The mammalian cochlea contains two types of mechanosensory hair cell that have different and critical functions in hearing. Inner hair cells (IHCs), which have an elaborate presynaptic apparatus, signal to cochlear neurons and communicate sound information to the brain. Outer hair cells (OHCs) mechanically amplify sound-induced vibrations, providing enhanced sensitivity to sound and sharp tuning. Cochlear hair cells are solely generated during development, and hair cell death—most often of OHCs—is the most common cause of deafness. OHCs and IHCs, together with supporting cells, originate in embryos from the prosensory region of the otocyst, but how hair cells differentiate into two different types is unknown1,2,3. Here we show that Insm1, which encodes a zinc finger protein that is transiently expressed in nascent OHCs, consolidates their fate by preventing trans-differentiation into IHCs. In the absence of INSM1, many hair cells that are born as OHCs switch fates to become mature IHCs. To identify the genetic mechanisms by which Insm1 operates, we compared the transcriptomes of immature IHCs and OHCs, and of OHCs with and without INSM1. In OHCs that lack INSM1, a set of genes is upregulated, most of which are normally preferentially expressed by IHCs. The homeotic cell transformation of OHCs without INSM1 into IHCs reveals a mechanism by which these neighbouring mechanosensory cells begin to differ: INSM1 represses a core set of early IHC-enriched genes in embryonic OHCs and makes them unresponsive to an IHC-inducing gradient, so that they proceed to mature as OHCs. Without INSM1, some of the OHCs in which these few IHC-enriched transcripts are upregulated trans-differentiate into IHCs, identifying candidate genes for IHC-specific differentiation.

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All data are available from the corresponding authors upon reasonable request. RNA-seq data are available for public view at the gEAR portal (https://umgear.org/).

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NU core facilities used were NUSeq, TTML, CAM and FC (partially supported by CA060553 to RHLCCC) . We thank A. Groves for protocols and advice and D. He for original databases of microarray results. Supported by NIH grants DC015903, DC000089 and DC012483.

Reviewer information

Nature thanks S. Heller and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Author notes

    • Jorge A. Cantú

    Present address: Department of Biology, Northeastern Illinois University, Chicago, IL, USA

  1. These authors contributed equally: Teerawat Wiwatpanit, Sarah M. Lorenzen, Jorge A. Cantú

  2. These authors jointly supervised this work: Anne Duggan, Jaime García-Añoveros


  1. Department of Anesthesiology, Northwestern University, Feinberg School of Medicine, Chicago, IL, USA

    • Teerawat Wiwatpanit
    • , Sarah M. Lorenzen
    • , Jorge A. Cantú
    • , Chuan Zhi Foo
    • , Ann K. Hogan
    • , Freddie Márquez
    • , John C. Clancy
    • , Anne Duggan
    •  & Jaime García-Añoveros
  2. Driskill Graduate Program in Life Sciences, Northwestern University, Chicago, IL, USA

    • Teerawat Wiwatpanit
    • , Chuan Zhi Foo
    •  & Ann K. Hogan
  3. NUIN Graduate Program, Northwestern University, Chicago, IL, USA

    • Sarah M. Lorenzen
  4. Next Generation Sequencing Core, Northwestern University, Feinberg School of Medicine, Chicago, IL, USA

    • Matthew J. Schipma
  5. Department of Communication Sciences and Disorders, Northwestern University, Evanston, IL, USA

    • Mary Ann Cheatham
  6. Hugh Knowles Center for Clinical and Basic Science in Hearing and its Disorders, Northwestern University, Chicago, IL, USA

    • Mary Ann Cheatham
    •  & Jaime García-Añoveros
  7. Department of Neurology, Northwestern University, Feinberg School of Medicine, Chicago, IL, USA

    • Jaime García-Añoveros
  8. Department of Physiology, Northwestern University, Feinberg School of Medicine, Chicago, IL, USA

    • Jaime García-Añoveros


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J.G.-A. and A.D. conceived the project. T.W., S.M.L., J.A.C., C.Z.F., A.K.H., F.M., J.C.C., M.A.C., A.D. and J.G.-A. performed experiments. T.W., S.M.L., J.A.C., C.Z.F., M.J.S., M.A.C., A.D. and J.G.-A. analysed data. J.G.-A. and T.W. wrote the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Anne Duggan or Jaime García-Añoveros.

Extended data figures and tables

  1. Extended Data Fig. 1 Generation of a conditional KO allele of Insm1.

    a, We generated a targeting construct in which the sole exon of Insm1 (green rectangle, with the coding sequence in dark green and the UTRs in light green) has a loxP site (purple triangle) inserted in a poorly conserved area of its 5′UTR and another loxP site downstream of the Insm1 gene. The construct also incorporates a neomycin resistance cassette (NEO, blue) surrounded by Frt sites (red triangles) and a thymidine kinase cassette (HSV-TK; orange), which are used to select for recombination events after gene targeting. b, We screened 439 clones and identified 5 recombinants (1 non-recombinant wild type, B5, and 3 recombinants, B6, E10 and B3, are shown) with PCR using primers indicated in a (arrows). The expected size for the wild-type allele using primers WT to R2 is 6,163 bp. The expected size for recombinant clones using CKO-reverse is 6,145 bp. c, Selected embryonic stem (ES) cell clones were additionally screened for homologous recombination upstream of the first loxP site by Southern blotting after digestion with SpeI and using the 5′ probe indicated in a6. Southern blotting was performed twice. The expected band sizes for wild-type and conditional KO alleles are 18,162 bp and 14,333 bp, respectively. From one of these clones (B3) we generated first chimeric mice and then mice with floxed alleles of Insm1 (obtained by crossing the chimaeras with mice expressing the FlpE recombinase (B6-Tg(CAG-FLPe)36, which deleted the NEO cassette flanked by FRT sites). Homozygous Insm1F/F mice are viable, demonstrating that the loxP insertions do not interfere with the vital functions of Insm1 and hence may be used for its conditional ablation. Co-expression with Cre recombinase generates an Insm1 KO allele lacking its entire coding sequence.

  2. Extended Data Fig. 2 Conditional ablation of Insm1 in cochleae.

    In situ hybridization for Insm1 transcripts on cryosections of embryonic E16.5 and neonatal (P0 and P1) cochleae. ac, In control cochleae (top), Insm1 is expressed in all OHCs (72/72 OHCs from 3 animals) and spiral ganglion (SG; white arrows) at E16.5. However, no Insm1 was detected in the organs of Corti from apical turns, in which recognizable hair cells have not yet appeared (a, c; asterisks). By postnatal age P0–P1, Insm1 mRNA is present in 90% of OHCs (94/105 OHCs from 2 animals), and it is undetectable in spiral ganglion (b, d). a, b, Bottom, in TgPax2Cre/+;Insm1F/F mice, Insm1 mRNA is undetectable in spiral ganglion and in all OHCs from E16.5 (0/69 OHCs from 2 mice; a) and P0 (0/42 OHCs from 1 mouse; b) cochleae. c, d, Bottom, in Atoh1Cre/+;Insm1Flox/Flox cochleae, Insm1 mRNA is present in spiral ganglion and 43% of OHCs (18/42 OHCs from 1 animal) at E16.5 (c), reduced to 7% (4/54) at E17.5 (not shown), and entirely absent all OHCs (0/60 OHCs from 1 animal) and spiral ganglion by postnatal day P1 (d). For quantification at E16.5, we did not include organs of Corti from apical turns, which do not yet have recognizable hair cells. Filled arrowheads indicate organs of Corti with Insm1 expression, and empty arrowheads indicate organs of Corti without Insm1 expression. Scale bars, 200 µm.

  3. Extended Data Fig. 3 Conditional ablation of Insm1 in hair cells and spiral ganglia neurons using TgPax2Cre causes hearing impairment and the appearance of IHC-like cells in place of OHCs.

    a, b, Hearing thresholds determined by ABRs (a) and DPOAEs (b) of TgPax2Cre/+;Insm1F/F mice at age P35–P46 (black traces; n = 4, 4 females) and control littermates (red traces; n = 5, 2 males and 3 females). The fact that shifts in ABR threshold are larger than shifts in DPOAE threshold may indicate an additional contribution to hearing impairment of the spiral ganglion neurons lacking INSM1 in TgPax2Cre/+;Insm1F/F cochlea. c, Despite the prevalence of OHCs with IHC characteristics (oc-IHCs) in TgPax2Cre/+;Insm1F/F cochleae (46.0 ± 5.64% (mean ± s.d.), n = 3 mice; Fig. 1m, n), these mice have the same density of IHCs (9.87 ± 2.41 cells per 100 µm along the organ of Corti; n = 3) as littermate controls (TgPax2Cre/+;Insm1F/+ and Insm1F/F; 10.54 ± 1.67 cells per 100 µm; n = 3) suggesting that oc-IHCs are not IHCs displaced from the inner to the outer compartment. d, There is no OHC loss in TgPax2Cre/+;Insm1F/F mice at ~P14–P16. Densities of oc-HCs do not differ between TgPax2Cre/+;Insm1F/F mice (OHCs and oc-IHCs) and their littermate controls (OHCs only) (29.88 ± 7.45 cells per 100 µm along the organ of Corti of TgPax2Cre/+;Insm1F/F mice, n = 3; 34.3 ± 6.92 cells per 100 µm in TgPax2Cre/+;Insm1F/+ and Insm1F/F littermate controls, n = 3). e, The number of oc-HCs per IHC does not differ between TgPax2Cre/+;Insm1F/F mice and their littermate controls (3.03 ± 0.25 OHCs plus oc-IHCs per IHC in TgPax2Cre/+;Insm1F/F mice, n = 3; 3.24 ± 0.21 OHCs per IHC in TgPax2Cre/+;Insm1F/+ and Insm1F/F littermate controls, n = 3). One-tailed Student’s t-tests were used in ce, n is number of mice. Statistical significance is defined as P < 0.05. f, Immunofluorescence for the IHC-enriched calmodulin (green) and hair cell marker myosin VIIa (white) on whole-mount organs of Corti from mid-cochlear positions at ages ~P14–P16 confirmed that many TgPax2Cre/+;Insm1F/F oc-HCs had IHC characteristics, in addition to having the flask shape and large nuclei of IHCs (blue, DAPI, marked with asterisks), as well as lacking prestin and expressing VGLUT3 (Fig. 1m). Scale bars, 10 µm. Biological replicates were used for all experiments and similar immunohistochemistry results obtained from three or more mice per genotype.

  4. Extended Data Fig. 4 IHC-like cells in the outer compartment (oc-IHCs) result from OHC misdifferentiation in the absence of INSM1, not from IHC displacement or from trans-differentiation of supporting cells.

    a, Atoh1Cre/+;Insm1F/F mice have the same density of IHCs (11.2 ± 1.2 (mean ± s.d.) cells per 100 µm; n = 12) as littermate controls (Atoh1Cre/+;Insm1+/F and Insm1F/F; 11.5 ± 1.3 cells per 100 µm; n = 13). b, There is no loss of OHCs in Atoh1Cre/+;Insm1F/F mice up to P34. Densities of oc-HCs do not differ between Atoh1Cre/+;Insm1F/F mice (OHCs + oc-IHCs) and their littermate controls (OHCs only) (34.6 ± 3.8 cells per 100 µm in Atoh1Cre/+;Insm1F/F mice, n = 9; 37.3 ± 4.5 cells per 100 µm in Atoh1Cre/+;Insm1+/F and Insm1F/F littermate controls, n = 10). c, The number of oc-HCs per IHC do not differ significantly between Atoh1Cre/+;Insm1F/F mice and their littermate controls (3.1 ± 0.3 OHCs and oc-IHCs per IHC in Atoh1Cre/+;Insm1F/F mice, n = 9; 3.3 ± 0.2 OHCs per IHC in Atoh1Cre/+;Insm1+/F and Insm1F/F littermate controls, n = 10). The criteria for identification of oc-IHCs in Atoh1Cre/+;Insm1F/F mice were the presence in the outer compartment of hair cells expressing IHC markers (VGLUT3, high levels of calmodulin and/or nuclear CtBP2), lacking OHC markers (oncomodulin and/or prestin) and with a shape (determined by myosin VIIa immunoreactivity) like that of IHCs. Mice used for hair cell counts were P0–P34. One-tailed Student’s t-tests were used in ac; n is number of mice. Statistical significance is defined as P < 0.05. d, SOX2 immunoreactivity, which labels the nuclei of cochlear supporting cells and, under certain conditions, of hair cells trans-differentiated from them postnatally, was not present in cells of the OHC region in Atoh1Cre/+;Insm1F/F pups (0/95 OHCs at P0, 0/42 OHCs at P2, and 0/39 OHCs at P5). e, To track postnatal cell proliferation in the organ of Corti, neonatal mice were injected twice daily with the thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU) from P0 to P5 or P8. The lack of EdU in any hair cell from in Atoh1Cre/+;Insm1F/F mice (0/77 oc-HCs at P5 and 0/40 oc-HCs at P8) confirmed that these cells, including oc-IHCs, were not produced from postnatally dividing supporting cells. Unless otherwise noted, images are from mid cochlear positions. Hair cells were identified by myosin VIIa immunoreactivity, phalloidin, DAPI and Hoechst. Scale bars, 10 µm. Biological replicates were used for all experiments and similar immunohistochemistry results obtained from three or more mice per genotype.

  5. Extended Data Fig. 5 Conditional ablation of Insm1 in hair cells using Gfi1-Cre causes no hearing impairment, and results in only very few oc-IHCs.

    We generated a conditional knockout of Insm1 in hair cells using Gfi1-Cre, in which the expression of cre recombinase coincided with that of Insm1. a, b, Hearing thresholds determined by ABRs (a) and DPOAEs (b) of Gfi1Cre/+;Insm1F/F mice at age P30–P35 (black traces; n = 6, 4 males and 2 females) and control littermates (red traces; n = 6; 1 Insm1F/F, 1 Insm1F/+ and 4 Gfi1Cre/+;Insm1F/+; 4 males and 2 females). There is no significant difference in ABR and DPOAE thresholds at any frequency tested between Gfi1Cre/+;Insm1F/F mice and their control littermates. c, Immunohistochemistry in whole-mount organs of Corti from mid-cochlear positions of P34 mice tested for hearing in a, b revealed that Gfi1Cre/+;Insm1F/F mice had normal IHCs expressing high levels of calmodulin. However, very few oc-HCs also expressed calmodulin at high levels, oncomodulin at low levels, and had a round, flask shape similar to that of IHCs (0.78%; 5/526 OHCs from 2 mice). Because in these Gfi1Cre/+;Insm1F/F mice the onset of Cre recombinase expression coincides with that of Insm1 (E15.5–E17.5)43, their nascent OHCs will express Insm1 for at least several hours. This result indicates that brief expression of Insm1 is sufficient to promote proper OHC differentiation. Scale bars, 10 µm. Biological replicates were used for all experiments and similar immunohistochemistry results obtained from three or more mice per genotype.

  6. Extended Data Fig. 6 FACS purification of and RNA extraction from OHCs from an E18.5 Insm1GFP.Cre/+ embryo.

    ac, Forward and side light scattering were used to exclude dead cells and debris (a) and aggregates (≥2 cells) (b, c). d, Live cells were gated in green and red (PerCP-Cy5, to assess autofluorescence) channels to define the GFP+ (green dots) and GFP (red dots) sorting windows. e, Myosin VIIa immunoreactivity and DAPI stain of cells collected through cytospinning after FACS confirm that most of the 547 sorted GFP+ cells are hair cells. This verification was done on all hair cell pools sorted (three pools per genotype). Inset is a representative merged image of one sorted OHC at high magnification. In this pool, no autofluorescent cells were collected. f, RT–qPCR after cell sorting (mean ± s.e.m.) reveals that, compared with GFP cells, GFP+ cells express the hair cell marker gene Myo7a and not the supporting cell marker genes S100 and Hes5. g, To ensure the quality of the extracted RNA, the RIN score was determined using a BioAnalyzer. g, Similar RIN scores were obtained from all pools of OHCs examined (including the three per genotype used for RNA-seq in Fig. 4a).

  7. Extended Data Table 1 Confirmed genes misregulated in OHCs lacking INSM1

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