Gene miles-apart is required for formation of otic vesicle and hair cells in zebrafish

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.

Autosomal-Recessive Hearing Impairment Due to Rare Missense Variants within S1PR2

“…Within the mapped region lies S1PR2 (MIM: 605111), a gene for which knockout mice and zebrafish models were previously shown to have inner ear structural alterations and hearing loss. [3][4][5]10 Prior to study onset, approval was obtained from the Institutional Review Boards of the Baylor College of Medicine and Affiliated Hospitals, Combined Neuroscience Institutional Review Board at the NIH, National Centre of Excellence in Molecular Biology, University of the Punjab, and Quaid-I-Azam University. Written informed consent was obtained from all family members who participated in the study.…”

“…[3][4][5] Likewise, in the zebrafish morphant in which translation is inhibited for milesapart, a homolog for S1PR2, lateral line hair cells were absent or shrunken. 10 Structural defects were also identified within the otic vesicle, the semicircular canals, otoliths, utricle, and saccule of morphant zebrafish. 10 In the S1pr2 À/À mouse, progressive deterioration of vestibular epithelium and loss or deformity of utricular and saccular otoconia were evident.…”

“…10 Structural defects were also identified within the otic vesicle, the semicircular canals, otoliths, utricle, and saccule of morphant zebrafish. 10 In the S1pr2 À/À mouse, progressive deterioration of vestibular epithelium and loss or deformity of utricular and saccular otoconia were evident. The utricular otoconia abnormalities were evident as early as P7 prior to manifestation of behavioral balance abnormalities.…”

“…23,24 In morphant zebrafish, knockdown of the S1PR2 homolog resulted in reduced expression of several homologs for genes involved in mouse and human HI, namely hmx2, fgf3, pax2, and tmie. 10 Independent mutations in these four downstream genes in mice or humans result in otolith abnormalities, aplastic inner ears, or hair cell defects. [25][26][27][28] Taken together, this body of evidence implies that S1PR2 can exert its influence on inner ear function through regulation of inner ear fluid homeostasis or through changes in expression of other essential inner ear proteins.…”

“…From these modeling predictions, it is hypothesized that S1PR2 variants result in either loss of activation of S1PR2 by S1P ligand or non-formation of the S1PR2-G protein complex. Both events would result in failed phosphorylation of the MAPK/ERK pathway, which would (1) affect transcription or translation of several inner ear proteins and thereby cause structural defects of the cochlea, otoconia, or hair cells [3][4][5]10 and (2) result in non-phosphorylation of ERM proteins, which colocalize with S1PR2-activated RhoA in order to maintain tight cell junctions. 21,22 Loss of S1PR2 function is expected to result in inflammation and hyperpermeability of the stria vascularis at onset of hearing function, 21 which can then cause loss of endocochlear potential, hearing loss, and eventually degeneration of inner ear structures.…”