Glycosaminoglycans are Critical Regulators for Limb Bud Outgrowth

Objectives: Limb development occurs through continuous interaction between mesodermal and ectodermal components of the limb bud. Glycosaminoglycans (GAGs) on the cell surface and in the extracellular matrix facilitate docking of signaling molecules and help shape morphogen gradients. This study aims to investigate the role of GAGs in limb development by inactivating FAM20B, a newly identified xylose kinase essential for GAG assembly.
Methods: K14-Cre and Prx1-Cre mice were crossbred with Fam20B-flox mice to inactivate FAM20B in limb epithelium and mesenchyme. Resultant mice were characterized by X-ray, micro-CT, and microscopy. In-situ hybridization (ISH), immunohistochemistry (IHC) and single cell RNA-Seq were performed to determine signaling changes in Fam20B-conditional knockout mice. Signaling changes were confirmed by crossbreeding with BMP-indicator mice (BRE-LacZ) and hedgehog-indicator mice (Gli1-LacZ and Patched-LacZ). GAG profiling was performed to characterize GAG type, amount, sulfation and disaccharide composition in limb bud mesenchyme using LCMS/MS MRM (liquid chromatography mass spectrometry with multiple reaction monitoring). Chondrogenesis was evaluated by micromass culture. Apoptosis and proliferation were examined by TUNEL and EdU assays.
Results: K14^Cre;Fam20B^fl/fl mice showed no limb abnormalities, while Prx1^Cre;Fam20B^fl/fl mice developed extremely short limbs. At E10.5-E13.5, Fam20B-deficient limb buds showed aberrant outgrowth. Signaling analysis revealed stronger and extended Shh expression at E11.5 and E12.5 in ZPA (zone of polarizing activity) mesenchyme compared with the wild type (WT); confirmed by Gli1-LacZ and Patched-LacZ indicators. Fgf10 was downregulated in GAG-deficient limb bud mesenchyme of Prx1^Cre;Fam20B^fl/fl mice, whereas Fgf8 was upregulated in the AER (apical ectodermal ridge). GAG profiling showed remarkably reduced GAG amounts in Fam20B-deficient mesenchyme, while GAG sulfation and composition showed no significant changes. Micromass revealed aberrant chondrogenesis of GAG-deficient mesenchymal cells.
Conclusions: Results indicate that GAGs in mesenchyme, but not epithelium, are essential for limb bud outgrowth and chondrogenesis; likely through SHH-FGF signal regulation.
Supported by NIH DE026461(XW), DE028345 (XW), DK111958 (RJL), and Baylor Oral Health Foundation.