Analysis of FGF20-regulated genes in differentiating cochlear hair and supporting cell progenitors via ribosome affinity purification

ABSTRACT: Background: Understanding the developmental mechanisms that regulate hair cell differentiation in the cochlea is essential to designing genetic therapies for acquired hearing loss due to hair cell loss or damage. We have previously identified Fibroblast Growth Factor 20 (FGF20) as having a key role in hair cell and supporting cell differentiation and patterning in the mouse cochlear sensory epithelium. To investigate the genetic landscape regulated by FGF20 signaling in hair cell and supporting cell progenitors, we employ Translating Ribosome Affinity Purification (TRAP) combined with Next Generation mRNA Sequencing (TRAPseq). Methods: In mice, we used Fgf20-Cre to activate ROSA-fsTRAP in hair cell and supporting cell progenitors, and then collected translating mRNA using TRAP from Fgf20+/- (control) and Fgf20-/- cochleae. Library preparation and sequencing were done in two separate experiments, each with 12 samples that included 2 pre-TRAP (pre-immunoprecipitation) Fgf20+/- samples, 2 TRAP Fgf20+/- samples, 4 pre-TRAP Fgf20-/- samples, and 4 TRAP Fgf20-/- samples. Samples were sequenced via Illumina HiSeq 3000. We compared pre-TRAP (pre-immunoprecipitation) samples with TRAP mRNA samples to validate the TRAP technique as well as identify genes enriched in our target cell population. We also compared Fgf20+/- and Fgf20-/- TRAP mRNA samples to identify differentially expressed genes downstream of FGF20 during hair cell and supporting cell differentiation. Results: TRAPseq targeting the prosensory cell population effectively enriches for translating mRNA within this rare cell population. TRAPseq comparing Fgf20+/- and Fgf20-/- samples identified differentially expressed genes downstream of FGF20. These included FGF-response genes Etv4, Etv5, Etv1, and Dusp6, as well as genes associated with cochlea development and hearing, such as Hey1, Hey2, Heyl, Tectb, Fat3, Cpxm2, Sall1, and cell cycle regulators such as Cdc20.

ORGANISM(S): Mus musculus

PROVIDER: GSE148380 | GEO | 2020/04/10

REPOSITORIES: GEO