Heparin

Heparin (Toronto Research Chemicals, North York, ON, Canada) was depolymerized with Heparinase I. Specifically, 1 g of Heparin was digested using 0.8 IU of Heparinase I in 20 mL of 50 mM NH₄CH₃CO₂, 5 mM CaCl₂, pH 7.0 buffer at 30 °C, until depolymerization was approximately 30% complete, as estimated using A₂₃₂. Digested fragments were separated via size exclusion chromatography on a 2.5 × 175 cm Bio-Rad Biogel P10 column with a flow rate of 0.2 mL/min. Heparin dp6 were further separated using SAX HPLC with a 4 × 250 mm Waters Spherisorb SAX column to produce pure DP6-C oligosaccharides. Selectively desulfated Heparin dp6 was purchased from Iduron (Alderly Edge, UK). Concentrations of all GAG samples were quantified using the carbazole assay [35 (link)].

Bead implantations were performed according to published methods6 (link) with the following modifications: Heparin acrylic (Sigma) and Affigel-Blue (Bio-Rad) beads were size-selected at 250 μm. Affigel-Blue beads were soaked in SHH-N protein (human origin, R&D Systems) diluted to 1 mg ml⁻¹ in PBS with 0.1% BSA. Heparin beads were soaked in cyclopamine (Toronto Research Chemicals) diluted to 4 mg ml⁻¹ in DMSO. Control experiments were performed using the respective vehicle-only beads. Stage 30 embryos were dissected from their egg cases, anaesthetized and the bead was surgically implanted into the posterior region of the pelvic fin using tungsten needles. For all bead implants, L. erinacea embryos were sorted by sex and then randomly allocated into either control or experimental treatment groups. After manipulation, animals were returned to their tank for 24-h before tissue-harvesting for qRT–PCR, or they were allowed to develop for an additional ∼10 weeks for phenotypic analysis.