Genetic background of Prop1(df) mutants provides remarkable protection against hypothyroidism-induced hearing impairment

Abstract

Hypothyroidism is a cause of genetic and environmentally induced deafness. The sensitivity of cochlear development and function to thyroid hormone (TH) mandates understanding TH action in this sensory organ. Prop1(df) and Pou1f1(dw) mutant mice carry mutations in different pituitary transcription factors, each resulting in pituitary thyrotropin deficiency. Despite the same lack of detectable serum TH, these mutants have very different hearing abilities: Prop1(df) mutants are mildly affected, while Pou1f1(dw) mutants are completely deaf. Genetic studies show that this difference is attributable to the genetic backgrounds. Using embryo transfer, we discovered that factors intrinsic to the fetus are the major contributor to this difference, not maternal effects. We analyzed Prop1(df) mutants to identify processes in cochlear development that are disrupted in other hypothyroid animal models but protected in Prop1(df) mutants by the genetic background. The development of outer hair cell (OHC) function is delayed, but Prestin and KCNQ4 immunostaining appear normal in mature Prop1(df) mutants. The endocochlear potential and KCNJ10 immunostaining in the stria vascularis are indistinguishable from wild type, and no differences in neurofilament or synaptophysin staining are evident in Prop1(df) mutants. The synaptic vesicle protein otoferlin normally shifts expression from OHC to IHC as temporary afferent fibers beneath the OHC regress postnatally. Prop1(df) mutants exhibit persistent, abnormal expression of otoferlin in apical OHC, suggesting delayed maturation of synaptic function. Thus, the genetic background of Prop1(df) mutants is remarkably protective for most functions affected in other hypothyroid mice. The Prop1(df) mutant is an attractive model for identifying the genes that protect against deafness.

FIG. 1

Prop1 ^df mutants have a mild hearing deficit. ABR tests were performed on sets of normal mice (white bars) and mutant mice (black bars) at ages of 4 weeks, 6–7 weeks, and 12 weeks. N = 3 per genotype for 4- and 12-week-old mice, n = 9 for 6–7-week-old wild type, and n = 8 for 6–7-week-old mutants.

FIG. 2

Gestational and neonatal environments do not account for different hearing abilities of Prop1 ^df and Pou1f1 ^dw mutants. Pups born from surrogate mothers (designated as “Genotype_S”) were tested by ABR. The hearing deficits of Prop1 ^df/df_S and Pou1f1 ^dw/dw_S are significantly different (P < 0.001). For each group, four mice were tested (n = 4).

FIG. 3

Prop1 ^df mutants exhibit mild OHC dysfunction with normal expression of KCNQ4 and prestin. A DPOAEs were measured in live 4-week and 7-week-old wild-type and Prop1 ^df mutant mice (black circles and white squares, respectively), and compared with DPOAEs of postmortem animals (dotted and dashed line). Data are shown for the 12 and 24 kHz frequencies. N = 3 for each genotype group of 4-week-old mice and n = 6 for 7-week-old ones. B, C KCNQ4 immunoreactivity is normal in OHCs (arrows) of mutant mice relative to wild type. Frozen sections obtained from P28 wild-type and mutant mice were stained for KCNQ4 (red). Nuclei were stained with DAPI (blue). D, E Prestin expression and localization was analyzed by staining frozen sections from wild-type and Prop1 ^df mutants at P28 with prestin-specific antibodies (red). Nuclei were labeled using DAPI (blue). Arrows identify rows of outer hair cells. Scale bars: 10 μm.

FIG. 4

Endocochlear potential (EP) is normal and KCNJ10 expression is developmentally delayed in Prop1 ^df mutants. A EP was measured in P42 wild-type and age-matched mutant animals. Levels of EP (mV) for littermate control and Prop1 ^df mutants are shown. B Frozen sections of the organ of Corti of wild-type and mutant mice collected at P28 and P42 were stained for KCNJ10 (red). Nuclei are marked by DAPI (blue). KCNJ10 expression is detected in the intermediate cells of the stria vascularis.

FIG. 5

Neurite growth and synaptogenesis of OHCs are grossly unaffected in Prop1 ^df mutants. A Synaptophysin, a presynaptic marker of efferent fibers, is stained on whole mount preparations of cochlear epithelia with an anti-synaptophysin antibody (green). B Neurofilament protein (NF-200) immunostaining was used to detect the neurite outgrowth in cochlea whole mounts of P28 mutants as well as wild-type controls. Prestin immunostaining was used to indicate the position of OHCs (red).

FIG. 6

Prolonged presence of otoferlin at apical OHCs in Prop1 ^df mutants. A, C Otoferlin immunostaining (red) was done on whole mount preparations of sensory epithelia from the apical turn of Prop1 ^df mutants and wild type. B, D Frozen sections of organ of Corti were collected and stained by anti-Otoferlin antibody (red). Arrowheads indicate the rows of OHCs. Arrows point to the IHCs. Nuclei are blue in all stainings.