In vivo function of Otopetrin 1 in the Vestibular Sensory Epithelium

Otopetrin family genes encode multi-transmembrane domain proteins with three highly conserved domains. In mice, three Otopetrin paralogues are found. One of its members, Otopetrin 1: Otop1) has been previously shown to be essential for the formation of otoconia in the vestibular system of the inner ear. Otoconia are calcium carbonate biominerals that are required for normal balance and the sensation of linear acceleration with respect to gravity. The mechanism by which OTOP1 mediates otoconia biosynthesis is not known, but the ability of OTOP1 to modulate [Ca2+]i in response to purinergic signals in heterologous systems suggest that OTOP1 may be involved in concentrating [Ca2+] within the sensory epithelium and/or the globular substance vesicles, which are secreted from the epithelium. In this study, we generated null alleles of Otop1: Otop1βgal/βgal) and Otop2: Otop2nβgal/nβgal) in mice to investigate the in vivo functions of these genes. The otoconial agenesis phenotype in Otop1βgal/βgal mice suggests that the most sensitive and important role for OTOP1 during development is mediating otoconia formation. X-gal staining and immunohistochemical analysis reveal that Otop1 is expressed in the developing and adult supporting cells in the non-striola region of the maculae. Within these sensory epithelia, OTOP1 localizes near the apical end. In primary utricular macular cultures, endogenous OTOP1 is necessary to inhibit P2Y function and the influx of extracellular Ca2+ in a Ca2+- and ATP-dependent manner. The two different recessive missense mutations of Otop1, tilted: tlt) and mergulhador: mlh), result in the same phenotype as in Otop1βgal/βgal, suggesting that they have inactivated essential OTOP1 activity. Localization studies and ratiometric Ca2+ imaging in COS7 cells and primary utricular macular cultures show that both tlt and mlh alter normal localization of OTOP1 and that the mlh mutation has a greater effect on modulating [Ca2+]i compared to tlt. When examined in heterologous systems, OTOP2 and 3 can modulate [Ca2+]i in a similar manner to OTOP1, suggesting possible functional redundancy between Otopetrins. However, the double knockout allele for Otop1 and Otop2 does not exhibit additional phenotypes, suggesting that these genes may have more prominent functional redundancy with OTOP3. Alternatively, a subtler phenotype may exist but will require a more careful analysis to be revealed. Because otoconia formation is affected by altered gravity it is possible that the genes important for otoconia biosynthesis, such as Otoconin90 and Otop1, may also be affected. However, a quantitative RT-PCR analysis of quail embryo development under constant hypergravity conditions shows that expression of these genes is not significantly affected by altered gravity treatment. This result indicates that gravity is unlikely to serve as an epigenetic factor for these two genes during development of otoconia

Discrete Functions of TRAF1 and TRAF2 in Drosophila melanogaster Mediated by c-Jun N-Terminal Kinase and NF-κB-Dependent Signaling Pathways

Two Drosophila tumor necrosis factor receptor-associated factors (TRAF), DTRAF1 and DTRAF2, are proposed to have similar functions with their mammalian counterparts as a signal mediator of cell surface receptors. However, their in vivo functions and related signaling pathways are not fully understood yet. Here, we show that DTRAF1 is an in vivo regulator of c-Jun N-terminal kinase (JNK) pathway in Drosophila melanogaster. Ectopic expression of DTRAF1 in the developing eye induced apoptosis, thereby causing a rough-eye phenotype. Further genetic interaction analyses revealed that the apoptosis in the eye imaginal disc and the abnormal eye morphogenesis induced by DTRAF1 are dependent on JNK and its upstream kinases, Hep and DTAK1. In support of these results, DTRAF1-null mutant showed a remarkable reduction in JNK activity with an impaired development of imaginal discs and a defective formation of photosensory neuron arrays. In contrast, DTRAF2 was demonstrated as an upstream activator of nuclear factor-κB (NF-κB). Ectopic expression of DTRAF2 induced nuclear translocation of two Drosophila NF-κBs, DIF and Relish, consequently activating the transcription of the antimicrobial peptide genes diptericin, diptericin-like protein, and drosomycin. Consistently, the null mutant of DTRAF2 showed immune deficiencies in which NF-κB nuclear translocation and antimicrobial gene transcription against microbial infection were severely impaired. Collectively, our findings demonstrate that DTRAF1 and DTRAF2 play pivotal roles in Drosophila development and innate immunity by differentially regulating the JNK- and the NF-κB-dependent signaling pathway, respectively

Regulation of Cellular Calcium in Vestibular Supporting Cells by Otopetrin 1

Otopetrin 1 (OTOP1) is a multitransmembrane domain protein, which is essential for mineralization of otoconia, the calcium carbonate biominerals required for vestibular function, and the normal sensation of gravity. The mechanism driving mineralization of otoconia is poorly understood, but it has been proposed that supporting cells and a mechanism to maintain high concentrations of calcium are critical. Using Otop1 knockout mice and a utricular epithelial organ culture system, we show that OTOP1 is expressed at the apex of supporting cells and functions to increase cytosolic calcium in response to purinergic agonists, such as adenosine 5′-triphosphate (ATP). This is achieved by blocking mobilization of calcium from intracellular stores in an extracellular calcium-dependent manner and by mediating influx of extracellular calcium. These data support a model in which OTOP1 acts as a sensor of the extracellular calcium concentration near supporting cells and responds to ATP in the endolymph to increase intracellular calcium levels during otoconia mineralization