Signaling pathways (Notch, Wnt, Bmp and Fgf) have additive effects on hair cell regeneration in the chick basilar papilla after streptomycin injury in vitro: Additive effects of signaling pathways on hair cell regeneration.

Hair cells can be regenerated after damage by transdifferentiation in which a supporting cell directly differentiates into a hair cell without mitosis. However, such regeneration is at the cost of exhausting the support cells in the mammalian mature cochlea. Thus, more effective methods should be found to promote mitotic regeneration but partially preserve support cells after damage. To address the issue, we first injured hair cells in the chick basilar papillae (BP) by treatment with streptomycin in vitro. We then compared the mitotic regeneration on the neural side in the middle part of BP after treatment with a pharmacological inhibitor or agonist of the Notch (DAPT), Wnt (LiCl), Bmp (Noggin) or Fgf (SU5402) signaling pathway, with that after treatment with combinations of two or three inhibitors or agonist of these pathways. Our results indicate that treatments with a single inhibitor or agonist of the Notch, Wnt, Bmp or Fgf signaling pathway could significantly increase mitotic regeneration as well as direct transdifferentiation. The results also show that hair cells (Myosin 7a+), support cells (Sox2+) and mitotically regenerated hair cells (Myosin 7a+/Sox2+/BrdU+) increased significantly on the neural side in the middle part of BP after two or three combinations of the inhibition of Notch, Bmp or Fgf signaling pathway or the activation of Wnt signaling pathway, besides the reported coregulatory effects of Notch and Wnt signaling. The study of the effects of systematic combinations of pathway modulators provided more insight into hair cell regeneration from mitosis.

Transcriptomic analysis of mouse cochleae suffering from gentamicin damage reveals the signalling pathways involved in hair cell regeneration.

There is a strong capacity for hair cell regeneration after damage in the inner ear of non-mammals. However, mammalian hair cells are substantially unable to regenerate. To obtain insights into the mechanism of this difference, we analyzed the transcriptomic changes in the mouse cochleae suffered from gentamicin damage and compared them with those in the chick cochleae suffered from the same damage. The results indicated that 2,230 genes had significantly differential expression between the gentamicin- and saline-treated mouse cochleae. Some of the differentially expressed genes were grouped into 265 signaling pathways, including the Notch, Wnt (Wingless and INT-1), Bmp (bone morphogenetic protein), FGF (fibroblast growth factor) and Shh (sonic hedgehog) pathways. Using pharmacological inhibitors or agonists of these pathways, the effects of these pathways on hair cell regeneration were further studied. The results indicated that Bmp alone and its coregulation with the Notch or Wnt signaling pathways increased the numbers of generated cells from transdifferentiation or proliferation in the mouse cochlea after damage, in addition to the reported coregulation of Notch and Wnt. Thus, this work indicates a new signaling pathway (Bmp) and its synergetic coregulation in mammalian hair cell regeneration, providing potential therapeutic targets to increase mammalian hair cell regeneration.

Study of the Mechanisms by Which Aminoglycoside Damage Is Prevented in Chick Embryonic Hair Cells.

A major side effect of aminoglycoside antibiotics is mammalian hair cell death. It is thus intriguing that embryonic chick hair cells treated with aminoglycosides at embryonic day (E) 12 are insensitive to ototoxicity. To exclude some unknown factors in vivo that might be involved in preventing aminoglycoside damage to embryonic hair cells, we first cultured chick embryonic basilar papilla (BP) with an aminoglycoside antibiotic in vitro. The results indicated that the hair cells were almost intact at E12 and E14 and were only moderately damaged in most parts of the BP at E16 and E18. Generally, hair cells residing in the approximate and abneural regions were more susceptible to streptomycin damage. After incubation with gentamicin-conjugated Texas Red (GTTR), which is typically used to trace the entry route of aminoglycosides, GTTR fluorescence was not remarkable in hair cells at E12, was weak at E14, but was relatively strong in the proximal part of BP at E18. This result indicates that the amounts of GTTR that entered the hair cells are related to the degrees of aminoglycoside damage. The study further showed that the fluorescence intensity of GTTR decreased to a low level at E14 to E18 after disruption of mechanotransduction machinery, suggesting that the aminoglycoside entry into hair cells was mainly through mechanotransduction channels. In addition, most of the entered GTTR was not found to be colocalized with mitochondria even at E18. This finding provides another reason to explain why embryonic chick hair cells are insensitive to aminoglycoside damage.

Transcriptomic analysis of chicken cochleae after gentamicin damage and the involvement of four signaling pathways (Notch, FGF, Wnt and BMP) in hair cell regeneration.

Unlike mammalian hair cells, which are essentially unable to regenerate after damage, avian hair cells have a robust capacity for regeneration. The prerequisite for understanding the above difference is knowing the genetic programming of avian hair cell regeneration. Although the major processes have been known, the precise molecular signaling that induces regeneration remains unclear. To address this issue, we performed a high-throughput transcriptomic analysis of gene expression during hair cell regeneration in the chick cochlea after antibiotic injury in vivo. A total of 16,588 genes were found to be expressed in the cochlea, of which about 1000 genes were differentially expressed among the four groups studied, i.e., 2 days (d) or 3 d post-treatment with gentamicin or physiological saline. The differentially expressed genes were distributed across approximately one hundred signaling pathways, including the Notch, MAPK (FGF), Wnt and TGF-ß (BMP) pathways that have been shown to play important roles in embryonic development. Some differentially expressed genes (2-3 in each pathway) were further verified by qRT-PCR. After blocking Notch, FGF or BMP signaling, the number of regenerating hair cells and mitotic supporting cells increased. However, the opposite effect was observed after suppressing the Wnt pathway or enhancing BMP signaling. To our knowledge, the present study provided a relatively complete dataset of candidate genes and signaling pathways most likely involved in hair cell regeneration and should be a useful start in deciphering the genetic circuitry for inducing hair cell regeneration in the chick cochlea.