Retinoic acid signaling is essential for maintenance of the blood-retinal barrier.

The predominant function of the blood-retinal barrier (BRB) is to maintain retinal homeostasis by regulating the influx and efflux between the blood and retina. Breakdown of the BRB occurs in a number of ocular diseases that result in vision loss. Understanding the molecular and cellular pathways involved in the development and maintenance of the BRB is critical to developing therapeutics for these conditions. To visualize the BRB in vivo, we used the transgenic Tg(l-fabp:DBP-EGFP:flk1:mCherry) zebrafish model that expresses vitamin D binding protein (a member of the albumin gene family) tagged to green fluorescent protein. Retinoic acid (RA) plays a number of important roles in vertebrate development and has been shown to play a protective role during inflammation-induced blood-brain barrier disruption. The role of RA in BRB development and maintenance remains unknown. To disrupt RA signaling, Tg(l-fabp:DBP-EGFP:flk1:mCherry) zebrafish were treated with N, N-diethylaminobenzaldehyde and 4-[(1 E)-2-[5,6-dihydro-5,5-dimethyl-8-(2-phenylethynyl)-2-naphthalenyl]ethenyl]benzoic acid, which are antagonists of retinal dehydrogenase and the RA receptor, respectively. Treatment with either compound resulted in BRB disruption and reduced visual acuity, whereas cotreatment with all- trans RA effectively rescued BRB integrity. Additionally, transgenic overexpression of Cyp26a1, which catalyzes RA degradation, resulted in breakdown of the BRB. Our results demonstrate that RA signaling is critical for maintenance of the BRB and could play a role in diseases such as diabetic macular edema.-Pollock, L. M., Xie, J., Bell, B. A., Anand-Apte, B. Retinoic acid signaling is essential for maintenance of the blood-retinal barrier.

Mutations in KARS, encoding lysyl-tRNA synthetase, cause autosomal-recessive nonsyndromic hearing impairment DFNB89.

Previously, DFNB89, a locus associated with autosomal-recessive nonsyndromic hearing impairment (ARNSHI), was mapped to chromosomal region 16q21-q23.2 in three unrelated, consanguineous Pakistani families. Through whole-exome sequencing of a hearing-impaired individual from each family, missense mutations were identified at highly conserved residues of lysyl-tRNA synthetase (KARS): the c.1129G>A (p.Asp377Asn) variant was found in one family, and the c.517T>C (p.Tyr173His) variant was found in the other two families. Both variants were predicted to be damaging by multiple bioinformatics tools. The two variants both segregated with the nonsyndromic-hearing-impairment phenotype within the three families, and neither mutation was identified in ethnically matched controls or within variant databases. Individuals homozygous for KARS mutations had symmetric, severe hearing impairment across all frequencies but did not show evidence of auditory or limb neuropathy. It has been demonstrated that KARS is expressed in hair cells of zebrafish, chickens, and mice. Moreover, KARS has strong localization to the spiral ligament region of the cochlea, as well as to Deiters' cells, the sulcus epithelium, the basilar membrane, and the surface of the spiral limbus. It is hypothesized that KARS variants affect aminoacylation in inner-ear cells by interfering with binding activity to tRNA or p38 and with tetramer formation. The identification of rare KARS variants in ARNSHI-affected families defines a gene that is associated with ARNSHI.

The cuticular plate: a riddle, wrapped in a mystery, inside a hair cell.

The mechanosensitive hair cells of the inner ear are crucial to hearing and vestibular function. Each hair cell detects the mechanical stimuli associated with sound or head movement with a hair bundle at the apical surface of the cell, consisting of a precise array of actin-based stereocilia. Each stereocilium inserts as a rootlet into a dense filamentous actin mesh known as the cuticular plate. Disruption of the parallel actin bundles forming the stereocilia results in hearing impairments and balance defects. The cuticular plate is thought to be involved in holding the stereocilia in place. However, the precise role of the cuticular plate in hair bundle development, maintenance, and hearing remains unknown. Ultrastructural studies have revealed a complex cytoskeletal architecture, but a lack of knowledge of proteins that inhabit the cuticular plate and a dearth of mutations that perturb relevant proteins have hindered our understanding of the functions of the cuticular plate. Here, we discuss what is known about the structure and development of this unique and poorly-understood actin-rich organelle.

ACF7 is a hair-bundle antecedent, positioned to integrate cuticular plate actin and somatic tubulin.

The precise morphology of the mechanosensitive hair bundle requires seamless integration of actin and microtubule networks. Here, we identify Acf7a (actin crosslinking family protein 7a) as a protein positioned to bridge these distinct cytoskeletal networks in hair cells. By imaging Acf7a-Citrine fusion protein in zebrafish and immunolabeling of vestibular and cochlear mouse hair cells, we show that Acf7a and ACF7 circumscribe, underlie, and are interwoven into the cuticular plate (CP), and they also encircle the basal body of the kinocilium. In cochlear hair cells, ACF7 localization is graded, with the highest concentration near each fonticulus--an area free of F-actin in the region of the CP that contains the basal body. During hair-cell development and regeneration, Acf7a precedes formation of the hair bundle and CP. Finally, electron tomography demonstrates that the ends of microtubules insert into the CP and are decorated with filamentous linkers connecting microtubules to the CP. These observations are consistent with ACF7 being a linker protein, which may shape the cytoskeleton of the hair cell early during hair-bundle genesis.

Predisposition to cancer caused by genetic and functional defects of mammalian Atad5.

ATAD5, the human ortholog of yeast Elg1, plays a role in PCNA deubiquitination. Since PCNA modification is important to regulate DNA damage bypass, ATAD5 may be important for suppression of genomic instability in mammals in vivo. To test this hypothesis, we generated heterozygous (Atad5(+/m)) mice that were haploinsuffficient for Atad5. Atad5(+/m) mice displayed high levels of genomic instability in vivo, and Atad5(+/m) mouse embryonic fibroblasts (MEFs) exhibited molecular defects in PCNA deubiquitination in response to DNA damage, as well as DNA damage hypersensitivity and high levels of genomic instability, apoptosis, and aneuploidy. Importantly, 90% of haploinsufficient Atad5(+/m) mice developed tumors, including sarcomas, carcinomas, and adenocarcinomas, between 11 and 20 months of age. High levels of genomic alterations were evident in tumors that arose in the Atad5(+/m) mice. Consistent with a role for Atad5 in suppressing tumorigenesis, we also identified somatic mutations of ATAD5 in 4.6% of sporadic human endometrial tumors, including two nonsense mutations that resulted in loss of proper ATAD5 function. Taken together, our findings indicate that loss-of-function mutations in mammalian Atad5 are sufficient to cause genomic instability and tumorigenesis.

ADAMTS1, MPDZ, MVD, and SEZ6: candidate genes for autosomal recessive nonsyndromic hearing impairment.

Hearing impairment (HI) is a common disorder of sensorineural function with a highly heterogeneous genetic background. Although substantial progress has been made in the understanding of the genetic etiology of hereditary HI, many genes implicated in HI remain undiscovered. Via exome and Sanger sequencing of DNA samples obtained from consanguineous Pakistani families that segregate profound prelingual sensorineural HI, we identified rare homozygous missense variants in four genes (ADAMTS1, MPDZ, MVD, and SEZ6) that are likely the underlying cause of HI. Linkage analysis provided statistical evidence that these variants are associated with autosomal recessive nonsyndromic HI. In silico analysis of the mutant proteins encoded by these genes predicted structural, conformational or interaction changes. RNAseq data analysis revealed expression of these genes in the sensory epithelium of the mouse inner ear during embryonic, postnatal, and adult stages. Immunohistochemistry of the mouse cochlear tissue, further confirmed the expression of ADAMTS1, SEZ6, and MPDZ in the neurosensory hair cells of the organ of Corti, while MVD expression was more prominent in the spiral ganglion cells. Overall, supported by in silico mutant protein analysis, animal models, linkage analysis, and spatiotemporal expression profiling in the mouse inner ear, we propose four new candidate genes for HI and expand our understanding of the etiology of HI.

Primary cilia are present on endothelial cells of the hyaloid vasculature but are not required for the development of the blood-retinal barrier.

Endothelial cilia are found in a variety of tissues including the cranial vasculature of zebrafish embryos. Recently, endothelial cells in the developing mouse retina were reported to also possess primary cilia that are potentially involved in vascular remodeling. Fish carrying mutations in intraflagellar transport (ift) genes have disrupted cilia and have been reported to have an increased rate of spontaneous intracranial hemorrhage (ICH), potentially due to disruption of the sonic hedgehog (shh) signaling pathway. However, it remains unknown whether the endothelial cells forming the retinal microvasculature in zebrafish also possess cilia, and whether endothelial cilia are necessary for development and maintenance of the blood-retinal barrier (BRB). In the present study, we found that the endothelial cells lining the zebrafish hyaloid vasculature possess primary cilia during development. To determine whether endothelial cilia are necessary for BRB integrity, ift57, ift88, and ift172 mutants, which lack cilia, were crossed with the double-transgenic zebrafish strain Tg(l-fabp:DBP-EGFP;flk1:mCherry). This strain expresses a vitamin D-binding protein (DBP) fused to enhanced green fluorescent protein (EGFP) as a tracer in the blood plasma, while the endothelial cells forming the vasculature are tagged by mCherry. The Ift mutant fish develop a functional BRB, indicating that endothelial cilia are not necessary for early BRB integrity. Additionally, although treatment of zebrafish larvae with Shh inhibitor cyclopamine results in BRB breakdown, the Ift mutant fish were not sensitized to cyclopamine-induced BRB breakdown.

My oh my(osin): Insights into how auditory hair cells count, measure, and shape.

The mechanisms underlying mechanosensory hair bundle formation in auditory sensory cells are largely mysterious. In this issue, Lelli et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201509017) reveal that a pair of molecular motors, myosin IIIa and myosin IIIb, is involved in the hair bundle's morphology and hearing.

Supervillin Is a Component of the Hair Cell's Cuticular Plate and the Head Plates of Organ of Corti Supporting Cells.

The organ of Corti has evolved a panoply of cells with extraordinary morphological specializations to harness, direct, and transduce mechanical energy into electrical signals. Among the cells with prominent apical specializations are hair cells and nearby supporting cells. At the apical surface of each hair cell is a mechanosensitive hair bundle of filamentous actin (F-actin)-based stereocilia, which insert rootlets into the F-actin meshwork of the underlying cuticular plate, a rigid organelle considered to hold the stereocilia in place. Little is known about the protein composition and development of the cuticular plate or the apicolateral specializations of organ of Corti supporting cells. We show that supervillin, an F-actin cross-linking protein, localizes to cuticular plates in hair cells of the mouse cochlea and vestibule and zebrafish sensory epithelia. Moreover, supervillin localizes near the apicolateral margins within the head plates of Deiters' cells and outer pillar cells, and proximal to the apicolateral margins of inner phalangeal cells, adjacent to the junctions with neighboring hair cells. Overall, supervillin localization suggests this protein may shape the surface structure of the organ of Corti.

Sequencing of candidate chromosome instability genes in endometrial cancers reveals somatic mutations in ESCO1, CHTF18, and MRE11A.

Most endometrial cancers can be classified histologically as endometrioid, serous, or clear cell. Non-endometrioid endometrial cancers (NEECs; serous and clear cell) are the most clinically aggressive of the three major histotypes and are characterized by aneuploidy, a feature of chromosome instability. The genetic alterations that underlie chromosome instability in endometrial cancer are poorly understood. In the present study, we used Sanger sequencing to search for nucleotide variants in the coding exons and splice junctions of 21 candidate chromosome instability genes, including 19 genes implicated in sister chromatid cohesion, from 24 primary, microsatellite-stable NEECs. Somatic mutations were verified by sequencing matched normal DNAs. We subsequently resequenced mutated genes from 41 additional NEECs as well as 42 endometrioid ECs (EECs). We uncovered nonsynonymous somatic mutations in ESCO1, CHTF18, and MRE11A in, respectively, 3.7% (4 of 107), 1.9% (2 of 107), and 1.9% (2 of 107) of endometrial tumors. Overall, 7.7% (5 of 65) of NEECs and 2.4% (1 of 42) of EECs had somatically mutated one or more of the three genes. A subset of mutations are predicted to impact protein function. The co-occurrence of somatic mutations in ESCO1 and CHTF18 was statistically significant (P = 0.0011, two-tailed Fisher's exact test). This is the first report of somatic mutations within ESCO1 and CHTF18 in endometrial tumors and of MRE11A mutations in microsatellite-stable endometrial tumors. Our findings warrant future studies to determine whether these mutations are driver events that contribute to the pathogenesis of endometrial cancer.

Fascin 2b is a component of stereocilia that lengthens actin-based protrusions.

Stereocilia are actin-filled protrusions that permit mechanotransduction in the internal ear. To identify proteins that organize the cytoskeleton of stereocilia, we scrutinized the hair-cell transcriptome of zebrafish. One promising candidate encodes fascin 2b, a filamentous actin-bundling protein found in retinal photoreceptors. Immunolabeling of zebrafish hair cells and the use of transgenic zebrafish that expressed fascin 2b fused to green fluorescent protein demonstrated that fascin 2b localized to stereocilia specifically. When filamentous actin and recombinant fusion protein containing fascin 2b were combined in vitro to determine their dissociation constant, a K(d)≈0.37 µM was observed. Electron microscopy showed that fascin 2b-actin filament complexes formed parallel actin bundles in vitro. We demonstrated that expression of fascin 2b or espin, another actin-bundling protein, in COS-7 cells induced the formation of long filopodia. Coexpression showed synergism between these proteins through the formation of extra-long protrusions. Using phosphomutant fascin 2b proteins, which mimicked either a phosphorylated or a nonphosphorylated state, in COS-7 cells and in transgenic hair cells, we showed that both formation of long filopodia and localization of fascin 2b to stereocilia were dependent on serine 38. Overexpression of wild-type fascin 2b in hair cells was correlated with increased stereociliary length relative to controls. These findings indicate that fascin 2b plays a key role in shaping stereocilia.