Deletion of the Notch ligand Jagged1 during cochlear maturation leads to inner hair cell defects and hearing loss

Abstract

The mammalian cochlea is an exceptionally well-organized epithelium composed of hair cells, supporting cells, and innervating neurons. Loss or defects in any of these cell types, particularly the specialized sensory hair cells, leads to deafness. The Notch pathway is known to play a critical role in the decision to become either a hair cell or a supporting cell during embryogenesis; however, little is known about how Notch functions later during cochlear maturation. Uniquely amongst Notch ligands, Jagged1 (JAG1) is localized to supporting cells during cell fate acquisition and continues to be expressed into adulthood. Here, we demonstrate that JAG1 in maturing cochlear supporting cells is essential for normal cochlear function. Specifically, we show that deletion of JAG1 during cochlear maturation disrupts the inner hair cell pathway and leads to a type of deafness clinically similar to auditory neuropathy. Common pathologies associated with disruptions in inner hair cell function, including loss of hair cells, synapses, or auditory neurons, were not observed in JAG1 mutant cochleae. Instead, RNA-seq analysis of JAG1-deficient cochleae identified dysregulation of the Rho GTPase pathway, known to be involved in stereocilia development and maintenance. Interestingly, the overexpression of one of the altered genes, Diaph3, is responsible for autosomal dominant auditory neuropathy-1 (AUNA1) in humans and mice, and is associated with defects in the inner hair cell stereocilia. Strikingly, ultrastructural analyses of JAG1-deleted cochleae revealed stereocilia defects in inner hair cells, including fused and elongated bundles, that were similar to those stereocilia defects reported in AUNA1 mice. Taken together, these data indicate a novel role for Notch signaling in normal hearing development through maintaining stereocilia integrity of the inner hair cells during cochlear maturation.

Jag1 represses Notch activation in lateral supporting cells and inhibits an outer hair cell fate in the medial compartment of the developing cochlea

“…Jag1 Ndr/Ndr IHC transcriptomes demonstrated very few dysregulated genes. The ABR-related hearing defects in Jag1 Ndr/Ndr mice are thus unlikely to be caused by IHC defects, as reported for mice with SC-specific Jag1 deletion 9 . Instead, reduced cochlear amplification, middle ear bone malformation, and a reduction in OHCs accompanied by pronounced transcriptomic changes in OHCs, could together explain the severe hearing loss in Jag1 Ndr/Ndr mice.…”

“…Table 4 , GO:0048839 and GO:0042471, such as Sox2 ). Jag1 Ndr/Ndr dysregulated genes overlapped with genes dysregulated upon Jag1 silencing in SCs 9,10 , specifically upregulation of the medial SC marker Cdh4 26 in all models, and downregulation of Agr3 , which is most strongly expressed in lateral SCs 27 ( Fig 2e-f ). Therefore, the Jag1 Ndr/Ndr inner ear exhibits dysregulated gene signature associated with inner ear development, morphogenesis, and hearing, and recapitulates aspects of published Jag1 loss-of-function transcriptomic data.…”

“…Hearing loss is reported in ALGS 5,7,15,16 , and in heterozygous mutant and Jag1 conditional loss-of-function mice 9–12 ( Supp. Table 2 ).…”

“…To compare the scRNAseq with previously published Jag1 -related bulk RNAseq datasets 9,10 , we first performed a pseudo-bulk analysis of the Jag1 Ndr/Ndr and Jag1 +/+ populations. Differential gene expression analysis for the cochlear epithelium dataset revealed 458 dysregulated genes (avg.…”

“…Both conductive and sensorineural hearing loss are reported in patients with Alagille syndrome 5 , as well as vestibular defects 6 . Similarly, Jag1 -deficiency results in hearing loss in mice 9–12 ( Supp Table2 ), and Jag1 missense mutant mice are nicknamed for overt balance defects, including Headturner 18 , Ozzy 11 and Slalom 17 . ABR measurements indicated severe hearing loss in Jag1 Ndr/Ndr mice, as well as reduced cochlear amplification by DPOAE.…”