DEV007674 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Generation of Sulf1-knockout ES cells. (A) Sequence alignment of the enzymatic domain of SULF1 (SULF1E) and SULF2E (amino acids 41-416). The sequences encoded by the second coding exon (exon 2) are shown in the black box. The essential Cys residue in exon 2 is marked by a red star. The two sulfatase signature sequences are in red. The homologous amino acids are in black. Sequences that are not conserved between SULF1E and SULF2E are in blue. (B) Schematic gene targeting strategy for Sulf1. The Sulf1 targeting vector contains a 4.5 kb 5′ homologous arm and a 3.9 kb 3′ homologous arm. A loxP site was inserted 3′ to the Sulf1 exon 2 by a NheI site. Two external probes, located 5′ and 3′ to the homologous arms, were used for southern blot. The probe shown is the 5′ external probe. The Sulf1 targeted allele was generated by homologous recombination in ES cells. The floxed exon 2 was subsequently removed by Cre recombinase to generate the Sulf1 mutant allele. (C) Southern blot to identify the recombinant Sulf1 ES cell clones. Genomic DNA was extracted from individual ES cell clones and cut by KpnI. Homologous recombination between the Sulf1 genomic sequences and the targeting vector was detected by southern blot using a radiolabeled 5′ external probe. The Sulf1 wild-type allele generates a 12.7 kb fragment, whereas the targeted Sulf1 allele generates a 10.3 kb fragment. The southern blot results using SacI and the 3′ external probe are not shown. (D) Genotyping to detect the wild-type allele and the mutant allele of Sulf1. (E) RT-PCR assay to detect the expression of Sulf1 mRNA and Sulf2 mRNA in Sulf1^−/− embryos. RT-PCR using Sulf1 primers located in the exon 3 did not detect any Sulf1 mRNA transcript in Sulf1^−/− embryos, whereas a partial Sulf1 mRNA transcript was detected in Sulf1^−/− embryos using primers located in downstream exons 11 and 15. By contrast, Sulf1^+/− embryos expressed full-length Sulf1 mRNA and the expression of Sulf2 mRNA was unaffected in Sulf1^−/− embryos. Gapdh mRNA expression was used as the control of the total amount of mRNA in the assay.

• Supplemental Figure S2 -

  Fig. S2. Generation of Sulf2-knockout ES cells. (A) Schematic gene targeting strategy for Sulf2. The Sulf2 targeting vector contains a 2.5 kb 5′ homologous arm and a 3.1 kb 3′ homologous arm. A loxP site was inserted 3′ to the Sulf2 exon 2 by an AatII site. Two external probes, located 5′ and 3′ to the homologous arms, were generated for southern blot. The probe shown is the 3′ external probe. The Sulf2 targeted allele was generated by homologous recombination in ES cells. The floxed exon 2 was removed by Cre recombinase to generate the Sulf2 mutant allele. (B) Southern blot to identify the recombinant ES cell clones. Genomic DNA was extracted from the individual ES cell clones and cut with BamHI. Homologous recombination between the Sulf2 genomic sequences and the targeting vector was detected by southern blot using a radiolabeled 3′ external probe. The Sulf2 wild-type allele generates a 12 kb fragment, whereas the targeted Sulf2 allele generates a 10.5 kb fragment. The results of southern blot using BglI and the 5′ external probe are not shown. (C) Genotyping to detect the wild-type allele and the mutant allele of Sulf2. (D) RT-PCR assays to detect the expression of Sulf1 mRNA and Sulf2 mRNA in Sulf2^−/− embryos. RT-PCR using primers located in targeted exon 2 of Sulf2 did not detect any Sulf2 mRNA transcript in Sulf2^−/− embryos, whereas a partial Sulf2 mRNA transcript was detected in Sulf2^−/− embryos using primers located in downstream exons 6 and 8. Sulf2^+/− embryos expressed full-length Sulf2 mRNA and the expression of Sulf1 mRNA was unaffected in Sulf2^−/− embryos. Gapdh mRNA expression was used as the control of the total amount of mRNA in the assay.

• Supplemental Figure S3 -

  Fig. S3. Antibodies against SULF1 and SULF2 identify specific Sulfs. (A) The antisera against the hydrophilic domain of SULF1 (MS1HD) and the hydrophilic domain of SULF2 (MS2HD) detect selectively the corresponding antigens by western blot assays. Purified MS1HD and MS2HD were resolved by SDS-PAGE and detected with the antisera against MS1HD and MS2HD, respectively, by western blot assay. (B) Antibodies against MS1HD and MS2HD selectively label transfected cells using immunocytochemistry. The HB12317 neuroblastoma cells were transfected with the expression vectors of SULF1 or SULF2. After 24 hours, the HB12317 cells were fixed followed by immunocytochemistry using specific antibodies against SULF1HD and SULF2HD. Scale bar: 40 μm. (C) Characterization of mRNA and protein expression of SULF1 and SULF2 at E16.5 in the neural tube (NT), lung, dorsal root ganglian (DRG), bone, cartilage and skeletal muscle (SK). In situ hybridization and immunohistochemistry were performed on cross-sections using specific Sulf RNA in situ probes and antibodies against MS1HD and MS2HD, respectively. The expression patterns of Sulf mRNA and protein completely overlapped. OL, oligodendricyte. (D,E) Enlarged images of Fig. 2E,N. Scale bar: 100 μm.

• Supplemental Figure S4 -

  Fig. S4. The esophagi of Sulf1^−/−;Sulf2^−/− adult mice have diminished neuronal innervation of the smooth muscle and enteric glial cells. (A) Detection of neuronal innervation and enteric glial cells in the esophageal smooth muscle of control and Sulf1^−/−;Sulf2^−/− mice. The cross-sections of the lower-half thoracic segment of the adult esophagi were immunolabeled with the TuJ1 antibody to detect neuronal innervation of the smooth muscle (arrowhead) and the antibody against GFAP to label enteric glial cells (arrows) in the esophagus (n=4). Serial sections 200 μm apart were used to quantify the total number of TuJ1⁺ axons innervating the smooth muscle of the muscularis mucosae and the GFAP⁺ enteric glial cells on each section. At least five serial sections were quantified for each esophagus. Innervation density was calculated by dividing the total number of innervating neurites by the circumference of the smooth muscle in muscularis mucosae on cross-sections. The circumstance of the smooth muscle was ∼85% longer in the Sulf1^−/−;Sulf2^−/− adult esophagi than in the littermate controls. A minimum of 20 sections from four independent controls or Sulf1^−/−;Sulf2^−/− mice were counted. Scale bars: 100 μm. (B) Quantification of the smooth muscle innervation in the adult esophagi and the number of enteric glial cells.

• Supplemental Figure S5 -

  Fig. S5. SULF2 enhances the GDNF signaling activity in neuroblastoma cells. NG-108-15 cells that were stably transfected with the control vector or the SULF2 expression vector were stimulated by GDNF at various concentrations for 5 minutes (A), or by GDNF (5 ng/ml) for various lengths of time (B). The activation of the GDNF signaling pathway was analyzed by assaying the phosphorylation of downstream ERK kinase by western blot. Total ERK was used as the loading control. Data shown are controlled for the amount of cell lysates and then normalized to the basal level of the control cells. Data presented are mean and standard deviation of a minimum of three independent experiments. *, P<0.05 (two-tailed Student’s t-test).

• Supplemental Figure S6 -

  Fig. S6. GDNF selectively induces the neurite outgrowth of E11.5 esophageal explants in a heparin-dependent manner. (A) The esophagi (∼400 μm in length) were dissected out from E11.5 embryos and cultured on collagen containing control BSA or signaling ligands. After 4 days, the neurite outgrowth was assayed by immunostaining with TuJ1 antibody. GDNF (20 ng/ml) induces neurite outgrowth from the esophageal explants, which was completely abolished by heparin (5 μg/ml), but not by dermatan sulfate (5 μg/ml and 10 μg/ml). Other HS-dependent growth factors, including FGF2, HGF and VGEF165, have no effect on neurite outgrowth at 5 μg/ml and 20 μg/ml. In addition, LiCl (10 mM), which activates the canonical Wnt signaling pathway, does not induce neurite outgrowth of the explants. Furthermore, BMP2 (20 μg/ml) primarily induces the migration of enteric neurons and neurite fasciculation, rather than the neurite sprouting of the esophageal explants. Lastly, sonic hedgehog (SHH) (5 μg/ml), which functions as a chemoattractant for commissural neurons, has no effect on neurite outgrowth of the enteric neurons in the esophageal explants. However, SHH induces the growth of the non-neuronal tissues in explant cultures. We observed a few neurons outside of the explants, which was likely to be due to the growth of non-neuronal tissues from the explants. All ligands and LiCl were tested for their signaling potency. Explants that were not attached to the collagen gel were excluded from the assay. Six explants from two independent experiments were analyzed for each condition. Scale bar: 200 μm. (B,C) Quantification of the neurite outgrowth.

• Supplemental Figure S7 -

  Fig. S7. Embryonic colon expresses abundant GDNF and Sulf1^−/−;Sulf2^−/− colon has apparently normal innervation and enteric glial cell formation. (A) The levels of GDNF expression in E14.5 esophagus and in E16.5 colon were compared by semiquantitative immunofluorescent staining. The local GDNF concentration in the colon is 2- to 3-fold higher than that in the esophagus. (B) Whole-mount staining of the E18.5 colon with the TuJ1 antibody. (C) Immunohistochemistry on sections of the adult colon using the TuJ1 antibody to label the nerve innervation and the anti-GFAP antibody to identify the enteric glial cells. Sulf1^−/−;Sulf2^−/− colon has normal TuJ1 staining and GFAP expression at E18.5 and in the adult. Scale bars: 50 μm.