Bud specific N-sulfation of heparan sulfate regulatesShp2-dependent FGF signaling during lacrimal gland induction

Abstract

Preferential outgrowth of the bud cells forms the basis of branching morphogenesis. Here, we show that lacrimal gland development requires specific modification of heparan sulfates by Ndst genes at the tip of the lacrimal gland bud. Systemic and conditional knockout experiments demonstrate the tissue specific requirement of Ndst1 and Ndst2 in the lacrimal gland epithelial, but not mesenchymal, cells, and the functional importance of Ndst1 in Fgf10-Fgfr2b, but not of Fgf1-Fgfr2b, complex formation. Consistent with this, Fgf10-induced ectopic lacrimal gland budding in explant cultures is dependent upon Ndst gene dose, and epithelial deletion of Fgfr2 abolishes lacrimal gland budding, its specific modification of heparan sulfate and its phosphorylation of Shp2 (Ptpn11 - Mouse Genome Informatics). Finally, we show that genetic ablation of Ndst1, Fgfr2 or Shp2 disrupts ERK signaling in lacrimal gland budding. Given the evolutionarily conserved roles of these genes, the localized activation of the Ndst-Fgfr-Shp2 genetic cascade is probably a general regulatory mechanism of FGF signaling in branching morphogenesis.

Lacrimal Gland Development and Fgf10-Fgfr2b Signaling Are Controlled by 2-O- and 6-O-sulfated Heparan Sulfate

“…Similarly, we have shown in lens development that a lack of heparan sulfate N-sulfation disrupts its interaction with Fgf8b-Fgfr3c but not with the Fgf8b-Fgfr3b pair (28). We recently showed that N-sulfated heparan sulfate is highly enriched in the lacrimal gland bud, which potentiates a restricted activation of FGF signaling during lacrimal gland outgrowth (29). Taken together, these findings suggest that FGF signaling is sensitive to the positional distribution of sulfate groups in the heparan sulfate chains.…”

“…Immunohistochemistry and RNA in Situ Hybridization-Immunohistochemistry was performed on cryosections or paraffin sections as previously described (28,29 A series of phage-display derived antibodies with VSV-tag were used to detect specific modifications of heparan sulfate in tissue sections (33). Cryo-sections were hydrated in PBS for 10 min, quenched of peroxidase activity by 3% H 2 O 2 and 10% methanol in PBS solution, and then blocked with 2% BSA in PBS at room temperature for 1 h followed by incubation with phage-display-derived antibodies (1:1-1:5 diluted with 0.2% BSA/PBS) at 4°C overnight.…”

“…RNA in situ hybridization on cryosections was performed according to a standard protocol (29). The following probes were used: Erm (from Dr. Bridget Hogan, Duke University Medical Center, Durham, NC), Hs6st1, Hs6st2, and Hs6st3 (34).…”

“…FGF-FGFR Complex Binding Assay-The binding of the FGF-FGFR complex with heparan sulfate was examined using the LACE assay as previously described (28,29). Briefly, 10 m paraffin sections of the mutant embryos and their matched littermates were deparaffinized and rehydrated followed by incubation in 0.5 mg/ml sodium borohydride for 10 min and in 0.1 M glycine for 30 min and then blocked with 2% BSA for 1 h at room temperature.…”

“…Explant Culture-Lacrimal gland explant cultures were carried out with E13.5-E14.5 embryos as described (29). Briefly, 80 -120-m diameter heparin acrylic beads (Sigma) were washed in PBS and incubated with 250 g/ml recombinant FGF10 (R&D Systems) or 5 mg/ml BSA in PBS at 4°C overnight.…”