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

Abstract

Background: Understanding 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 RNA Sequencing (TRAPseq) in the Fgf20 lineage.

Results: We show that TRAPseq targeting OC progenitors effectively enriched for RNA from this rare cell population. TRAPseq identified differentially expressed genes (DEGs) 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-null and Sall1-ΔZn2-10 mice, which harbor a mutation that causes Townes-Brocks syndrome, identified a decrease in outer hair cell number.

Conclusions: We present two datasets: genes with enriched expression in OC progenitors, and DEGs downstream of FGF20 in the embryonic day 14.5 cochlea. We validate select DEGs via in situ hybridization and in vivo functional studies in mice.

Keywords: RNAseq; SALL1; Townes-Brocks syndrome; cochlea; hair cell; hearing loss.

Figure 1.. Fgf20^Cre targets L10a-eGFP expression to the prosensory domain and Kölliker’s organ

(A) Schematic representing cross-sectional view through the E14.5 and P0 cochlear duct. At E14.5, the epithelium at the cochlear duct floor can be divided into three regions: outer sulcus (OS), prosensory domain (PD), and Kölliker’s organ (KO). Cells from these three regions contribute to the lesser epithelial ridge (LER), organ of Corti (OC), and greater epithelial ridge (GER), respectively, at P0. Double-headed arrow indicates medial (neural) and lateral (abneural) directions. In all figures, sections through the cochlear duct are presented in this orientation. (B) Sections through the middle turn of E14.5 and P0 Fgf20^Cre/+;ROSA^fsTRAP/+ cochlear ducts, showing L10a-eGFP (green) expression. At E14.5, L10a-eGFP is found in the prosensory domain (PD; bracket), Kölliker’s organ and medial wall, and spiral ganglion (SG). At P0, it is found in the organ of Corti (OC; bracket), greater epithelial ridge, and medial wall. DAPI, nuclei (blue); scale bar, 100 μm. (C) Section through the middle turn of E14.5 cochlear ducts from Fgf20^Cre/+;ROSA^mTmG/+ and Fgf20^Cre/βgal;ROSA^mTmG/+ embryos. Cells of the Fgf20^Cre lineage express mGFP (mG, green); non-lineage cells express mTomato (mT, red). DAPI, nuclei (blue); scale bar, 100 μm. (D) Schematic showing an overview of the TRAPseq protocol (see Experimental Procedures). 1) Ventral otocysts containing the cochlea were dissected from E14.5 embryos. 2) Otocysts from each litter were pooled according to genotype to increase RNA yield. 3) Otocysts were then homogenized and centrifuged to make polysomes (pre-TRAP samples were collected at this stage). This was followed by immunoprecipitation with anti-GFP antibodies to collect L10a-eGFP labelled polysomes. 4) This produced TRAP samples, which were then purified for RNA along with pre-TRAP samples and used for downstream applications. (E) Schematic of a cross-sectional view of the E14.5 ventral otocyst, showing three turns of the cochlear duct, surrounded by periotic mesenchyme and otic capsule. Pre-TRAP RNA (representing total input tissue) comes from the cochlear duct epithelium, periotic mesenchyme, and otic capsule (gray). TRAP RNA (representing Fgf20^Cre-lineage tissue, which expresses L10a-eGFP) comes from the prosensory domain, Kölliker’s organ, medial wall of the cochlear duct, and some cells of the spiral ganglion (green). (F) qRT-PCR showing fold change in Twist2 and Id2 expression (normalized to Gadph) in TRAP RNA samples compared to pre-TRAP samples from Fgf20^Cre/+;ROSA^mTmG/+ E14.5 cochleae. Each dot represents an RNA sample pooled from at least 3 embryos.

Figure 2.. Fgf20^Cre TRAPseq enriched for prosensory domain RNA

(A) Principal Component Analysis (PCA) on 24 TRAPseq samples (8 pre-TRAP samples – 4 Fgf20^−/+, 4 Fgf20^−/−; 16 TRAP samples – 8 Fgf20^−/+, 8 Fgf20^−/−) showing separation of pre-TRAP and TRAP samples along PC1, but not of Fgf20^−/+ and Fgf20^−/− samples. (B) PCA on the 16 TRAP samples (excluding the 8 pre-TRAP samples) also did not show separation between Fgf20^−/+ and Fgf20^−/− samples along the first two principal components. (C) Volcano plot showing TRAP vs. pre-TRAP differentially expressed genes. Positive Log₂ Fold Change value indicates enrichment by TRAP; negative Log₂ Fold Change value indicates depletion by TRAP. Labeled genes represent markers of the prosensory domain, Kölliker’s organ, spiral ganglion, outer sulcus, periotic mesenchyme, and otic capsule. Padj, adjusted p-value for multiple comparisons (Benjamini-Hochberg method). The p-value plotted on y-axis is unadjusted. Arrowheads indicate genes above y-axis range.

Figure 3.. TRAPseq revealed known FGF target genes during organ of Corti differentiation downstream of Fgf20

(A) Volcano plot showing Fgf20^−/+ vs. Fgf20^−/− differentially expressed genes. Fgf20, transcripts associated with FGF signaling, and transcripts meeting the criteria padj < 0.1 and Log₂ Fold Change < −1 or > 1 are labeled, with the exception of predicted genes and unnamed transcripts. padj, adjusted p-value for multiple comparisons (Benjamini-Hochberg method). The p-value plotted on the y-axis is unadjusted. Arrowheads indicate genes above the y-axis range. (B) RNA in situ hybridization for known FGF target genes Dusp6, Etv1, Spry1, and Spry4 on sections through the middle turn of E14.5 Fgf20^−/+ (Fgf20^Cre/+) and Fgf20^−/− (Fgf20^Cre/βgal) cochlear ducts. Bracket, prosensory domain. Arrowhead, increased expression of Etv1 in the outer sulcus of Fgf20^−/− cochleae. Scale bar, 100 μm.

Figure 4.. Expression analysis of genes identified by TRAPseq associated with cochlea development or hearing loss downstream of Fgf20

RNA in situ hybridization on sections through the middle turn of E14.5 Fgf20^−/+ (Fgf20^Cre/+) and Fgf20^−/− (Fgf20^Cre/βgal) cochlear ducts. Bracket, prosensory domain. Scale bar, 100 μm. (A) Genes expressed within the Fgf20^Cre lineage: Tectb, Tecta, Smpx, Epyc, Fat3, and Heyl (B) Genes expressed outside of the Fgf20^Cre lineage: Gata2, Meis2, Lmx1a, and Bmp4

Figure 5.. Conditional deletion of Cdc20 with Fgf20^Cre resulted in a shorter cochlea

(A) The largest protein-protein interaction network identified via the STRING database consisted of genes involved in cell cycle regulation. Lines represent known and predicted protein-protein interactions of high or very high confidence (minimum required interaction score = 0.700). (B) RNA in situ hybridization for Cdc20 on sections through the middle turn of E12.5 and E14.5 Fgf20^−/+ (Fgf20^Cre/+) and Fgf20^−/− (Fgf20^Cre/βgal) cochlear ducts. Bracket, prosensory domain. Scale bar, 100 μm. (C) Section through the middle turn of E12.5 Fgf20^Cre/+;ROSA^mTmG/+ cochlear duct. Cells of the Fgf20^Cre lineage express mGFP (mG, green); non-lineage cells express mTomato (mT, red). DAPI, nuclei (blue); scale bar, 100 μm. (D) Dissected inner ears from E18.5 Cdc20^CHet (Fgf20^Cre/+;Cdc20^flox/+) and Cdc20^CKO (Fgf20^Cre/+;Cdc20^flox/flox) embryos with the otic capsule removed to reveal the coiled cochlea (dotted lines). Scale bar, 0.5 mm. (E) Whole mount cochlea from E18.5 Cdc20^CHet and Cdc20^CKO embryos showing one row of inner hair cells (IHC) and three rows of outer hair cells (OHC) marked by phalloidin (green) and separated by inner pillar cells (p75NTR, red). Representative regions from the basal, mid-basal, and mid-apical turns, and apical tip of the cochlea are shown. See schematic showing locations of the turns of the cochlea. At the apical tip, four or more rows of OHCs were frequently observed in Cdc20^CHet cochleae. Scale bar, 100 μm. (F) Quantification of cochlea length and total number of inner and outer hair cells (IHCs and OHCs) in E18.5 Cdc20^CHet (n = 4) and Cdc20^CKO (n = 5) cochleae. Error bars represent mean ± std. Results were analyzed by Student’s t-test; p-values are shown.

Figure 6.. Sall1^−/− cochleae exhibit an outer hair cell phenotype

(A) RNA in situ hybridization for Sall1, Sall2, and Sall3 on sections through the middle turn of E14.5 Fgf20^−/+ (Fgf20^Cre/+) and Fgf20^−/− (Fgf20^Cre/βgal) cochlear ducts. Bracket, prosensory domain. Scale bar, 100 μm. (B) Whole mount cochlea from E18.5 Sall1^+/+, Sall1^−/+, and Sall1^−/− embryos showing inner hair cells and outer hair cells marked by phalloidin (green) and separated by inner pillar cells (p75NTR, red). Representative regions from the basal (5% of total length from the basal tip), mid-basal (33%), and mid-apical (67%) turns, and apical tip (90%) of the cochlea are shown. See schematic showing locations of the turns of the cochlea. Numerous ectopic inner hair cells were found throughout Sall1^−/− cochleae, especially towards the apex (arrowheads). Scale bar, 100 μm. (C) Quantification of cochlea length, total number of inner hair cells (IHCs) and outer hair cells (OHCs), and total number of ectopic IHCs in E18.5 Sall1^+/+ (n = 4), Sall1^−/+ (n = 3), and Sall1^−/− (n = 5) cochleae. Error bars represent mean ± std. Results were analyzed by one-way ANOVA. P-values shown are from the ANOVA. * indicates p < 0.05 from Tukey’s HSD (ANOVA post-hoc).

Figure 7.. Sall1-ΔZn2-10 mutant cochleae exhibit a more severe phenotype than Sall1^−/− cochleae

(A) Whole mount cochlea from E18.5 Sall1^+/+, Sall1^Δ/+, and Sall1^Δ/Δ embryos showing inner hair cells and outer hair cells marked by phalloidin (green) and separated by inner pillar cells (p75NTR, red). Representative regions from the basal (5% of total length from the basal tip), mid-basal (33%), and mid-apical (67%) turns, and apical tip (90%) of the cochlea are shown. See schematic to the right showing locations of the turns of the cochlea. Inset: 3.8x magnified image of a representative OHC showing stereocilia bundle formation (arrows in mid-apical region). Numerous ectopic inner hair cells were found throughout Sall1^Δ/Δ cochleae, especially towards the apex (arrowheads). Scale bar, 100 μm. (B) Quantification of cochlea length, total number of inner hair cells (IHCs) and outer hair cells (OHCs), and total number of ectopic IHCs in E18.5 Sall1^+/+ (n = 8), Sall1^Δ/+ (n = 9), and Sall1^Δ/Δ (n = 3) cochleae. Error bars represent mean ± std. Results were analyzed by one-way ANOVA. P-values shown are from the ANOVA. * indicates p < 0.05 from Tukey’s HSD (ANOVA post-hoc).