Heparan sulfate proteoglycans are essential for biological processes regulated by fibroblast growth factors (FGFs). Heparan sulfate (HS) regulates the activity of FGFs by acting as a coreceptor at the cell surface, enhancing FGF-FGFR affinity, and being a storage reservoir for FGFs in the extracellular matrix (ECM). Here we demonstrate a critical role for heparanase during mouse submandibular gland (SMG) branching morphogenesis. Heparanase, an endoglycosidase, colocalized with perlecan in the basement membrane and in epithelial clefts of SMGs. Inhibition of heparanase activity in organ culture decreased branching morphogenesis, and this inhibition was rescued specifically by FGF10 and not by other FGFs. By contrast, exogenous heparanase increased SMG branching and MAPK signaling and, surprisingly, when isolated epithelia were cultured in a three-dimensional ECM with FGF10, it increased the number of lateral branches and end buds. In a solid-phase binding assay, an FGF10-FGFR2b complex was released from the ECM by heparanase. In addition, surface plasmon resonance (SPR) analysis showed that FGF10 and the FGF10-FGFR2b complex bound to purified perlecan HS and could be released by heparanase. We used the FGF10-FGFR2b complex as a probe for HS in SMGs, and it colocalized with perlecan in the basement membrane and partly colocalized with syndecan 1 in the epithelium, and binding was reduced by treatment with heparanase. In summary, our results show heparanase releases FGF10 from perlecan HS in the basement membrane, increasing MAPK signaling, epithelial clefting, and lateral branch formation, which results in increased branching morphogenesis.

FGF10, a heparan sulfate (HS)-binding growth factor, is required for branching morphogenesis of mouse submandibular glands (SMGs). HS increases the affinity of FGF10 for FGFR2b, which forms an FGF10.FGFR2b.HS ternary signaling complex, and results in diverse biological outcomes, including proliferation and epithelial morphogenesis. Defining the HS structures involved in specific FGF10-mediated events is critical to understand how HS modulates growth factor signaling in specific developmental contexts. We used HS-deficient BaF3/FGFR2b cells, which require exogenous HS to proliferate, to investigate the HS requirements for FGF10-mediated proliferation and primary SMG epithelia to investigate the structural requirements of HS for FGF10-mediated epithelial morphogenesis. In BaF3/FGFR2b cells, heparin with at least 10 saccharides and 6-O-, 2-O-, and N-sulfates were required for maximal proliferation. During FGF10-mediated SMG epithelial morphogenesis, HS increased proliferation and end bud expansion. Defined heparin decasaccharide libraries showed that 2-O-sulfation with either an N-or 6-O-sulfate induced end bud expansion, whereas decasaccharides with 6-O-sulfation alone induced duct elongation. End bud expansion resulted from increased FGFR1b signaling, with increased FGFR1b, Fgf1, and Spry1 as well as increased Aqp5 expression, a marker of end bud differentiation. Duct elongation was associated with expression of Cp2L1, a marker of developing ducts. Collectively, these findings show that the size and sulfate patterns of HS modulate specific FGF10-mediated events, such as proliferation, duct elongation, end bud expansion, and differentiation, and provide mechanistic insight as to how the developmental localization of specific HS structures in tissues influences FGF10-mediated morphogenesis and differentiation.