Growth Factor/Extracellular Matrix Interactions During Branching Morphogenesis
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The exquisite control of growth factor signaling by HS is dictated by the tremendous structural heterogeneity of its sulfated modifications. Heparan sulfotransferase enzymes generate highly 3-O-sulfated epitopes on cell surfaces and in the extracellular matrix. Functional redundancy exists among the family of seven enzymes, but Hs3st3a1 and Hs3st3b1 sulfated HS increases epithelial FGFR signaling and morphogenesis. Single-cell RNAseq analysis of control SMGs identifies increased expression of Hs3st3a1 and Hs3st3b1 in endbud and myoepithelial cells, both of which are progenitor cells during development and regeneration. We previously generated both Hs3st3a1-/- and Hs3st3b1-/- single knockout mice. Salivary glands from both mice have impaired fetal epithelial morphogenesis and reduced 3-O-sulfated HS in the basement membrane. Analysis of adult KO gland function revealed there was an increase in frequency of drinking behavior in both KO mice, suggesting basal salivary hypofunction. We have also generated Hs3st3a1-/- ;Hs3st3b1-/- double knockout (DKO) mice, which are viable, but smaller, with disrupted acinar and myoepithelial cell development. Analysis of HS from the DKO SG shows a loss of specific highly 3-O-sulfated tetrasaccharides. We measured increased increased acinar phospho-Erk staining, with disrupted acinar polarity, suggesting 3-O-sulfated epitopes restrict FGF signaling and their loss increases FGF signaling disrupting acinar differentiation and function. There was also reduced development of myoepithelial cells, which contract to expel saliva. DKO mice have reduced saliva flow and increased drinking frequency. We used novel HS reagents with defined 3-O-sulfated modifications, primary organ cultures and organoids to investigate 3-O-HS-dependent mechanisms regulating acinar and myoepithelial development. We identified that HS-dependent FGFR signaling drives both acinar and MEC development. Thus, loss of s3-O-sulfated epitopes increases FGF signaling, disrupts acinar and MEC development resulting in hypofunction. Understanding how 3-O-sulfation regulates FGFR-dependent myoepithelial progenitor function will be important to manipulate HS-binding growth factors to enhance tissue function and regeneration. This information may be useful to manipulate cellular specificity of HS-binding growth factors and fine-tune biological responses, enhancing progenitor expansion for tissue regeneration. While we focus on the salivary gland, the mouse phenotypes direct us to investigate earlier stages of embryo development and to compare other organs that are affected. These mice are important tools to analyze 3-O-sulfation in salivary gland progenitors and to better understand the fine tuning of cellular responses to FGFRs and HS modifications.
