Transcriptomic Analysis of Mouse Cochlear Supporting Cell Maturation Reveals Large-Scale Changes in Notch Responsiveness Prior to the Onset of Hearing

Abstract

Neonatal mouse cochlear supporting cells have a limited ability to divide and trans-differentiate into hair cells, but this ability declines rapidly in the two weeks after birth. This decline is concomitant with the morphological and functional maturation of the organ of Corti prior to the onset of hearing. However, despite this association between maturation and loss of regenerative potential, little is known of the molecular changes that underlie these events. To identify these changes, we used RNA-seq to generate transcriptional profiles of purified cochlear supporting cells from 1- and 6-day-old mice. We found many significant changes in gene expression during this period, many of which were related to regulation of proliferation, differentiation of inner ear components and the maturation of the organ of Corti prior to the onset of hearing. One example of a change in regenerative potential of supporting cells is their robust production of hair cells in response to a blockade of the Notch signaling pathway at the time of birth, but a complete lack of response to such blockade just a few days later. By comparing our supporting cell transcriptomes to those of supporting cells cultured in the presence of Notch pathway inhibitors, we show that the transcriptional response to Notch blockade disappears almost completely in the first postnatal week. Our results offer some of the first molecular insights into the failure of hair cell regeneration in the mammalian cochlea.

Sox2 haploinsufficiency primes regeneration and Wnt responsiveness in the mouse cochlea

“…Thus, Sox2 haplo and damage may coordinate to dictate mitotic regeneration, transitional cell formation, and Wnt responsiveness via Notch signaling in the neonatal cochlea, but not in the mature cochlea ( Figure 8G) because of age-related changes of these factors. Our results are in agreement with previous reports noting a decline of Hes5 expression in the maturing cochlea (72,75). Furthermore, Hes5 and Atoh1 expression remained low in the noise-damaged mature cochlea (77).…”

“…All qPCR reactions were performed in triplicate, and the relative quantification of gene expression was analyzed using the ΔΔCt method (87). These and Notch inhibition increases Atoh1 expression and hair cell formation in undamaged (71)(72)(73) and damaged neonatal cochleae (74).…”

ADAM10 and γ-secretase regulate sensory regeneration in the avian vestibular organs

“…It is of further interest to compare our data to those of a recent study that employed similar RNA-Seq methods to profile DAPT-evoked changes in gene expression in supporting cells of the mammalian cochlea at two developmental time points – P1 (a stage at which inhibition of γ-secretase leads to enhanced hair cell differentiation) and P6 (when hair cell differentiation is no longer affected by γ-secretase inhibition – Maass et al, 2016). Among the genes whose expression levels were most down-regulated by DAPT treatment, the single commonality was Heyl , while a comparison of the lists of the genes whose expression was most enhanced by DAPT treatment revealed no commonalities (compare Tables 1 and 2 with Tables 9 and 10 in Maass et al, 2016). These differing responses are likely to reflect differences in cellular signaling mechanisms that mediate vestibular vs. auditory phenotype as well as the early developmental stages of the treated mammalian samples.…”

The acquisition of positional information across the radial axis of the cochlea

“…Supporting cells retain their Sox2 expression, and this may explain how they maintain a certain plasticity and can be stimulated to transdifferentiate into hair cells, which can be advantageous for regeneration. However, such evidence of transdifferentiation ceases near the end of the first postnatal week after birth …”

“…However, such evidence of transdifferentiation ceases near the end of the first postnatal week after birth. 58 Once the prosensory domain is specified, its cells must acquire positional information across the radial axis that drives the differential development of the medial and lateral sensory halves. One of the earliest indicators of this dichotomy is the differential expression of Prox1.…”