This proposal addresses the uncharacterized role of the mInsc-LGN-G?i protein complex in stereocilia elongation, and defines LGN-G?i as a long sought-after cargo of Myo15 required for differential stereocilia growth across rows during hair cell development. In the absence of LGN or G?i, postnatal stereocilia are stunted and form an immature-looking bundle that only retains a shallow staircase pattern. Interestingly, very similar defects have been reported in the absence of Myo15, Whirlin and Eps8, three proteins enriched at the tips of all stereocilia in amounts proportional to their height. We show here that LGN and G?i share the same tip localization, but are restricted to the first stereocilia row. Strikingly, LGN and G?i fail to traffic to tips in absence of Myo15. In contrast, Myo15-Whrn-Eps8 are found in similar low amounts across all stunted rows in LGN or G?i mutants, suggesting that LGN-G?i instruct differential stereocilia identity across rows by specifying the first row. By intercrossing mutants and using genetics, protein immunolocalization and protein binding biochemistry, we propose to solve the function of an extended Myo15 complex now including LGN-G?i where all proteins apparently have independent, complementary functions to shape the bundle. We previously showed that mInsc-LGN-G?i first occupy and generate the 'bare zone', the flat region of the hair cell apex where early microvilli exclusion outlines the V-shaped or semi-circular edge of the forming bundle. Based on evidence for dynamic and balanced LGN-G?i protein amounts between subcellular compartments, we propose that restriction of LGN-G?i to the first row could be instructed by their prior enrichment at the adjacent bare zone. LGN-G?i would promote higher amounts of Myo15-Whrn-Eps8 at the first row, its growth into the tallest row in turn influencing the height of shorter rows via oblique tip-links, as proposed previously. To test the novel idea that the staircase pattern is established by recycling planar polarity information, we propose stage-specific loss- and gain-of-function approaches, including exploratory experiments using cochlear explants aimed at elucidating and manipulating LGN-G?i function at the subcellular level. The staircase-like architecture of the hair bundle is essential for hearing and considered instrumental for direction-sensitivity to sound stimuli, but remains largely unexplained at the molecular level. By uncovering new members of the Myo15 complex and clarifying how asymmetry of growth is created across rows, this work notably improves our understanding of hereditary hearing loss in DFNB3 (MYO15), DFNB31/USH2D (WHRN), DFNB102 (EPS8) and DFNB82/Chudley-McCullough syndrome (LGN/GPSM2).
Hearing loss is the most common birth defect and the most prevalent sensory disorder in developed countries. Mouse models of genetic conditions affecting hearing frequently have defective development, maintenance or function of the stereocilia bundle, the specialized compartment processing sound in sensory hair cells. Accomplishment of the research goals proposed in this grant application will (1) advance our molecular comprehension of stereocilia bundle morphogenesis, (2) help diagnose and treat underlying pathologies where a defective structure impairs hearing, and (3) inform emerging therapies aimed at regenerating hair cells lost to injury or disease, where new bundles must be developed de novo.
|Tarchini, Basile; Longo-Guess, Chantal; Tian, Cong et al. (2018) A spontaneous mouse deletion in Mctp1 uncovers a long-range cis-regulatory region crucial for NR2F1 function during inner ear development. Dev Biol 443:153-164|
|Siletti, Kimberly; Tarchini, Basile; Hudspeth, A J (2017) Daple coordinates organ-wide and cell-intrinsic polarity to pattern inner-ear hair bundles. Proc Natl Acad Sci U S A 114:E11170-E11179|
|Tarchini, Basile; Tadenev, Abigail L D; Devanney, Nicholas et al. (2016) A link between planar polarity and staircase-like bundle architecture in hair cells. Development 143:3926-3932|