© 2020. Published by The Company of Biologists Ltd | Development (2020) 147, dev186015. doi:10.1242/dev.186015

RESEARCH REPORT GFI1 functions to repress neuronal expression in the developing inner ear hair cells Maggie S. Matern1, Beatrice Milon1, Erika L. Lipford1, Mark McMurray1, Yoko Ogawa1, Andrew Tkaczuk1, Yang Song2, Ran Elkon3 and Ronna Hertzano1,2,4,*

ABSTRACT Pou4f3 and Gfi1 mutants (Bermingham et al., 1999; Hertzano et al., −/− Despite the known importance of the transcription factors ATOH1, 2004; Wallis et al., 2003). In the Gfi1 mouse inner ears, first the POU4F3 and GFI1 in hair cell development and regeneration, their cochlear outer HCs (OHCs) and then the inner HCs (IHCs) downstream transcriptional cascades in the inner ear remain largely degenerate in a basal-to-apical gradient, while dysfunctional unknown. Here, we have used Gfi1cre;RiboTag mice to evaluate vestibular HCs survive up to 5 months of age, resulting in both changes to the hair cell translatome in the absence of GFI1. We auditory and vestibular deficits (Fiolka et al., 2006; Wallis et al., identify a systematic downregulation of hair cell differentiation , 2003). Additionally, studies that identified Gfi1 as a downstream concomitant with robust upregulation of neuronal genes in the target of POU4F3 found that Gfi1 expression was nearly GFI1-deficient hair cells. This includes increased expression of undetectable in POU4F3-deficient HCs, and similarities between neuronal-associated transcription factors (e.g. Pou4f1) as well as the OHC degradation pattern of Pou4f3 and Gfi1 mutant mice −/− transcription factors that serve dual roles in hair cell and neuronal suggest that the Pou4f3 OHC phenotype is mainly a result of Gfi1 development (e.g. Neurod1, Atoh1 and Insm1). We further show that deficiency (Hertzano et al., 2004). However, despite the known the upregulated genes are consistent with the NEUROD1 regulon importance of these transcription factors, little is known of their and are normally expressed in hair cells prior to GFI1 onset. downstream transcriptional cascades in developing HCs. Additionally, minimal overlap of differentially expressed genes in Here, we define the role of GFI1 in the developing mouse auditory and vestibular hair cells suggests that GFI1 serves different inner ear by analyzing the translatome of Gfi1 mutant HCs. Our roles in these systems. From these data, we propose a dual studies reveal that Gfi1 mutant HCs exhibit significantly mechanism for GFI1 in promoting hair cell development, consisting decreased expression of genes associated with normal HC of repression of neuronal-associated genes as well as activation of development and function, as well as significantly increased hair cell-specific genes required for normal functional maturation. expression of genes involved in neuronal differentiation. Further analysis of the upregulated genes during HC development KEY WORDS: GFI1, Hair cells, Inner ear indicates that this neuronal-associated pattern is normally expressed within the cochlear HCs early in INTRODUCTION development, and their downregulation corresponds to the Hair cells (HCs) are the sensory cells of the inner ear that are onset to Gfi1 expression. necessary for hearing and balance. Studies of HC development have demonstrated that progression of precursor cells to HCs relies on the RESULTS AND DISCUSSION transcription factors ATOH1, POU4F3 and GFI1 (Bermingham HC degeneration and TUBB3 expression in newborn outer et al., 1999; Costa et al., 2015; Hertzano et al., 2004; Wallis et al., but not inner or vestibular Gfi1cre/cre HCs 2003). During cochlear development, ATOH1 protein is expressed To analyze GFI1-deficient HCs, we used the Gfi1cre knock-in in a basal to apical strip of cells between embryonic days (E) 13.5 mouse in which the coding regions of Gfi1 exons 1-5 have been and 14.5, committing these cells to a HC fate and also activating replaced with Cre recombinase (Yang et al., 2011). Homozygotes expression of POU4F3 between E14.5 and E16 (Hertzano et al., display both loss of GFI1 and efficient recombination in auditory 2004; Mulvaney and Dabdoub, 2012). POU4F3 then activates the and vestibular HCs, as well as inner ear macrophages (Matern et al., expression of GFI1 at E16.5 (Hertzano et al., 2004; Wallis et al., 2017; Yang et al., 2011). Our earlier work showed that Gfi1cre/cre 2003). Mice deficient in these transcription factors exhibit severe mice are deaf and exhibit severe vestibular dysfunction, comparable defects in HC development, ranging from no HC formation in the with the phenotype reported for Gfi1−/− mice (Matern et al., 2017). inner ears of Atoh1 mutants, to delayed degeneration of HCs in Staining for the HC marker MYO6 further demonstrates that early postnatal (P0) Gfi1cre/cre cochlear OHCs but not IHCs degenerate in 1Department of Otorhinolaryngology Head and Neck Surgery, University of a basal to apical gradient, whereas mutant vestibular HCs are not Maryland School of Medicine, Baltimore, MD 21201, USA. 2Institute for Genome significantly different in density compared with controls (Fig. 1A- Sciences, University of Maryland School of Medicine, Baltimore, MD 21201, USA. 3Department of Human Molecular Genetics and Biochemistry, Sackler School of B,G). P0-P5 whole-mounted cochleae also show a gradient pattern Medicine, Tel Aviv University, Tel Aviv 69978, Israel. 4Department of Anatomy and of OHC degeneration, with progressive loss of OHCs from base to Neurobiology, University of Maryland School of Medicine, Baltimore, MD 21201, apex, while the IHCs are present but appear immature (more like USA. apical IHCs) and ultimately degenerate by P32 (Fig. 1C, Figs S1A *Author for correspondence ([email protected]) and S2A). We also observed an increase in the number of IHCs in mutants at P0 and P5 (Fig. 1D, Fig. S1B), which we attribute to R.E., 0000-0003-3440-1286; R.H., 0000-0002-8093-6567 an increased incidence of IHC doublets (Fig. 1E). In the P0, P5 Handling Editor: Paola Arlotta and P32 utricle, saccule and crista, stereociliary bundles appear

Received 11 November 2019; Accepted 24 July 2020 thinner compared with controls (Fig. 1F, Figs S1C and S2B). DEVELOPMENT

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Fig. 1. HC degeneration and TUBB3 expression in outer but not inner or vestibular HCs of newborn GFI1 mutant ears. (A-C) Gfi1cre/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 Gfi1cre/cre cochlea (Gfi1cre/+ n=3, Gfi1cre/cre n=4). (F) Gfi1cre/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 Gfi1cre/cre cochlea, which is indicative of OHC death by apoptosis, while no TUNEL staining is observed in the vestibular system (n=3). (I) Gfi1cre/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.

Terminal deoxynucleotidyl transferase dUTP nick end labeling Enrichment of the inner ear HC translatomes using RiboTag (TUNEL) revealed that mutant OHCs degenerate by apoptosis, As GFI1 is known to function as a transcriptional (Suzuki whereas no TUNEL-positive HCs were detected in the mutant et al., 2016), we hypothesized that GFI1 targets would be vestibular organs (Fig. 1H). Finally, it has previously been upregulated in Gfi1cre/cre HCs. We therefore took advantage of reported that Gfi1−/− OHCs aberrantly express the neuron- Cre recombinase expression in the Gfi1cre model to perform a specific marker TUBB3 at E17.5 (Wallis et al., 2003). Staining comprehensive analysis of GFI1-deficient HC gene expression of P0 Gfi1cre/cre inner ears showed that the OHCs of this model using the RiboTag mouse (see Materials and Methods). This model also aberrantly express TUBB3, while TUBB3 staining was not allows for Cre-dependent expression of hemagglutinin-tagged (HA) observed in the IHCs or vestibular HCs (Fig. 1I). When ribosomes that can then be used to immunoprecipitate actively combined, these results indicate that the Gfi1cre/cre mouse can translated mRNA from cell types of interest (i.e. the translatome) be used as a reliable model to study Gfi1 deficiency in the inner (Sanz et al., 2009). Cochlear and vestibular tissues (utricles, ear and suggest different functional roles for GFI1 in the saccules and cristae) were collected from newborn Gfi1cre/cre; HA/HA cre/+ HA/HA cochlear and vestibular HCs. Rpl22 mutants and Gfi1 ;Rpl22 controls for DEVELOPMENT

2 RESEARCH REPORT Development (2020) 147, dev186015. doi:10.1242/dev.186015 immunoprecipitation of HC mRNA (Fig. 2A). qPCR analysis mRNA levels of the mesenchymal marker Pou3f4 (Fig. 2B,C). indicated that immunoprecipitation from both mutants and controls Tubb3 mRNA was not enriched by RiboTag immunoprecipitation increased mRNA levels of the HC marker Myo6 while depleting compared with whole-tissue inputs across all samples (Fig. 2B,C).

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 Rpl22HA in HCs were collected separately for RiboTag immunoprecipitation. (B,C) In both cochlear (B, Gfi1cre/+;B’, Gfi1cre/cre) and vestibular (C, Gfi1cre/+;C’, Gfi1cre/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 Gfi1cre/cre cochlear and vestibular HCs. (F-H) Top 15 enriched (GO) terms from genes downregulated (F) or upregulated (G) in Gfi1cre/cre cochlear HCs, or upregulated in Gfi1cre/cre vestibular HCs (H). (I-K) qPCR validation of dysregulated genes in vestibular (I) and cochlear (J,K) Gfi1cre 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. DEVELOPMENT

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However, comparing transcript levels of Tubb3 between Gfi1cre/cre genes in the Gfi1cre/cre cochlear HCs: Lhx2 (LFC=7.1, FDR=1.22E- and Gfi1cre/+ immunoprecipitated mRNA identified a significant 125), Neurod1 (LFC=6.73, FDR=2.62E-132) and St18 (LFC=6.72, increase in Tubb3 in the mutant cochlear HCs (∼7-fold, P=0.0046), FDR=4.75E-133) (Kameyama et al., 2011; Matsushita et al., 2014; while the same comparison using Gfi1cre/cre and Gfi1cre/+ whole- Pataskar et al., 2016; Subramanian et al., 2011). Of particular cochlea input samples was not sufficiently sensitive to detect this interest is Neurod1, a known driver of neuronal fate that is also difference (Fig. 2D). This highlights the utility of RiboTag for important for early HC development (Jahan et al., 2010, 2013). studying HC-specific changes in gene expression, as differences in NEUROD1 has been shown to target and upregulate other HC transcripts that are also expressed in other cell types (e.g. Tubb3 transcriptional regulators of neuronal development, such as St18, in the cochleovestibular ganglion) may not be detected when which was also significantly upregulated in Gfi1cre/cre vestibular comparing mRNA from whole tissues. HCs (LFC=4.86, FDR=8.66E-42), and Myt1, which was significantly upregulated in Gfi1cre/cre cochlear HCs (LFC=1.1, GFI1-deficient HCs downregulate HC genes and upregulate FDR=8.43E-9) (Lizio et al., 2015; Pataskar et al., 2016). genes associated with neuronal differentiation Additionally, the gene Insm1, another To further assess global differences in Gfi1cre/cre HC gene NEUROD1 target important for spiral ganglion and OHC expression, we performed RNA sequencing (RNA-seq) of the HC development, was upregulated in the Gfi1cre/cre cochlear HCs translatomes (Tables S1 and S2). Overall, we detected 210 (LFC=1.16, FDR=1.36E-7), and Atoh1 was upregulated in the upregulated and 277 downregulated genes in the Gfi1cre/cre Gfi1cre/cre vestibular HCs (LFC=2.04, FDR=1.45E-17) (Jahan et al., cochlear HCs, and 223 upregulated and 76 downregulated genes 2010; Lizio et al., 2015; Lorenzen et al., 2015; Wiwatpanit et al., cre/cre in the Gfi1 vestibular HCs [log2 fold change (LFC) >1 or <−1, 2018). false discover rate (FDR) <0.001, full separation – see Materials and To validate the RNA-seq results, we chose 17 genes for analysis Methods) (Fig. 2E). The HC-expressed genes Chrna1, Atp2a3 and by qPCR in independent RiboTag samples. These showed that the Fcrlb were found to be downregulated in both systems, whereas the HC-expressed genes Cdh1, Sema5b, Slc26a5, Strc, Strip2 and Tmc1 HC-expressed gene Myo6, which normally precedes GFI1 in were downregulated in the Gfi1cre/cre cochleae, and Fcrlb and Ocm expression, was not significantly changed (Chessum et al., 2018; were downregulated in the Gfi1cre/cre vestibular system (Fig. 2I,J). Liu et al., 2014; Scheffer et al., 2007). This suggests that the Additionally, the neuronal-associated genes Gap43, Gfy, Myt1, differences in HC gene expression observed between Gfi1cre/cre and Ncam1, Insm1, Neurod1, St18 and Lhx2 were upregulated in the Gfi1cre/+ are not a result of differences in HC number. Consistent mutant cochlear HCs, and Atoh1 and Gfy were upregulated in the with our qPCR and immunostaining results, Tubb3 was upregulated mutant vestibular HCs (Fig. 2I,K). Of note, upregulation of St18 in in the Gfi1cre/cre cochlear HC translatome only (LFC=3.49, the mutant vestibular HCs did not reach statistical significance, most FDR=6.59E-40). Interestingly, among the genes detected as likely due to variability in fold change between the mutant samples upregulated or downregulated in either the cochlear or vestibular (St18 fold change ranged from 18-33× upregulated in Gfi1cre/cre, HCs, only 73 (25.17%) upregulated and 21 (6.33%) downregulated P=0.051) (Fig. 2I). In situ hybridization and immunostaining genes were shared between the two systems (Fig. 2E). Finally, as further revealed HC-specific differences in expression of the HC Gfi1cre also drives recombination in inner ear macrophages, we genes Fcrlb and Sema5b, as well as the neuronal-associated genes interrogated our dataset for changes to 360 previously defined Gfy, Neurod1, Lhx2, doublecortin (DCX) and POU4F1 (normally a macrophage-expressed genes (Matern et al., 2017). Of these, only specific marker of type IC neurons) in the mutant cochlear HCs four genes (Nceh1, Rnf128, Fgd3 and Spp1) were dysregulated in compared with controls (Fig. 3A-G, Figs S3 and S4) (Petitpré et al., either the Gfi1cre/cre cochlear or vestibular samples, suggesting that 2018; Shrestha et al., 2018; Sun et al., 2018). Interestingly, changes the observed differences in gene expression are likely a result of in mRNA and protein expression varied between mutant IHCs and global changes to HCs rather than macrophages. OHCs, confirming that GFI1 plays distinct roles in the development To identify major dysregulated biological processes in the Gfi1- of these two cell types. In the vestibular system, immunostaining deficient HCs, we performed Gene Ontology (GO) term enrichment revealed upregulation of the neuronal marker DCX (Fig. 3H), as analyses. These revealed that genes downregulated in the mutant well as downregulation of the striolar HC-expressed protein OCM cochlear HCs are significantly enriched for genes involved in (oncomodulin) in Gfi1cre/cre ears (Fig. 3I). These results further sensory perception of sound and inner ear development, such as validate that, in the absence of GFI1, HC-specific gene expression is Slc26a5 (LFC=−4.24, FDR=3.28E-33), Strc (LFC=−3.07, significantly disrupted, and HCs express multiple markers of FDR=6.07E-37) and Tmc1 (LFC=−1.28, FDR=1.74E-06), neuronal cells. suggesting Gfi1-deficient cochlear HCs have undergone a maturation arrest (Fig. 2F). A similar analysis of genes downregulated in the GFI1 plays a role in repressing early neuronal gene Gfi1cre/cre vestibular HCs revealed only one significant GO term expression in developing HCs (‘myeloid leukocyte migration’, enrichment=14.95, adjusted Given the upregulation of neuronal-associated genes in Gfi1cre/cre P=0.0347), possibly highlighting the limited published knowledge of HCs, we assessed whether these genes are aberrantly activated vestibular HC gene function. Supporting this, one gene within this GO in Gfi1cre/cre HCs or whether they are normally present in early term (Spp1, encoding osteopontin: LFC=−2.03, FDR=2.70E-13) was HCs but fail to be downregulated without GFI1. For this, we recently identified as a marker for type I HCs in the mouse utricle examined a dataset that recorded gene expression profiles in sorted (McInturff et al., 2018; Wang et al., 2019). cochlear and utricular HCs at four developmental time points (E16, Analysis of genes upregulated in the mutant cochlear and P0, P4 and P7) (data available via the gEAR portal; Scheffer et al., vestibular HCs revealed a striking enrichment for genes involved in 2015). Importantly, this dataset shows that the neuronal-associated neuronal processes, including GO terms such as ‘neuron genes upregulated in Gfi1cre/cre cochlear HCs are normally development’ and ‘positive regulation of neuron differentiation’ expressed at E16, preceding the onset of Gfi1 expression at E16.5, (Fig. 2G,H). Interestingly, three transcriptional regulators associated and are subsequently downregulated by P0 (Fig. 4A,C). This with neuronal development fall within the top five upregulated suggests that a neuronal transcriptional profile is indeed expressed DEVELOPMENT

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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 Gfi1cre/cre cochlea (n=3). Arrowheads indicate IHCs; arrows indicate OHCs; asterisks indicate nuclear POU4F1 staining. Scale bars: 20 µm. (H) Upregulation of DCX in Gfi1cre/cre vestibular HCs (n=3). Scale bar: 20 µm. (I,I′) Downregulation of OCM in the Gfi1cre/cre striolar vestibular HCs (n=3, arrows indicate loss of OCM expression). Scale bars: 50 µm. See also Figs S3 and S4. DEVELOPMENT

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Fig. 4. GFI1 represses a neuronal-associated transcriptional profile in developing HCs. (A,B) As a group, genes upregulated (↑) in the Gfi1cre/cre cochlear HCs (n=198) are normally repressed during HC development between E16 and P0 (A). Genes upregulated in Gfi1cre/cre vestibular HCs (n=207) did not show a difference in expression between E16 and P0 (B). Genes downregulated (↓)inGfi1cre/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 Gfi1cre/cre HCs. (D) Examples of HC genes upregulated during HC development but downregulated in Gfi1cre/cre HCs. (E) Neurod1 is the second most strongly upregulated gene in Gfi1cre/cre cochlear HCs. (F) Genes upregulated in Gfi1cre/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. in early differentiating cochlear HCs but fails to be repressed in the cochlear HCs between E16.5 and P0 (Fig. 4C; ∼1000-fold Gfi1cre/cre mice. Importantly, the expression of genes upregulated in repression), we investigated whether NEUROD1 or its targets Gfi1cre/cre vestibular HCs did not change between E16 and P0 in could be responsible for driving neuronal-associated gene utricular HCs (Fig. 4B), reflecting either a different mechanism of expression in Gfi1 mutant HCs. For this, we compared the genes vestibular HC development or the earlier onset of Gfi1 expression in upregulated in Gfi1cre/cre HCs with a dataset measuring changes in the vestibular system (E14.5, a time point not captured in the gene expression in mouse embryonic stem cells (mESCs) induced to interrogated dataset) (Wallis et al., 2003). Additionally, we found neurodifferentiate through NEUROD1 overexpression (Pataskar that genes downregulated in Gfi1cre/cre cochlear and vestibular HCs et al., 2016). We found that the neuronal-associated genes are normally upregulated during development from E16 to P0 upregulated in the Gfi1cre/cre HCs are also significantly induced by (Fig. 4A,B,D). Neurod1 expression in mESCs (Fig. 4F). Overall, these analyses NEUROD1 is a known transcriptional regulator of neuronal suggest that GFI1-deficient cochlear and vestibular HCs undergo a programs (Pataskar et al., 2016). As Neurod1 is detected in maturation arrest and maintain expression of a neuronal our dataset as the third highest upregulated gene in Gfi1cre/cre transcriptional profile that is likely normally present immediately cochlear HCs (Fig. 4E), and is also the most repressed gene in normal after HC specification. DEVELOPMENT

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Conclusions previously noted between Gfi1+/+ and Gfi1cre/+ at P8 (all attributed to We have used the Gfi1cre/cre mouse model to perform a potential sex bias in samples analyzed), suggesting that Gfi1cre/+ can serve +/+ comprehensive analysis of GFI1 loss on neonatal inner ear HC as a proxy for Gfi1 (Matern et al., 2017). For each biological sample, cochlear ducts or vestibular tissues (utricles, saccules and cristae) from five translatomes. Our gene expression results show that in the absence cre/+ HA/HA cre/cre HA/HA of GFI1, HCs within the cochlear and the vestibular organs undergo P0 Gfi1 ;Rpl22 or Gfi1 ;Rpl22 mice were pooled and homogenized in 1 ml of supplemented homogenization buffer [50 mM Tris- a maturation arrest, failing to upregulate known markers of mature HCl (pH 7.4), 100 mM KCl, 12 mM MgCl2, 1% Nonidet P-40, 1 mM 1,4- HCs, such as Strc, Tmc1 and Ocm. Additionally, our results suggest Dithiothreitol, 1× protease inhibitor cocktail, 200 U/ml RNAseOUT, that an important role of GFI1 in early HC differentiation is to 100 μg/ml cycloheximide and 1 mg/ml heparin]. Careful dissection was repress neuronal-associated gene expression (Fig. 4G), adding to the performed to minimize collection of neuronal tissues. Homogenates were accumulating evidence of GFI1s ability to serve this function (see then centrifuged to remove cell debris (9400 g for 10 min at 4°C) and 40 μl Lee et al., 2019). The abundant upregulation of neuronal-associated reserved as input control. HA antibody (5 μg; BioLegend, 901502, Lot# genes in the Gfi1cre/cre HCs is likely secondary to the maintained B220767) was added to the remaining homogenate and incubated under expression of key transcriptional drivers of neuronal fate, such as gentle rotation for 4-6 h at 4°C before adding 300 μl of rinsed Invitrogen Neurod1, which appear to be normally expressed in early Dynabeads Protein G magnetic beads (Thermo Fisher). Samples were then developing HCs. Finally, this identified role for GFI1 in incubated under gentle rotation overnight at 4°C. Bound beads were rinsed 3× with 800 μl high salt buffer [50 mM Tris-HCl (pH 7.4), 300 mM KCl, repressing neuronal-associated gene expression has an important 12 mM MgCl2, 1% Nonidet P-40, 1 mM 1,4-Dithiothreitol, 100 μg/ml translational impact, explaining part of its functional significance in cycloheximide] under gentle rotation at 4°C. Buffer RLT from the RNeasy directing stem cells towards the HC fate. Plus Micro kit (Qiagen) (350 μl) was used to release bound ribosomes and associated RNAs from the beads. RNA was extracted using the RNeasy Plus MATERIALS AND METHODS Micro kit (Qiagen), using 16 µl of nuclease free water for elution. RNA Animals quality was assessed using the Agilent RNA Pico kit (Agilent The RiboTag (C57BL/6N background) and Gfi1cre mice (C57BL/6J Technologies), and only samples with RIN >8 were used for RNA- background) have been described previously (Sanz et al., 2009; Yang sequencing. et al., 2011). To generate animals for the Gfi1cre;RiboTag RNA-seq dataset, RiboTag mice (Rpl22HA/HA) were crossed to Gfi1cre/+ mice to produce RNA-sequencing and data analysis Gfi1cre/+;Rpl22HA/+ mice. These mice were further crossed to obtain Gfi1cre/+; Gfi1cre;RiboTag IP RNA-seq libraries were prepared in biological triplicates Rpl22HA/HA breeding pairs, which were used to generate Gfi1cre/+;Rpl22HA/HA from cochlear tissues using the Ovation Ultralow Library Preparation Kit and Gfi1cre/cre;Rpl22HA/HA neonates for RiboTag immunoprecipitation. Both (NuGEN), and vestibular tissues using the NEBNext Ultra Directional RNA male and female mice were used for all experiments. All procedures Library Prep Kit (New England BioLabs) as per the manufacturers’ involving animals were carried out in accordance with the National instructions. Samples were then sequenced on a HiSeq 2500 system Institutes of Health Guide for the Care and Use of Laboratory Animals and (Illumina) using a 100 bp paired end read configuration at the University of have been approved by the Institutional Animal Care and Use Committee Maryland School of Medicine Institute for Genome Sciences. Reads were at the University of Maryland School of Medicine (protocol numbers aligned to the mouse reference genome (mm10) using TopHat (Trapnell 1015003 and 0918005). et al., 2009), and the number of reads aligning to predicted coding regions was quantified using HTSeq (Anders et al., 2015). See Table S3 for Immunostaining and image acquisition alignment statistics. Significant differential gene expression between Inner ears from Gfi1cre mice were dissected between P0 and P32 and fixed in Gfi1cre/+ and Gfi1cre/cre RiboTag IP samples was assessed using DEseq 4% paraformaldehyde (PFA) in PBS overnight at 4°C. Ears were then either (Anders and Huber, 2010). In addition to a cutoff of LFC >1 or <−1 and stored in PBS for further dissection or processed and embedded in OCT FDR<0.001, we required a full separation of normalized expression values compound (Tissue-Tek). Immunostaining on sectioned or whole-mounted (counts per million, CPM) between replicates to refer to a gene as tissues was performed using the following primary antibodies: mouse anti- differentially expressed. For example, for a gene to be referred to as TUBB3 (1:500, BioLegend, 801213); goat anti-oncomodulin N-19 (1:100, downregulated in the Gfi1cre/cre samples compared with Gfi1cre/+, the Santa Cruz Biotechnology, sc-7446, Lot L0814); mouse anti-POU4F1 normalized expression levels measured in each replicate of Gfi1cre/cre must (1:50, Millipore, MAB1585); guinea pig anti-DCX (1:5000, Millipore, be lower than the lowest expression level measured in a Gfi1cre/+ replicate. AB2253); rabbit anti-myosin-VI (1:1000, Proteus BioSciences, 25-6791). Gene ontology analyses were performed using the Gene Ontology database Complementary Alexa Fluor 488 and 546 (1:800, Invitrogen) antibodies (www.geneontology.org) (Harris et al., 2004). were used for secondary detection, Alexa Fluor 488 Phalloidin (1:1000, Invitrogen) was used to stain F-actin, and DAPI (25 µg/ml, Invitrogen) was Quantitative PCR used to stain cell nuclei. TUNEL staining was performed using the Click-iT RNA from independent Gfi1cre;RiboTag IP and input samples was reverse- Plus TUNEL Assay (Invitrogen) following the manufacturer’s instructions. transcribed using the Maxima First Strand cDNA Synthesis Kit (Thermo An average of between zero and one TUNEL-positive HC nuclei were Fisher), and preamplified using TaqMan PreAmp Master Mix (Applied observed per Gfi1cre/cre cochlear cross-section (∼12 cross-sections analyzed Biosystems). qPCR was performed using TaqMan Fast Advanced Master per animal, three animals per genotype), always within the middle and basal Mix (Applied Biosystems). In cases where no amplification was detected, turns where OHCs are actively degenerating. Images were acquired using threshold values were set to the maximum cycle (40). Cycle threshold levels either a Nikon Eclipse E600 microscope equipped with a Lumenera were normalized to averaged Actb and Tbp expression, with the exception of Infinity 3 camera or a Nikon CSU-W1 spinning disk confocal microscope Fig. 2J,K, where cochlear samples were normalized to Myo6 to control for located at the University of Maryland School of Medicine Center for possible differences in HC number. qPCR was performed in three biological Innovative Biomedical Resources, Confocal Microscopy Core, Baltimore, replicates, and statistical significance between samples was assessed by Maryland. Welch’s t-test. See Table S4 for TaqMan probes.

RiboTag immunoprecipitations In situ hybridization RiboTag immunoprecipitations from inner ear tissues were performed as In situ hybridization was performed as previously described (Chessum et al., previously described (Chessum et al., 2018). Of note, Gfi1cre/+ animals were 2018). Briefly, 10 µm inner ear sections mounted on positively charged used as controls in this experiment out of necessity to drive Cre glass slides were refixed with 4% PFA before a 10 min treatment with recombination, despite their age-related hearing loss phenotype (Matern 2 µg/ml Proteinase-K (New England Biolabs). The Proteinase-K reaction et al., 2017). Only minimal differences in gene expression have been was stopped using 4% PFA, and tissues were soaked in acetylation and DEVELOPMENT

7 RESEARCH REPORT Development (2020) 147, dev186015. doi:10.1242/dev.186015 permeabilization buffers. Digoxigenin-labeled probes for Fcrlb, Sema5b, Hertzano, R., Montcouquiol, M., Rashi-elkeles, S., Elkon, R., Yücel, R., Frankel, Gfy, Lhx2 and Neurod1 were hybridized to sections overnight at 65°C. See W. N., Rechavi, G., Möröy, T., Friedman, T. B., Kelley, M. W. et al. (2004). Table S4 for primer sequences. After washing, slides were incubated with Transcription profiling of inner ears from Pou4f3 ddl/ddl identifies Gfi1 as a target of the Pou4f3 deafness gene. Hum. Mol. Genet. 13, 2143-2153. doi:10.1093/hmg/ sheep anti-digoxigenin antibody conjugated to alkaline phosphatase (Sigma- ddh218 Aldrich, 11093274910, Lot# 14608125) diluted to 1:100 overnight at 4°C. Jahan, I., Pan, N., Kersigo, J. and Fritzsch, B. (2010). Neurod1 suppresses hair Colorimetric visualization of hybridized probes was performed using BM cell differentiation in ear ganglia and regulates hair cell subtype development in purple AP substrate precipitating solution (Roche). The colorimetric reaction the cochlea. PLoS ONE 5, e11661. doi:10.1371/journal.pone.0011661 was halted by soaking slides in 1× TBS, after which slides were reblocked and Jahan, I., Pan, N., Kersigo, J. and Fritzsch, B. (2013). Beyond generalized hair immunostained with the rabbit anti-myosin-VI antibody (1:1000, Proteus cells: molecular cues for hair cell types. Hear. Res. 297, 30-41. doi:10.1016/j. heares.2012.11.008 BioSciences, Cat# 25-6791) and a corresponding Alexa Fluor 488 or 546 Janky, R., Verfaillie, A., Imrichová, H., Van de Sande, B., Standaert, L., secondary (1:800, Invitrogen). Christiaens, V., Hulselmans, G., Herten, K., Sanchez, M. N., Potier, D. et al. (2014). iRegulon: from a gene list to a gene regulatory network using large motif Acknowledgements and track collections. PLoS Comput. Biol. 10, e1003731. doi:10.1371/journal. pcbi.1003731 The authors thank Drs M. K. Lobo and J. Zuo for providing the RiboTag and Gfi1cre Kameyama, T., Matsushita, F., Kadokawa, Y. and Marunouchi, T. (2011). Myt/ mouse models for this study, and Dr A. Cheng for critically reviewing the manuscript. NZF family transcription factors regulate neuronal differentiation of P19 cells. Neurosci. Lett. 497, 74-79. doi:10.1016/j.neulet.2011.04.033 Competing interests Lee, C., Rudneva, V. A., Erkek, S., Zapatka, M., Chau, L. Q., Tacheva-Grigorova, The authors declare no competing or financial interests. S. K., Garancher, A., Rusert, J. M., Aksoy, O., Lea, R. et al. (2019). Lsd1 as a therapeutic target in Gfi1-activated medulloblastoma. Nat. Commun. 10, 332. Author contributions doi:10.1038/s41467-018-08269-5 Liu, H., Pecka, J. L., Zhang, Q., Soukup, G. A., Beisel, K. W., David, X. and He, Conceptualization: M.S.M., B.M., R.E., R.H.; Methodology: B.M., R.H.; Software: Z. Z. (2014). Characterization of transcriptomes of cochlear inner and outer hair Y.S., R.E.; Validation: M.S.M.; Formal analysis: M.S.M., Y.S., R.E.; Investigation: cells. J. Neurosci. 34, 11085-11095. doi:10.1523/JNEUROSCI.1690-14.2014 M.S.M., B.M., E.L.L., M.M., Y.O., A.T., R.E.; Writing - original draft: M.S.M., R.H.; Lizio, M., Ishizu, Y., Itoh, M., Lassmann, T., Hasegawa, A., Kubosaki, A., Writing - review & editing: M.S.M., B.M., R.E., R.H.; Visualization: M.S.M., B.M.; Severin, J., Kawaji, H., Nakamura, Y., Suzuki, H. et al. (2015). Mapping Supervision: B.M., R.E., R.H.; Project administration: B.M., R.H.; Funding mammalian cell-type-specific transcriptional regulatory networks using KD-CAGE acquisition: R.E., R.H. and ChIP-seq data in the TC-YIK cell line. Front. Genet. 6, 331. doi:10.3389/ fgene.2015.00331 Funding Lorenzen, S. M., Duggan, A., Osipovich, A. B., Magnuson, M. A. and Garcıa-́ This work was supported by the National Institute on Deafness and Other Añoveros, J. (2015). Insm1 promotes neurogenic proliferation in delaminated otic Communication Disorders (NIDCD)/National Institutes of Health (R01DC013817 progenitors. Mech. Dev. 138, 233-245. doi:10.1016/j.mod.2015.11.001 Matern, M. S., Vijayakumar, S., Margulies, Z., Milon, B., Song, Y., Elkon, R., and R01DC03544 to R.H.; T32DC00046 and F31DC016218 to M.S.M.); and by the Zhang, X., Jones, S. M. and Hertzano, R. (2017). Gfi1Cre mice have early onset United States - Israel Binational Science Foundation (2017218 to R.H. and R.E.). progressive hearing loss and induce recombination in numerous inner ear non- Deposited in PMC for release after 12 months. hair cells. Sci. Rep. 7, 42079. doi:10.1038/srep42079 Matsushita, F., Kameyama, T., Kadokawa, Y. and Marunouchi, T. (2014). Data availability Spatiotemporal expression pattern of Myt/NZF family transcription RNA-seq data have been deposited in the GEO under accession number factors during mouse nervous system development. Dev. Dyn. 243, 588-600. GSE135760 and are also available for viewing and analysis on the gEAR portal doi:10.1002/dvdy.24091 (https://umgear.org/p?s=fd78e02b). McInturff, S., Burns, J. C. and Kelley, M. W. (2018). Characterization of spatial and temporal development of Type I and Type II hair cells in the mouse utricle using new cell-type-specific markers. Biol. Open 7, bio038083. doi:10.1242/bio.038083 Supplementary information Mulvaney, J. and Dabdoub, A. (2012). Atoh1, an essential transcription factor in Supplementary information available online at neurogenesis and intestinal and inner ear development: function, regulation, and https://dev.biologists.org/lookup/doi/10.1242/dev.186015.supplemental context dependency. J. Assoc. Res. Otolaryngol. 13, 281-293. doi:10.1007/ s10162-012-0317-4 Peer review history Pataskar, A., Jung, J., Smialowski, P., Noack, F., Calegari, F., Straub, T. and Tiwari, V. K. (2016). NeuroD1 reprograms chromatin and transcription factor The peer review history is available online at landscapes to induce the neuronal program. EMBO J. 35, 24-45. doi:10.15252/ https://dev.biologists.org/lookup/doi/10.1242/dev.186015.reviewer-comments.pdf. embj.201591206 Petitpré, C., Wu, H., Sharma, A., Tokarska, A., Fontanet, P., Wang, Y., References Helmbacher, F., Yackle, K., Silberberg, G., Hadjab, S. et al. (2018). Neuronal Anders, S. and Huber, W. (2010). Differential expression analysis for sequence heterogeneity and stereotyped connectivity in the auditory afferent system. Nat. count data. Genome Biol. 11, R106. doi:10.1186/gb-2010-11-10-r106 Commun. 9, 3691. doi:10.1038/s41467-018-06033-3 Anders, S., Pyl, P. T. and Huber, W. (2015). Genome analysis HTSeq — a Python Sanz, E., Yang, L., Su, T., Morris, D. R., Mcknight, G. S. and Amieux, P. S. (2009). framework to work with high-throughput sequencing data. Bioinformatics 31, Cell-type-specific isolation of ribosome-associated mRNA from complex tissues. 166-169. doi:10.1093/bioinformatics/btu638 Proc. Natl. Acad. Sci. USA 106, 13939-13944. doi:10.1073/pnas.0907143106 Bermingham, N. A., Hassan, B. A., Price, S. D., Vollrath, M. A., Ben-Arie, N., Scheffer, D., Sage, C., Plazas, P. V., Huang, M., Wedemeyer, C., Zhang, D.-S., Eatock, R. A., Bellen, H. J., Lysakowski, A. and Zoghbi, H. Y. (1999). Math1: an Chen, Z.-Y., Elgoyhen, A. B., Corey, D. P. and Pingault, V. (2007). The α1 essential gene for the generation of inner ear hair cells. Science 284, 1837-1841. subunit of nicotinic acetylcholine receptors in the inner ear: transcriptional doi:10.1126/science.284.5421.1837 regulation by ATOH1 and co-expression with the γ subunit in hair cells. Chessum, L., Matern, M. S., Kelly, M. C., Johnson, S. L., Ogawa, Y., Milon, B., J. Neurochem. 103, 2651-2664. doi:0.1111/j.1471-4159.2007.04980.x McMurray, M., Driver, E. C., Parker, A., Song, Y. et al. (2018). Helios is a key Scheffer, I., Shen, X. J., Corey, X. D. P. and Chen, X. Z. (2015). Gene expression transcriptional regulator of outer hair cell maturation. Nature 563, 696-700. doi:10. by mouse inner ear hair cells during development. J. Neurosci. 35, 6366-6380. 1038/s41586-018-0728-4 doi:10.1523/JNEUROSCI.5126-14.2015 Costa, A., Sanchez-Guardado, L., Juniat, S., Gale, J. E., Daudet, N. and Shrestha, B. R., Chia, C., Wu, L., Kujawa, S. G., Liberman, M. C. and Goodrich, Henrique, D. (2015). Generation of sensory hair cells by genetic programming L. V. (2018). Sensory neuron diversity in the inner ear is shaped by activity. Cell with a combination of transcription factors. Development 142, 1948-1959. doi:10. 174, 1229-1246.e17. doi:10.1016/j.cell.2018.07.007 1242/dev.119149 Subramanian, L., Sarkar, A., Shetty, A. S., Muralidharan, B., Fiolka, K., Hertzano, R., Vassen, L., Zeng, H., Hermesh, O., Avraham, K. B., Padmanabhan, H., Piper, M., Monuki, E. S., Bach, I., Gronostajski, Dührsen, U. and Möröy, T. (2006). Gfi1 and Gfi1b act equivalently in R. M., Richards, L. J. et al. (2011). Transcription factor Lhx2 is necessary , but have distinct, non-overlapping functions in inner ear and sufficient to suppress astrogliogenesis and promote neurogenesis in the development. EMBO Rep. 7, 326-333. doi:10.1038/sj.embor.7400618 developing hippocampus. Proc. Natl. Acad. Sci. USA 108, E265-E274. Harris, M., Clark, J., Ireland, A., Lomax, J., Ashburner, M., Foulger, R., Eilbeck, doi:10.1073/pnas.1101109108 K., Lewis, S., Marshal, L. B., Mungall, C. et al. (2004). The Gene Ontology (GO) Sun, S., Babola, T., Pregernig, G., So, K. S., Nguyen, M., Su, S. S.-M., Palermo, database and informatics resource. Nucleic Acids Res. 32, 258-261. doi:10.1093/ A. T., Bergles, D. E., Burns, J. C. and Müller, U. (2018). Hair cell

nar/gkh066 mechanotransduction regulates spontaneous activity and spiral ganglion DEVELOPMENT

8 RESEARCH REPORT Development (2020) 147, dev186015. doi:10.1242/dev.186015

subtype specification in the auditory system. Cell 174, 1247-1263.e15. doi:10. hair cell differentiation and survival. Development 130, 221-232. doi:10.1242/dev. 1016/j.cell.2018.07.008 00190 Suzuki, J., Maruyama, S., Tamauchi, H., Kuwahara, M., Horiuchi, M., Mizuki, M., Wang, T., Niwa, M., Sayyid, Z. N., Hosseini, D. K., Pham, N., Jones, S. M., Ricci, Ochi, M., Sawasaki, T., Zhu, J., Yasukawa, M. et al. (2016). Gfi1, a A. J. and Cheng, A. G. (2019). Uncoordinated maturation of developing and transcriptional repressor, inhibits the induction of the T helper type 1 regenerating postnatal mammalian vestibular hair cells. PLoS Biol. 17, e3000326. programme in activated CD4 T cells. Immunology 147, 476-487. doi:10.1111/ doi:10.1371/journal.pbio.3000326 imm.12580 Wiwatpanit, T., Lorenzen, S. M., Cantú, J. A., Foo, C. Z., Hogan, A. K., Márquez, Trapnell, C., Pachter, L. and Salzberg, S. L. (2009). TopHat: discovering splice F., Clancy, J. C., Schipma, M. J., Cheatham, M. A., Duggan, A. et al. (2018). junctions with RNA-Seq. Bioinformatics 25, 1105-1111. doi:10.1093/ Trans-differentiation of outer hair cells into inner hair cells in the absence of bioinformatics/btp120 INSM1. Nature 563, 691-695. doi:10.1038/s41586-018-0570-8 Wallis, D., Hamblen, M., Zhou, Y., Venken, K. J. T., Schumacher, A., Grimes, Yang, H., Gan, J., Xie, X., Deng, M., Feng, L., Chen, X., Gao, Z. and Gan, L. H. L., Zoghbi, H. Y., Orkin, S. H. and Bellen, H. J. (2003). The zinc finger (2011). Gfi1-Cre knock-in mouse line: a tool for inner ear hair cell-specific gene transcription factor Gfi1, implicated in lymphomagenesis, is required for inner ear deletion. Genesis 48, 400-406. doi:10.1002/dvg.20632 DEVELOPMENT

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