Analysis of FGF20-regulated genes in organ of Corti progenitors by translating ribosome affinity purification

Abstract

BackgroundUnderstanding the mechanisms that regulate hair cell (HC) differentiation in the organ of Corti (OC) is essential to designing genetic therapies for hearing loss due to HC loss or damage. We have previously identified Fibroblast Growth Factor 20 (FGF20) as having a key role in HC and supporting cell differentiation in the mouse OC. To investigate the genetic landscape regulated by FGF20 signaling in OC progenitors, we employ Translating Ribosome Affinity Purification combined with Next Generation mRNA Sequencing (TRAPseq) in the Fgf20 lineage.ResultsWe show that TRAPseq targeting OC progenitors effectively enriched for mRNA within this rare cell population. TRAPseq identified differentially expressed genes downstream of FGF20, including Etv4, Etv5, Etv1, Dusp6, Hey1, Hey2, Heyl, Tectb, Fat3, Cpxm2, Sall1, Sall3, and cell cycle regulators such as Cdc20. Analysis of Cdc20 conditional-null mice identified decreased cochlea length, while analysis of Sall1-ΔZn2-10 mice, which harbor a mutation that causes Townes-Brocks syndrome, identified a decrease in outer hair cell number.ConclusionsWe present two datasets: genes with enriched expression in OC progenitors, and genes regulated by FGF20 in the embryonic day 14.5 cochlea. We validate select differentially expressed genes via in situ hybridization and in vivo functional studies in mice.Key findingsTranslating Ribosome Affinity Purification (TRAP) with Fgf20-Cre enriches for prosensory cell mRNATRAP combined with RNAseq identifies genes downstream of FGF20 during prosensory cell differentiationFGF20 regulates Sall1, gene implicated in human sensorineural hearing lossGrant Sponsor and NumberNational Institute on Deafness and Other Communication Disorders – DC017042 (DMO) Washington University Institute of Clinical and Translational Sciences and National Center for Advancing Translational Sciences – CTSA grant UL1TR002345 (JIT471 to DMO) March of Dimes – 6-FY13-127 (MR)

New developments in the biology of fibroblast growth factors

“…Etv1 ( Er81 ), Etv4 ( Pea3 ), and Etv5 ( Erm ) are direct transcriptional effectors of FGF signaling (Brent & Tabin, 2004; DeSalvo et al, 2021; di Martino et al, 2019; Ebeid & Huh, 2020; Herriges et al, 2015; Jones et al, 2019; Peluso et al, 2017; Willardsen et al, 2014; Yamamoto‐Shiraishi et al, 2014; L. M. Yang, Stout, et al, 2020). Transgenic reporter mice have been developed in which a green fluorescent protein (GFP) reporter gene was inserted into the Etv4 locus in a large bacterial artificial chromosome (Lamballe et al, 2011) or in which a CreER‐RFP gene was inserted into the endogenous Etv5 gene locus (Jones et al, 2019).…”

“…FGF9 and FGF20 signal to mesenchymal FGFRs and activate ETV4 and ETV5 to indirectly regulate the size of the sensory progenitor population and overall cochlear length. These FGFs also signal to epithelial FGFR1 to regulate the differentiation of outer hair cells and supporting cells (Ebeid & Huh, 2020; Huh et al, 2015; L. M. Yang, Stout, et al, 2020). In mouse explant cultures, FGF20–FGFR1 signaling activates MAPK and PI3K signaling to regulate hair cell and support cell differentiation (Su et al, 2021).…”

“…FGFR1 has an FGF20‐independent, Sox2‐dependent role in specification, and Sox2 and Fgf20 interact to ensure that specification occurs before differentiation toward the cochlear base (Yang et al, 2019). Genes regulated by FGF20 in the developing cochlea include Etv4 , Etv5 , Etv1 , Dusp6 , Hey1 , Hey2 , Heyl , Tectb , Fat3 , Cpxm2 , Sall1 , Sall3 , and the cell cycle regulator Cdc20 (L. M. Yang, Stout, et al, 2020).…”

FGF20‐FGFR1 signaling through MAPK and PI3K controls sensory progenitor differentiation in the organ of Corti

“…During sensory progenitor differentiation, Etv1 , Etv4 ( Pea3 ) and Etv5 ( Erm ), known downstream effectors of FGF signaling, have decreased expression in the prosensory domain of Fgf20 −/− mice at E14.5 4,12,22 . Expression of Dusp6 , a negative feedback regulator of ERK signaling, 23 is also decreased in Fgf20 −/− cochlea 22 . Similarly, MEKK4 ( Map3k4 ), a MAP2K kinase expressed throughout the floor of the cochlea at E13 that becomes restricted to HCs and SCs by E16.5, is required for HC and SC development.…”

“…At slightly later stages, ERK1/2 signaling is active in unspecified cochlear epithelial progenitors at E10.5 and E11.5 and is reduced in cochleae with reduced FGFR1 signaling 10 . During sensory progenitor differentiation, Etv1 , Etv4 ( Pea3 ) and Etv5 ( Erm ), known downstream effectors of FGF signaling, have decreased expression in the prosensory domain of Fgf20 −/− mice at E14.5 4,12,22 . Expression of Dusp6 , a negative feedback regulator of ERK signaling, 23 is also decreased in Fgf20 −/− cochlea 22 .…”