Growth Factor/Extracellular Matrix Interactions During Branching Morphogenesis

National Institute Of Dental & Craniofacial Research

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Abstract

We investigate how heparan sulfate (HS) influences FGFR signaling in specific progenitor cell types in the epithelium. The exquisite control of growth factor function by HS is dictated by the tremendous structural heterogeneity of its sulfated modifications. We focus on the heparan sulfotransferase enzymes that generate highly 3-O-sulfated epitopes on cell surfaces and in the extracellular matrix. Previous ex vivo experiments suggested functional redundancy exists among the family of seven enzymes, but that 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. To analyze their in vivo functions, we generated both Hs3st3a1-/- and Hs3st3b1-/- single knockout mice, which are viable and fertile. Salivary glands from both mice have impaired fetal epithelial morphogenesis when cultured with FGF10. Hs3st3b1-/- mice have reduced intact SMG branching morphogenesis and reduced 3-O-sulfated HS in the basement membrane. Analysis of HS biosynthetic enzyme transcription highlighted some compensatory changes in sulfotransferases expression early in development. The overall glycosaminoglycan composition of adult control and KO mice were similar, although HS disaccharide analysis showed increased N- and non-sulfated disaccharides in Hs3st3a1-/- HS. Analysis of adult KO gland function revealed normal secretory innervation, but without stimulation there was an increase in frequency of drinking behavior in both KO mice, suggesting basal salivary hypofunction, possibly due to myoepithelial dysfunction. 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. We continue generating a genetic toolkit of Hs3st knockout mice to investigate how 3-O-sulfation influences organ development and homeostasis. Our goal is to use genetics to understand the 3-O-sulfated code of HS. This information will 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.