Structural defects in cilia of the choroid plexus, subfornical organ and ventricular ependyma are associated with ventriculomegaly

<p>Abstract</p> <p>Background</p> <p>Hydrocephalus is a heterogeneous disorder with multiple etiologies that are not yet fully understood. Animal models have implicated dysfunctional cilia of the ependyma and choroid plexus in the development of the disorder. In this report, we sought to determine the origin of the ventriculomegaly in four Bardet Biedl syndrome (BBS) mutant mouse strains as models of a ciliopathy.</p> <p>Methods</p> <p>Evans Blue dye was injected into the lateral ventricle of wild- type and BBS mutant mice to determine whether obstruction of intra- or extra-ventricular CSF flow contributed to ventriculomegaly. Transmission electron microscopy (TEM) was used to examine the ultrastructure of the choroid plexus, subfornical organ (SFO), subcommisural organ (SCO), and ventricular ependyma to evaluate their ultrastructure and the morphology of their primary and motile cilia.</p> <p>Results and discussion</p> <p>No obstruction of intra- or extra-ventricular CSF flow was observed, implying a communicating form of hydrocephalus in BBS mutant mice. TEM analyses of the mutants showed no evidence of choroidal papillomas or breakdown of the blood:CSF barrier. In contrast, structural defects were observed in a subpopulation of cilia lining the choroid plexus, SFO, and ventricular ependyma. These included disruptions of the microtubular structure of the axoneme and the presence of electron-dense vesicular-like material along the ciliary shaft and at the tips of cilia.</p> <p>Conclusions</p> <p>Abnormalities in cilia structure and function have the potential to influence ciliary intraflagellar transport (IFT), cilia maintenance, protein trafficking, and regulation of CSF production. Ciliary structural defects are the only consistent pathological features associated with CSF-related structures in BBS mutant mice. These defects are observed from an early age, and may contribute to the underlying pathophysiology of ventriculomegaly.</p

A Mutation in the Mouse <i>Ttc26</i> Gene Leads to Impaired Hedgehog Signaling

<div><p>The phenotype of the spontaneous mutant mouse hop-sterile (hop) is characterized by a hopping gait, polydactyly, hydrocephalus, and male sterility. Previous analyses of the hop mouse revealed a deficiency of inner dynein arms in motile cilia and a lack of sperm flagella, potentially accounting for the hydrocephalus and male sterility. The etiology of the other phenotypes and the location of the <i>hop</i> mutation remained unexplored. Here we show that the <i>hop</i> mutation is located in the <i>Ttc26</i> gene and impairs Hedgehog (Hh) signaling. Expression analysis showed that this mutation led to dramatically reduced levels of the Ttc26 protein, and protein-protein interaction assays demonstrated that wild-type Ttc26 binds directly to the Ift46 subunit of Intraflagellar Transport (IFT) complex B. Although IFT is required for ciliogenesis, the Ttc26 defect did not result in a decrease in the number or length of primary cilia. Nevertheless, Hh signaling was reduced in the hop mouse, as revealed by impaired activation of Gli transcription factors in embryonic fibroblasts and abnormal patterning of the neural tube. Unlike the previously characterized mutations that affect IFT complex B, <i>hop</i> did not interfere with Hh-induced accumulation of Gli at the tip of the primary cilium, but rather with the subsequent dissociation of Gli from its negative regulator, Sufu. Our analysis of the hop mouse line provides novel insights into Hh signaling, demonstrating that Ttc26 is necessary for efficient coupling between the accumulation of Gli at the ciliary tip and its dissociation from Sufu.</p></div

The <i>hop</i> mutation does not impair ciliogenesis or ciliary localization of the Ttc26-interacting protein Ift46.

<p>(<b>A</b>) Color test of protein-protein interactions in yeast transformed with the indicated combination of Ift46 and Ttc26^wt or Ttc26^hop. Blue staining of the yeast colony (upper patch) is indicative of a protein-protein interaction; a lack thereof indicates the absence of a protein-protein interaction (lower patch). (<b>B</b>) Immunoprecipitation analysis of the Ttc26^wt-Ift46 interaction. HEK293 cells were transfected with the indicated combinations of HA-tagged Ift46 and flag (FL)-tagged Ttc26^wt or Ttc26^hop. The two upper panels show immunoblot analysis of tagged proteins pulled down with an anti-flag antibody, and the two lower panels show immunoblot analysis of input controls. (<b>C</b>) Percentages of ciliated <i>hop/+</i> and <i>hop/hop</i> MEFs following 2 days of serum starvation. MEFs were isolated from 4 embryos per genotype, and 150 MEFs per embryo were analyzed. (<b>D</b>) Statistical analysis of cilium length in the primary cultures of serum-starved <i>hop/+</i> and <i>hop/hop</i> MEFs (mean ± SEM, n = 200 cilia per genotype, unpaired <i>t</i>-test with Welch correction, ***<i>P</i><0.0001). (<b>E,F</b>) Immunofluorescence analysis of (<b>E</b>) Ttc26 and (<b>F</b>) Ift46 expression in the cilia of <i>hop/+</i> and <i>hop/hop</i> MEFs. The axoneme was visualized by immunolabeling of acetylated-α-tubulin (AαT).</p

The hop mouse exhibits patterning defects and hearing impairment.

<p>(<b>A</b>) Representative images of <i>hop/+</i> and <i>hop/hop</i> mice at postnatal day 4. (<b>B</b>) Comparison of Alcian Blue-stained fore and hind limbs of <i>hop/+</i> and <i>hop/hop</i> mice. Extra digits are indicated by arrows. (<b>C</b>) Statistical analysis of FoxA2-positive cells in the lumbar neural tubes of <i>hop/+</i> and <i>hop/hop</i> mice (E10.5). Each symbol represents the average number of FoxA2⁺ cells per focal plane in a single embryo (for each embryo, 12 focal planes in 4 sections were analyzed, Mann-Whitney test: *<i>P</i> = 0.01). (<b>D</b>) Immunostaining of the lumbar neural tube of <i>hop/+</i> and <i>hop/hop</i> mice (E10.5) with antibodies against the V3 progenitor marker Nkx2.2 (white contrast), the floor plate marker FoxA2 (red), and the motor neuron protein HB9 (white contrast). The <i>hop/hop</i> genotype is associated with reduced FoxA2 expression and ventralization of the Nkx2.2- and HB9-expressing cells. Scale bar: 50 µm. (<b>E</b>) Representative ABR waveforms for 3–4 week-old <i>hop/+</i> and <i>hop/hop</i> mice. Broadband click stimuli were applied at the indicated sound pressure levels (in dB). (<b>F</b>) Statistical analysis of ABR thresholds measured in 3–4 week-old <i>hop/+</i> and <i>hop/hop</i> mice. Broadband click stimuli between 30 and 90 dB sound pressure level (SPL) were used. Each symbol represents the value for a single mouse (Mann-Whitney test: ***<i>P</i><0.0001).</p

The <i>Ttc26</i> gene of the hop mouse contains a nonsense mutation.

<p>(<b>A</b>) Schematic representation of genomic positions of genes that both fall within the 16-mega base pair (Mbp) interval to which the <i>hop</i> mutation was mapped and had a known association with the ciliome. (<b>B</b>) Comparison of the 15^th exon of the <i>Ttc26</i> gene in wild-type and <i>hop/hop</i> mice. Horizontal lines represent introns, and black and white rectangles represent the coding and non-coding regions of exons, respectively. A deoxycytidine nucleotide (C) of wild-type <i>Ttc26</i> (upper chromatogram) is replaced with a deoxyadenosine (A) in the hop mouse, as indicated by an arrow in the lower chromatogram. The point mutation changes the tyrosine (Tyr) at position 430 of Ttc26 to a stop codon (Stop), as shown in the amino-acid sequence lines. (<b>C</b>) Schematic representation of the Ttc26 protein. Blue boxes indicate the predicted TPR motifs. The bracket indicates the C-terminal 125-amino acid region of the protein that is predicted to be missing in <i>hop/hop</i> cells. The asterisk indicates the position of the epitope that is recognized by the anti-Ttc26 antibody. (<b>D</b>) Immunoblot analysis of Ttc26 expression in transfected HEK293 cells, airway epithelial cells (AEC) and testis of wild type (wt) and <i>hop/hop</i> (hop) mice. HEK293 cells were transfected with the indicated Ttc26-encoding construct or an empty expression vector. Arrowheads indicate the positions of the 64, 49, and 37 kDa standards. The antibodies used for immunoblotting are indicated next to the upper and lower panels.</p

The <i>Ttc26</i> mutation in the hop mouse impairs Shh signaling.

<p>(<b>A</b>) Induction of an adenovirus-delivered Gli^x8-luciferase reporter gene in wild-type (+/+) and <i>hop/hop</i> MEFs following 2-day incubation with DMSO (0.02%), Shh-conditioned medium, or SAG (400 nM) as indicated (mean ± SEM, n = 3–7). The increase in luciferase expression is shown relative to that in the DMSO control. Constitutive GFP expression from the Gli^x8-luciferase-encoding viral vector was used for normalization. (<b>B</b>) SAG-dependent induction of the Gli^x8-luciferase gene in <i>hop/hop</i> MEFs following adenoviral delivery of Ttc26^hop or Ttc26^wt (mean ± SEM, n = 3–7). The control group of MEFs was not transduced with a Ttc26-encoding virus (no Ttc26). The luciferase signal was normalized to GFP expression as described in panel A. (<b>C</b>) Immunoblot analyses of expression of Gli3-F and Gli3-R in wild-type and <i>hop/hop</i> MEFs, following SAG treatment (400 nM) for the indicated times. ß-actin serves as a loading control (lower panel). (<b>D</b>) Statistical analysis of Gli3-F band intensities in the immunoblot experiments described in panel C (mean ± SEM, n = 3; two-way ANOVA, <i>P</i><0.006 for the genotype variable; <i>post-hoc</i> Bonferroni test, **<i>P</i><0.01). (<b>E</b>) Statistical analysis of the ratio of the Gli3-F and Gli3-R bands at the 0 time point in the experiments described in panel C (mean ± SEM, n = 3; unpaired <i>t</i>-test, *<i>P</i> = 0.014).</p

Comparative Genomics and Gene Expression Analysis Identifies BBS9, a New Bardet-Biedl Syndrome Gene

Bardet-Biedl syndrome (BBS) is an autosomal recessive, genetically heterogeneous, pleiotropic human disorder characterized by obesity, retinopathy, polydactyly, renal and cardiac malformations, learning disabilities, and hypogenitalism. Eight BBS genes representing all known mapped loci have been identified. Mutation analysis of the known BBS genes in BBS patients indicate that additional BBS genes exist and/or that unidentified mutations exist in the known genes. To identify new BBS genes, we performed homozygosity mapping of small, consanguineous BBS pedigrees, using moderately dense SNP arrays. A bioinformatics approach combining comparative genomic analysis and gene expression studies of a BBS-knockout mouse model was used to prioritize BBS candidate genes within the newly identified loci for mutation screening. By use of this strategy, parathyroid hormone-responsive gene B1 (B1) was found to be a novel BBS gene (BBS9), supported by the identification of homozygous mutations in BBS patients. The identification of BBS9 illustrates the power of using a combination of comparative genomic analysis, gene expression studies, and homozygosity mapping with SNP arrays in small, consanguineous families for the identification of rare autosomal recessive disorders. We also demonstrate that small, consanguineous families are useful in identifying intragenic deletions. This type of mutation is likely to be underreported because of the difficulty of deletion detection in the heterozygous state by the mutation screening methods that are used in many studies

Glucocorticoid Induction of the Glaucoma Gene MYOC in Human and Monkey Trabecular Meshwork Cells and Tissues

PURPOSE. To examine the intracellular and extracellular expression of myocilin in the human and primate trabecular meshwork (TM) in the presence and absence of glucocorticoids. METHODS. Myocilin expression was examined in cultured human TM cells by Northern blot analysis and myocilin antibody– mediated immunoprecipitation. Myocilin expression was quantified using high-resolution two-dimensional polyacrylamide gel electrophoresis of radiolabeled proteins from human TM cells, TM tissue explants, and perfused human anterior segments cultured with and without dexamethasone (DEX) for 14 to 21 days, as well as TM tissue from pigtailed monkeys treated orally for 1 year with cortisone acetate. Immunofluorescence with anti-myocilin antibodies was used to localize cellular and extracellular expression of myocilin in cultured human TM cells. RESULTS. Glucocorticoid treatment caused a significant induction of myocilin mRNA, a tetrad of cell-associated proteins, and 8 to 20 secreted proteins (molecular mass [Mr] 56 and 59 kDa and isoelectric point [pI] 5.2 and 5.3) in some, but not all the cultured human TM cells and explanted tissues. Western immunoblot analysis using anti-myocilin peptide antibodies identified these proteins as encoded by the MYOC gene. There was significant induction of the myocilin proteins in three perfusion- cultured human eyes, in which DEX-induced elevated intraocular pressure developed. Monkeys treated 1 year with cortisol acetate showed steroid glaucoma-like morphologic changes in the TM that correlated with the induction of myocilin in the TM. Immunofluorescence analysis of cultured TM cells localized myocilin intracellularly in discrete perinuclear and cytoplasmic vesicular deposits as well as extracellularly on the cell surface associated with the extracellular matrix. In several DEX-treated TM cell lines, there were significant levels of myocilin secreted into the media. Enzymatic deglycosylation of proteins in the TM media converted the higher molecular weight isoforms of myocilin (;57 kDa) to the lower molecular weight isoforms (;55 kDa). CONCLUSIONS. Although the function of myocilin is unknown, induction of these TM proteins was found in eyes in which glucocorticoid-induced ocular hypertension developed. Therefore, myocilin may play an important pathogenic role in ocular hypertension in addition to its role in certain forms of POAG. (Invest Ophthalmol Vis Sci. 2001;42:1769–1780