Highlights d TubeMap enables fast construction of labeled vascular graphs from TB-sized images d Automated arterio-venous annotations are based on iDISCO+ immunolabeling d We measure regional variations in vessel topology and arterio-venous distances d We study the plasticity of this network in stroke and sensorydeprivation models

Several lines of evidence indicate that very large G-protein-coupled receptor 1 (Vlgr1) makes up the ankle links that connect the stereocilia of hair cells at their base. Here, we show that the transmembrane protein usherin, the putative transmembrane protein vezatin, and the PDZ (postsynaptic density-95/Discs large/zona occludens-1) domain-containing submembrane protein whirlin are colocalized with Vlgr1 at the stereocilia base in developing cochlear hair cells and are absent in Vlgr1 Ϫ/Ϫ mice that lack the ankle links. Direct in vitro interactions between these four proteins further support their involvement in a molecular complex associated with the ankle links and scaffolded by whirlin. In addition, the delocalization of these proteins in myosin VIIa defective mutant mice as well as the myosin VIIa tail direct interactions with vezatin, whirlin, and, we show, Vlgr1 and usherin, suggest that myosin VIIa conveys proteins of the ankle-link complex to the stereocilia. Adenylyl cyclase 6, which was found at the base of stereocilia, was both overexpressed and mislocated in Vlgr1 Ϫ/Ϫ mice. In postnatal day 7 Vlgr1 Ϫ/Ϫ mice, mechanoelectrical transduction currents evoked by displacements of the hair bundle toward the tallest stereocilia (i.e., in the excitatory direction) were reduced in outer but not inner hair cells. In both cell types, stimulation of the hair bundle in the opposite direction paradoxically resulted in significant transduction currents. The absence of ankle-linkmediated cohesive forces within hair bundles lacking Vlgr1 may account for the electrophysiological results. However, because some long cadherin-23 isoforms could no longer be detected in Vlgr1 Ϫ/Ϫ mice shortly after birth, the loss of some apical links could be involved too. The premature disappearance of these cadherin isoforms in the Vlgr1 Ϫ/Ϫ mutant argues in favor of a signaling function of the ankle links in hair bundle differentiation.

Defects in myosin XVa and the PDZ domain-containing protein, whirlin, underlie deafness in humans and mice. Hair bundles of mutant mice defective for either protein have abnormally short stereocilia. Here, we show that whirlin, like myosin XVa, is present at the very tip of each stereocilium in the developing and mature hair bundles of the cochlear and vestibular system. We found that myosin XVa SH3-MyTH4 region binds to the short isoform of whirlin (PR-PDZ3) that can rescue the stereocilia growth defect in whirlin defective mice. Moreover, the C-terminal MyTH4-FERM region of myosin XVa binds to the PDZ1 and PDZ2 domains of the long whirlin isoform. We conclude that a direct myosin XVa-whirlin interaction at the stereocilia tip is likely to control the elongation of stereocilia. Whirlin, unlike myosin XVa, is also transiently localized in the basal region of developing stereocilia in rat vestibular and cochlear hair cells until P4 and P12, respectively. Notably, whirlin also interacts with myosin VIIa that is present along the entire length of the stereocilia. Finally, we show that the transmembrane netrin-G1 ligand (NGL-1) binds to the PDZ1 and PDZ2 domains of whirlin and has an extracellular region that homophilically self-interacts in a Ca2+-dependent manner. The interaction between whirlin and NGL-1 might be involved in the stabilization of interstereociliar links.

Usher syndrome type IIa (USH2A) combines moderate to severe congenital hearing impairment and retinitis pigmentosa. It is the most common genetic form of USH. USH2A encodes usherin, which was previously defined as a basement membrane protein. A much larger USH2A transcript predicted to encode a transmembrane (TM) isoform was recently reported. Here, we address the role of TM usherin in the inner ear. Analysis of the usherin alternative transcripts in the murine inner ear revealed the existence of several predicted TM usherin isoforms with modular ectodomains of different lengths. In addition, we identified in the usherin cytoplasmic region a predicted 24 amino acid peptide, derived from a newly defined exon that is predominantly expressed in the inner ear but not in the retina. In mouse and rat inner ears, we show that TM usherin is present at the base of the differentiating stereocilia, which make up the mechanosensitive hair bundles receptive to sound. The usherin immunolabeling is transient in the hair bundles of cochlear hair cells (HCs), but persists in mature hair bundles of vestibular HCs. Several lines of evidence support the involvement of TM usherin in the composition of the ankle links, a subset of filamentous lateral links connecting stereocilia at the base. By co-immunoprecipitation and in vitro binding assays, we establish that the usherin cytodomain can bind to whirlin and harmonin, two PDZ domain-containing proteins that are defective in genetic forms of isolated deafness and USH type I, respectively. These PDZ proteins are suitable to provide the anchoring of interstereocilia lateral links to the F-actin core of stereocilia. Our results strongly suggest that congenital deafness in USH type I and type II shares similar pathogenic mechanisms, i.e. the disruption of hair bundle links-mediated adhesion forces that are essential for the proper organization of growing hair bundles.