Figure 1.

Workflow for time-resolved proteome analysis of otic morphogenesis

Xenopus larvae and tadpoles from stages 34–47 were selected for otic proteome analysis. These stages were chosen because they include the simplest structure displaying onset of tubular duct growth (st 34), the initial polarization and compartmentalization of the OV into distinct functional regions and the appearance of otoliths (st 40), the onset of semicircular canal formation (st 42/43), the onset of sensory hair cell formation (st 45) and the completion of semicircular canal formation, sensory patch formation and hair cell differentiation (st 47). Insets show stage associated optical coherence tomography-based images of the otic vesicle. Otolith (o), pouch and protrusions for the horizontal canal (h), anterior canal (a), sacculus (s). Bar indicates 100 μm. Otic vesicles were dissected and processed using a bottom-up proteomic workflow. Peptides were tagged with isobaric mass tags to quantify relative changes in protein abundances during the course of otic morphogenesis. Tagged peptides were fractionated offline using RP spin columns at a high pH and then analyzed by LC-HRMS. Quantification of select proteins was verified using targeted (PRM) HRMS.