Abstract
Hearing loss is a serious burden to physical and mental health worldwide. Aberrant development and damage of hearing organs are recognized as the causes of hearing loss, the molecular mechanisms underlining these pathological processes remain elusive. Investigation of new molecular mechanisms involved in proliferation, differentiation, migration and maintenance of neuromast primordium and hair cells will contribute to better understanding of hearing loss pathology. This knowledge will enable the development of protective agents and mechanism study of drug ototoxicity. In this study, we demonstrate that the zebrafish gene miles-apart, a homolog of sphingosine-1-phosphate receptor 2 (s1pr2) in mammals, has an important role in the development of otic vesicle, neuromasts and survival of hair cells. Whole-mount in situ hybridization of embryos showed that miles-apart expression occurred mainly in the encephalic region and the somites at 24 h.p.f. (hour post fertilization), in the midbrain/hindbrain boundary, the brainstem and the pre-neuromast of lateral line at 48 h.p.f. in a strict spatiotemporal regulation. Both up- and downregulation of miles-apart led to abnormal otoliths and semicircular canals, excess or few hair cells and neuromasts, and their disarranged depositions in the lateral lines. Miles-apart (Mil) dysregulation also caused abnormal expression of hearing-associated genes, including hmx2, fgf3, fgf8a, foxi1, otop1, pax2.1 and tmieb during zebrafish organogenesis. Moreover, in larvae miles-apart gene knockdown significantly upregulated proapoptotic gene zBax2 and downregulated prosurvival gene zMcl1b; in contrast, the level of zBax2 was decreased and of zMcl1b enhanced by miles-apart overexpression. Collectively, Mil activity is linked to organization and number decision of hair cells within a neuromast, also to deposition of neuromasts and formation of otic vesicle during zebrafish organogenesis. At the larva stage, Mil as an upstream regulator of bcl-2 gene family has a role in protection of hair cells against apoptosis by promoting expression of prosurvival gene zMcl1b and suppressing proapoptotic gene zBax2.
Highlights
Sphingosine-1-phosphate (S1P) is a lipid-active mediator and signal molecule, in addition to being a component of the cell membrane
The miles-apart expression was mainly aggregated in the encephalic region, in the midbrain/hindbrain boundary and the ventral hindbrain; and weak expression was observed in somites but not in notochord at 24 h.p.f. (Figures 1a, a0 and a00)
The results show that in the 6 d.p.f. morphants at the posterior lateral line several neuromasts were absent or shrunken (Figure 3b); and at the anterior lateral line number of the hair cells in neuromast orbital 1 (o1), middle lateral line 1, middle lateral line 1, orbital 2 (o2) and infraorbital 4, which resided at the otic vesicles around, were counted and found that the hair cells were obviously diminished in neuromast o1 and o2 (Figures 3g and j), Sacculus/Utricle
Summary
Sphingosine-1-phosphate (S1P) is a lipid-active mediator and signal molecule, in addition to being a component of the cell membrane. As in S1p2-knockout mice, significant and progressive degeneration of auditory and vestibular organs occurred in S1p2/S1p3 double knockout mice with age, culminating in complete hearing loss.[1,2] These results indicate that S1p receptors have important roles in maintaining hair cell functions in mice. Loss of Mil function caused edematous pericardial sac and epithelium-like blistering in the tail.[5,8,10] there are no reports for zebrafish on the role of Mil in the development of auditory and vestibular organs, such as hair cells and neuromasts. Zebrafish hair cells are similar to their mammalian counterparts in both morphology and function, and reside inside the otic vesicles and in the neuromasts at the lateral line system on the body surface. Our findings will contribute to a better understanding of the processes of hearing development and facilitate research of otoprotective agents
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