GFI1 functions to repress neuronal gene expression in the developing inner ear hair cells

Abstract

Despite the known importance of the transcription factors ATOH1, POU4F3 and GFI1 in hair cell development and regeneration, their downstream transcriptional cascades in the inner ear remain largely unknown. Here, we have used Gfi1^cre;RiboTag mice to evaluate changes to the hair cell translatome in the absence of GFI1. We identify a systematic downregulation of hair cell differentiation genes, concomitant with robust upregulation of neuronal genes in the GFI1-deficient hair cells. This includes increased expression of neuronal-associated transcription factors (e.g. Pou4f1) as well as transcription factors that serve dual roles in hair cell and neuronal development (e.g. Neurod1, Atoh1 and Insm1). We further show that the upregulated genes are consistent with the NEUROD1 regulon and are normally expressed in hair cells prior to GFI1 onset. Additionally, minimal overlap of differentially expressed genes in auditory and vestibular hair cells suggests that GFI1 serves different roles in these systems. From these data, we propose a dual mechanism for GFI1 in promoting hair cell development, consisting of repression of neuronal-associated genes as well as activation of hair cell-specific genes required for normal functional maturation.

Keywords: GFI1; Hair cells; Inner ear.

Fig. 1.

HC degeneration and TUBB3 expression in outer but not inner or vestibular HCs of newborn GFI1 mutant ears. (A-C) Gfi1^cre/cre cochlear OHCs degenerate in a basal to apical gradient at P0 (A,C), whereas vestibular HCs persist (B) (n=6). (D,E) Inner hair cell (IHC) counts at P0 revealed an increase in IHCs between 8 and 32 kHz (D), as well as increased IHC doublets at 16 kHz (E) in the Gfi1^cre/cre cochlea (Gfi1^cre/+ n=3, Gfi1^cre/cre n=4). (F) Gfi1^cre/cre vestibular HCs possess thinner stereocilia bundles (n=3). (G) P0 vestibular HC counts revealed no significant difference in HC number between genotypes (n=3). (H) Positive TUNEL staining is present in the P0 Gfi1^cre/cre cochlea, which is indicative of OHC death by apoptosis, while no TUNEL staining is observed in the vestibular system (n=3). (I) Gfi1^cre/cre OHCs abnormally expressed the neuronal marker TUBB3 (n=3). Scale bars: 20 µm (A,C,E,H,I; cochlea); 50 µm (B,F,H,I; vestibule). Data are mean±s.d. *P<0.05, **P<0.01; ns, not significant. Statistical significance assessed using a two-tailed Welch's t-test.

Fig. 2.

Translatome analysis reveals significant upregulation of neuronal mRNA in the HCs of GFI1 mutant mice. (A) P0 cochlear and vestibular tissues from mice expressing Rpl22^HA in HCs were collected separately for RiboTag immunoprecipitation. (B,C) In both cochlear (B, Gfi1^cre/+; B', Gfi1^cre/cre) and vestibular (C, Gfi1^cre/+; C', Gfi1^cre/cre) samples, immunoprecipitates (IPs) had higher levels of transcripts for the HC-expressed gene Myo6 compared with input (IN), but had lower levels of the transcripts for the mesenchymal-expressed gene Pou3f4. Immunoprecipitates were not enriched with Tubb3 (n=3). Dots represent individual replicates. Data are mean±s.d. (D) Tubb3 is upregulated in the mutant cochlear IPs compared with control IPs (fold change=7.87, P=0.0046), but not in mutant cochlear IN compared with control IN (n=3). (E) Number of genes upregulated and downregulated in the Gfi1^cre/cre cochlear and vestibular HCs. (F-H) Top 15 enriched gene ontology (GO) terms from genes downregulated (F) or upregulated (G) in Gfi1^cre/cre cochlear HCs, or upregulated in Gfi1^cre/cre vestibular HCs (H). (I-K) qPCR validation of dysregulated genes in vestibular (I) and cochlear (J,K) Gfi1^cre RiboTag immunoprecipitation samples (n=3). *P<0.05, **P<0.01, ***P<0.001, ns, not significant. Statistical significance assessed by a two-tailed Welch's t-test. Data are mean±s.d.

Fig. 3.

Downregulation of HC differentiation genes and upregulation of neuronal-associated genes in the GFI1 mutant HCs. (A-F) HC-specific downregulation of Fcrlb (A) and Sema5b (B), and upregulation of the neuronal markers Gfy (C), Lhx2 (D), Neurod1 (E), POU4F1 (normally Type IC neuron-specific, F,F′) and DCX (doublecortin, normally neuron-specific, G) in the Gfi1^cre/cre cochlea (n=3). Arrowheads indicate IHCs; arrows indicate OHCs; asterisks indicate nuclear POU4F1 staining. Scale bars: 20 µm. (H) Upregulation of DCX in Gfi1^cre/cre vestibular HCs (n=3). Scale bar: 20 µm. (I,I′) Downregulation of OCM in the Gfi1^cre/cre striolar vestibular HCs (n=3, arrows indicate loss of OCM expression). Scale bars: 50 µm. See also Figs S3 and S4.

Fig. 4.

GFI1 represses a neuronal-associated transcriptional profile in developing HCs. (A,B) As a group, genes upregulated (↑) in the Gfi1^cre/cre cochlear HCs (n=198) are normally repressed during HC development between E16 and P0 (A). Genes upregulated in Gfi1^cre/cre vestibular HCs (n=207) did not show a difference in expression between E16 and P0 (B). Genes downregulated (↓) in Gfi1^cre/cre cochlear (n=248) and vestibular (n=73) HCs are normally upregulated during HC development between E16 and P0 (A,B). (C) Examples of neuronal genes downregulated during HC development but upregulated in Gfi1^cre/cre HCs. (D) Examples of HC genes upregulated during HC development but downregulated in Gfi1^cre/cre HCs. (E) Neurod1 is the second most strongly upregulated gene in Gfi1^cre/cre cochlear HCs. (F) Genes upregulated in Gfi1^cre/cre cochlear (n=194) and vestibular (n=189) HCs are upregulated by mESCs overexpressing Neurod1. Statistical significance for A-C,F was assessed by a two-tailed Wilcoxon's test, comparing the Log₂ fold change (FC) values of each gene group to the Log₂ FC values of background (BG), which is all other genes expressed: B and C, cochlea↑BG, n=20,009, cochlea↓BG, n=19,959, vestibule↑BG, n=20,000, vestibule↓BG, n=20,134; and D, cochlea↑BG, n=21,743, vestibule↑BG, n=21,748. Center line represents median Log2 FC, gray box demarcates first and third quartiles, whiskers demarcate first and third quartiles±1.5× interquartile range values, dots represent single outliers. (G) The proposed mechanism by which GFI1 promotes HC fate is by repressing a neuronal-associated transcriptional profile during development.