The Notch ligand Jagged1 plays a dual role in cochlear hair cell regeneration

Hair cells (HCs) within the inner ear cochlea are highly specialized mechano-sensory cells that enable us to detect sound. In humans and other mammals, HC loss is permanent and a leading cause of deafness. Recent studies in newborn mice revealed that supporting cells (SCs) have the capacity to form cochlear HCs, and that inhibition of Notch signaling dramatically increases the otherwise low rate of SC-to-HC conversion. It has been proposed that in the absence of HCs, the SC-specific Notch ligand Jagged1 (JAG1) mediates the HC-repressive role of Notch signaling. Here we show that conditional deletion of Jag1 at postnatal day2 (P2) increases the rate of HC formation/regeneration in cochlear tissue and organoids. However, Jag1 deficiency also reduces the expression of key progenitor and metabolic genes and attenuates PI3K-Akt-mTOR signaling in cochlear SCs and Kölliker’s cells and we show that Notch1 and Notch2 are critical for mediating these pro-growth functions of Jag1. Conversely, we show that increasing JAG1/Notch signaling enhances the mitotic capacity of cochlear SCs/ Kölliker’s cells. Finally, we show that JAG1 expression declines in SCs as they undergo maturation, and that stimulation of JAG1 signaling boosts the ability of maturing cochlear SCs to form HCs in an mTOR-dependent manner. We established organoid culture with postnatal day 2 cochlear epithelilal cells. Then we added doxycycline in the culture medium at the beginning of plating the cells. At 7 days of expansion, organoid culture was harvested with cell recovery solution to dissolve the matrigel in the 3D culture. Finally the cells were washed with PBS for three times, and total RNA from organoids was extracted using the miRNeasy Micro Kit as described previously. Then the total RNA samples were used for RNA-seq analysis.