electrical stimuli and ion-channel blocks as the control esophagi. (C) Physiological tests of esophageal smooth muscle contractility. The smooth muscle of the control and the Sulf1-/-;Sulf2-/- mutant esophagi were dissected and their contractile forces in response to various stimuli measured (n=3). The Sulf1-/-;Sulf2-/- esophagi showed diminished smooth muscle contractility in response to carbachol, and partially reduced contractility in response to other chemicals. (D) Quantification of the esophageal smooth muscle contractility induced by various stimuli as shown in C.

View larger version (64K): [in this window] [in a new window]   Fig. 4. The esophagi of Sulf1-/-;Sulf2-/- mice have diminished neuronal innervation and enteric glial cells. The lower-half thoracic segments of the esophagus were immunolabeled either on cross-sections or by whole-mounts with various antibodies to examine neural innervation. (A) Comparable TuJ1 staining and expression of GFR1, p75 and GDNF in Sulf1+/-;Sulf2+/- control esophagi and Sulf1-/-;Sulf2-/- esophagi at E14.5 (n=3). (B) Reduced neural innervation at the esophageal smooth muscle layer in Sulf1-/-;Sulf2-/- esophagi at E18.5 (n=4). The whole esophagus was mounted on the glass slide after whole-mount immunohistochemistry with the TuJ1 antibody. One side of the flattened esophagus was photographed. The number of neurons (cell body marked by *) on one side of a 1-mm longitudinal segment was counted and four non-overlapping segments were counted for each esophagus. Numbers presented are the averages of the neural number per 1-mm segment. The innervation density at the smooth muscle layer was calculated by dividing the total number of innervating TuJ1+ neurites (white arrowheads) on cross-sections by the circumference of smooth muscle. The circumference of the smooth muscle was not different between control and Sulf1-/-;Sulf2-/- esophagi at E18.5 (control, 0.81±0.09 mm; mutant, 0.83±0.07 mm). A minimum of five serial cross-sections (200 µm apart) of each esophagus were quantified. The smooth muscle innervation was also shown by p75 staining (white arrowheads). Sulf1-/-;Sulf2-/- esophagi had the same number of neurons as the control, whereas the smooth muscle innervation was reduced in Sulf1-/-;Sulf2-/- esophagi. (C) Reduced esophageal innervation and enteric glial cells in Sulf1-/-;Sulf2-/- esophagi at P15. Arrowheads indicate the TuJ1+ neurites innervating the smooth muscle of muscularis mucosae. Arrows point to the enteric glial cells located between the skeletal muscle layers. The circumference of smooth muscle on cross-sections of Sulf1-/-;Sulf2-/- esophagi (1.37±0.18 mm) was 20% longer than that of littermate controls (1.15±0.13 mm). A minimum of 28 sections from four independent controls or Sulf1-/-;Sulf2-/- mice were counted. Data shown in the bar graphs represent the number of TuJ1+ neurites innervating smooth muscle, innervation density and the average number of enteric glial cells per cross-section. Statistics were calculated by two-tailed Student's t-test. Scale bars: 100 µm.

View larger version (76K): [in this window] [in a new window]   Fig. 5. Sulf1-/-;Sulf2-/- esophagi have defective GDNF-dependent neurite outgrowth. Esophagi (400 µm) were dissected from E11.5 embryos and plated on collagen gel containing BSA, GDNF or neurotrophins at various concentrations. After 4 days, the whole explant was immunostained with the TuJ1 antibody. Explants that failed to attach to collagen gel were not included in the assay. (A,B) Neurite outgrowth of E11.5 esophageal explants was selectively dependent on GDNF, but not on neurotrophin. Sulf1-/-;Sulf2-/- esophagi failed to extend neurites at 10 ng/ml GDNF and showed reduced neurite outgrowth at 20 ng/ml and 50 ng/ml GDNF. (C) Quantification of the neurite outgrowth shown in A and B. The length of the extended neurite was measured along six axes, 30° apart and the average was calculated to represent the neurite outgrowth of one explant. Data represent the mean and the standard deviation of a minimum of four individual cultures. (D,E) Quantification of the total number of neurons in the explants. The neurons in the explants (dark cell-body staining by the TuJ1 antibody, indicated by arrows) were quantified using the bright field at low magnitude. Neurons were scattered, or even migrated out of the control explants in the presence of 10 ng/ml GDNF. In control explants cultured in the presence of BSA or NGF and in GDNF-treated Sulf1-/-;Sulf2-/- explants, neurons tended to form clusters. The large clusters of neurons in Sulf1-/-;Sulf2-/- explants were quantified by summing the neuronal numbers at different focal planes. **, P<0.01 (two-tailed Student's t-test). Scale bars: 250 µm in A,B; 100 µm in D.

View larger version (48K): [in this window] [in a new window]   Fig. 6. SULF1 and SULF2 regulate GDNF binding to heparin and the GDNF signaling activity. (A) SULF1 reduces GDNF binding to heparin. Heparin conjugated to agarose beads was digested by SULF1 or inactive QSULF1(C-A) control. Various amounts of GDNF were incubated with enzyme-digested heparin beads (20 µl) to allow binding. The amount of GDNF bound to the beads was assayed by western blot. The intensity of individual bands was quantified by Multi-analysis software (Bio-Rad). Numbers listed beneath each lane are normalized quantification of the individual bands from three independent experiments. (B) SULF1 has no effect on GDNF binding to GFR1. Heparin predigested either by SULF1 or inactive QSULF1(C-A) was added to a mixture of GDNF (10 ng) and GFR1-Fc (1 µg) to allow GDNF-heparin-GFR1-Fc ternary complex formation. The complex was pulled down by protein A-agarose beads. The amount of GDNF bound to GFR1 was assayed by western blot and normalized to the amount of GFR1. (C-E) SULF2 enhances the GDNF signaling activity. NG108-15 cells that were stably transfected with the control vector or the SULF2 expression vector were stimulated by GDNF for 5 minutes, or by GDNF (5 ng/ml) for various lengths of time. The activation of GDNF signaling pathway was analyzed by assaying the phosphorylation of RET (at tyrosine, p-RET) and of the downstream AKT (p-AKT) by western blot. Total RET or Erks were used as loading control. Data shown are controlled for loading and then normalized to the basal level of control cells. (F) SULF2 had no effect on NGF signaling in PC12 cells. Serum-starved PC12 cells that stably expressed SULF2 or inactive QSULF1(C-A) were treated with NGF. The activation of NGF signaling was analyzed by assaying the phosphorylation of downstream Erks (p-Erk). Data presented are the mean and standard deviation of a minimum of three independent experiments. **, P<0.01, * P<0.05 (two-tailed Student's t-test). (G) Sulf double-mutant esophagi have reduced MAPK phosphorylation in the intrinsic neurons. E14.5 esophageal sections were immunostained with an antibody against phosphorylated MAPK. Arrowheads point to phosphorylated MAPK immunoreactivity in neuronal cell bodies within the muscle layers. Asterisks mark the endothelial cells with phosphorylated MAPK immunoreactivity.