Helen Hilton

Pkd1l1 establishes left-right asymmetry and physically interacts with Pkd2

In mammals, left-right (L-R) asymmetry is established by posteriorly oriented cilia driving a leftwards laminar flow in the embryonic node, thereby activating asymmetric gene expression. The two-cilia hypothesis argues that immotile cilia detect and respond to this flow through a Pkd2-mediated mechanism; a putative sensory partner protein has, however, remained unidentified. We have identified the Pkd1-related locus Pkd1l1 as a crucial component of L-R patterning in mouse. Systematic comparison of Pkd1l1 and Pkd2 point mutants reveals strong phenocopying, evidenced by both morphological and molecular markers of sidedness; both mutants fail to activate asymmetric gene expression at the node or in the lateral plate and exhibit right isomerism of the lungs. Node and cilia morphology were normal in mutants and cilia demonstrated typical motility, consistent with Pkd1l1 and Pkd2 activity downstream of nodal flow. Cell biological analysis reveals that Pkd1l1 and Pkd2 localise to the cilium and biochemical experiments demonstrate that they can physically interact. Together with co-expression in the node, these data argue that Pkd1l1 is the elusive Pkd2 binding partner required for L-R patterning and support the two-cilia hypothesis.

Dominant β-catenin mutations cause intellectual disability with recognizable syndromic features.

The recent identification of multiple dominant mutations in the gene encoding β-catenin in both humans and mice has enabled exploration of the molecular and cellular basis of β-catenin function in cognitive impairment. In humans, β-catenin mutations that cause a spectrum of neurodevelopmental disorders have been identified. We identified de novo β-catenin mutations in patients with intellectual disability, carefully characterized their phenotypes, and were able to define a recognizable intellectual disability syndrome. In parallel, characterization of a chemically mutagenized mouse line that displays features similar to those of human patients with β-catenin mutations enabled us to investigate the consequences of β-catenin dysfunction through development and into adulthood. The mouse mutant, designated batface (Bfc), carries a Thr653Lys substitution in the C-terminal armadillo repeat of β-catenin and displayed a reduced affinity for membrane-associated cadherins. In association with this decreased cadherin interaction, we found that the mutation results in decreased intrahemispheric connections, with deficits in dendritic branching, long-term potentiation, and cognitive function. Our study provides in vivo evidence that dominant mutations in β-catenin underlie losses in its adhesion-related functions, which leads to severe consequences, including intellectual disability, childhood hypotonia, progressive spasticity of lower limbs, and abnormal craniofacial features in adults.

Scribble is required for normal epithelial cell-cell contacts and lumen morphogenesis in the mammalian lung

During lung development, proper epithelial cell arrangements are critical for the formation of an arborized network of tubes. Each tube requires a lumen, the diameter of which must be tightly regulated to enable optimal lung function. Lung branching and lumen morphogenesis require close epithelial cell–cell contacts that are maintained as a result of adherens junctions, tight junctions and by intact apical–basal (A/B) polarity. However, the molecular mechanisms that maintain epithelial cohesion and lumen diameter in the mammalian lung are unknown. Here we show that Scribble, a protein implicated in planar cell polarity (PCP) signalling, is necessary for normal lung morphogenesis. Lungs of the Scrib mouse mutant Circletail (Crc) are abnormally shaped with fewer airways, and these airways often lack a visible, ‘open’ lumen. Mechanistically we show that Scrib genetically interacts with the core PCP gene Vangl2 in the developing lung and that the distribution of PCP pathway proteins and Rho mediated cytoskeletal modification is perturbed in ScribCrc/Crc lungs. However A/B polarity, which is disrupted in Drosophila Scrib mutants, is largely unaffected. Notably, we find that Scrib mediates functions not attributed to other PCP proteins in the lung. Specifically, Scrib localises to both adherens and tight junctions of lung epithelia and knockdown of Scrib in lung explants and organotypic cultures leads to reduced cohesion of lung epithelial cells. Live imaging of Scrib knockdown lungs shows that Scrib does not affect bud bifurcation, as previously shown for the PCP protein Celsr1, but is required to maintain epithelial cohesion. To understand the mechanism leading to reduced cell–cell association, we show that Scrib associates with β-catenin in embryonic lung and the sub-cellular distribution of adherens and tight junction proteins is perturbed in mutant lung epithelia. Our data reveal that Scrib is required for normal lung epithelial organisation and lumen morphogenesis by maintaining cell–cell contacts. Thus we reveal novel and important roles for Scrib in lung development operating via the PCP pathway, and in regulating junctional complexes and cell cohesion.

HIF-VEGF Pathways Are Critical for Chronic Otitis Media in Junbo and Jeff Mouse Mutants

Otitis media with effusion (OME) is the commonest cause of hearing loss in children, yet the underlying genetic pathways and mechanisms involved are incompletely understood. Ventilation of the middle ear with tympanostomy tubes is the commonest surgical procedure in children and the best treatment for chronic OME, but the mechanism by which they work remains uncertain. As hypoxia is a common feature of inflamed microenvironments, moderation of hypoxia may be a significant contributory mechanism. We have investigated the occurrence of hypoxia and hypoxia-inducible factor (HIF) mediated responses in Junbo and Jeff mouse mutant models, which develop spontaneous chronic otitis media. We found that Jeff and Junbo mice labeled in vivo with pimonidazole showed cellular hypoxia in inflammatory cells in the bulla lumen, and in Junbo the middle ear mucosa was also hypoxic. The bulla fluid inflammatory cell numbers were greater and the upregulation of inflammatory gene networks were more pronounced in Junbo than Jeff. Hif-1α gene expression was elevated in bulla fluid inflammatory cells, and there was upregulation of its target genes including Vegfa in Junbo and Jeff. We therefore investigated the effects in Junbo of small-molecule inhibitors of VEGFR signaling (PTK787, SU-11248, and BAY 43-9006) and destabilizing HIF by inhibiting its chaperone HSP90 with 17-DMAG. We found that both classes of inhibitor significantly reduced hearing loss and the occurrence of bulla fluid and that VEGFR inhibitors moderated angiogenesis and lymphangiogenesis in the inflamed middle ear mucosa. The effectiveness of HSP90 and VEGFR signaling inhibitors in suppressing OM in the Junbo model implicates HIF–mediated VEGF as playing a pivotal role in OM pathogenesis. Our analysis of the Junbo and Jeff mutants highlights the role of hypoxia and HIF–mediated pathways, and we conclude that targeting molecules in HIF–VEGF signaling pathways has therapeutic potential in the treatment of chronic OM.

Regulation of TGF-β signalling by Fbxo11, the gene mutated in the Jeff otitis media mouse mutant

BackgroundJeff is a dominant mouse mutant displaying chronic otitis media. The gene underlying Jeff is Fbxo11, a member of the large F-box family, which are specificity factors for the SCF E3 ubiquitin ligase complex. Jeff homozygotes die shortly after birth displaying a number of developmental abnormalities including cleft palate and eyes open at birth. TGF-β signalling is involved in a number of epithelial developmental processes and we have investigated the impact of the Jeff mutation on the expression of this pathway.ResultsPhospho-Smad2 (pSmad2) is significantly upregulated in epithelia of Jeff homozygotes. Moreover, there was a significant increase in nuclear localization of pSmad2 in contrast to wild type. Mice heterozygous for both Jeff and Smad2 mutations recapitulate many of the features of the Jeff homozygous phenotype. However, tissue immunoprecipitations failed to detect any interaction between Fbxo11 and Smad2. Fbxo11 is known to neddylate p53, a co-factor of pSmad2, but we did not find any evidence of genetic interactions between Jeff and p53 mutants. Nevertheless, p53 levels are substantially reduced in Jeff mice suggesting that Fbxo11 plays a role in stabilizing p53.ConclusionOverall, our findings support a model whereby Fbxo11, possibly via stabilization of p53, is required to limit the accumulation of pSmad2 in the nucleus of epithelial cells of palatal shelves, eyelids and airways of the lungs. The finding that Fbxo11 impacts upon TGF-β signalling has important implications for our understanding of the underlying disease mechanisms of middle ear inflammatory disease.

Gelsolin plays a role in the actin polymerization complex of hair cell stereocilia.

A complex of proteins scaffolded by the PDZ protein, whirlin, reside at the stereocilia tip and are critical for stereocilia development and elongation. We have shown that in outer hair cells (OHCs) whirlin is part of a larger complex involving the MAGUK protein, p55, and protein 4.1R. Whirlin interacts with p55 which is expressed exclusively in outer hair cells (OHC) in both the long stereocilia that make up the stereocilia bundle proper as well as surrounding shorter microvilli that will eventually regress. In erythrocytes, p55 forms a tripartite complex with protein 4.1R and glycophorin C promoting the assembly of actin filaments and the interaction of whirlin with p55 indicates that it plays a similar role in OHC stereocilia. However, the components directly involved in actin filament regulation in stereocilia are unknown. We have investigated additional components of the whirlin interactome by identifying interacting partners to p55. We show that the actin capping and severing protein, gelsolin, is a part of the whirlin complex. Gelsolin is detected in OHC where it localizes to the tips of the shorter rows but not to the longest row of stereocilia and the pattern of localisation at the apical hair cell surface is strikingly similar to p55. Like p55, gelsolin is ablated in the whirler and shaker2 mutants. Moreover, in a gelsolin mutant, stereocilia in the apex of the cochlea become long and straggly indicating defects in the regulation of stereocilia elongation. The identification of gelsolin provides for the first time a link between the whirlin scaffolding protein complex involved in stereocilia elongation and a known actin regulatory molecule.

ATMIN is a transcriptional regulator of both lung morphogenesis and ciliogenesis

Initially identified in DNA damage repair, ATM-interactor (ATMIN) further functions as a transcriptional regulator of lung morphogenesis. Here we analyse three mouse mutants, Atmingpg6/gpg6, AtminH210Q/H210Q and Dynll1GT/GT, revealing how ATMIN and its transcriptional target dynein light chain LC8-type 1 (DYNLL1) are required for normal lung morphogenesis and ciliogenesis. Expression screening of ciliogenic genes confirmed Dynll1 to be controlled by ATMIN and further revealed moderately altered expression of known intraflagellar transport (IFT) protein-encoding loci in Atmin mutant embryos. Significantly, Dynll1GT/GT embryonic cilia exhibited shortening and bulging, highly similar to the characterised retrograde IFT phenotype of Dync2h1. Depletion of ATMIN or DYNLL1 in cultured cells recapitulated the in vivo ciliogenesis phenotypes and expression of DYNLL1 or the related DYNLL2 rescued the effects of loss of ATMIN, demonstrating that ATMIN primarily promotes ciliogenesis by regulating Dynll1 expression. Furthermore, DYNLL1 as well as DYNLL2 localised to cilia in puncta, consistent with IFT particles, and physically interacted with WDR34, a mammalian homologue of the Chlamydomonas cytoplasmic dynein 2 intermediate chain that also localised to the cilium. This study extends the established Atmin-Dynll1 relationship into a developmental and a ciliary context, uncovering a novel series of interactions between DYNLL1, WDR34 and ATMIN. This identifies potential novel components of cytoplasmic dynein 2 and furthermore provides fresh insights into the molecular pathogenesis of human skeletal ciliopathies.

A Novel Mouse Fgfr2 Mutant, Hobbyhorse (hob), Exhibits Complete XY Gonadal Sex Reversal

The secreted molecule fibroblast growth factor 9 (FGF9) plays a critical role in testis determination in the mouse. In embryonic gonadal somatic cells it is required for maintenance of SOX9 expression, a key determinant of Sertoli cell fate. Conditional gene targeting studies have identified FGFR2 as the main gonadal receptor for FGF9 during sex determination. However, such studies can be complicated by inefficient and variable deletion of floxed alleles, depending on the choice of Cre deleter strain. Here, we report a novel, constitutive allele of Fgfr2, hobbyhorse (hob), which was identified in an ENU-based forward genetic screen for novel testis-determining loci. Fgr2hob is caused by a C to T mutation in the invariant exon 7, resulting in a polypeptide with a mis-sense mutation at position 263 (Pro263Ser) in the third extracellular immunoglobulin-like domain of FGFR2. Mutant homozygous embryos show severe limb and lung defects and, when on the sensitised C57BL/6J (B6) genetic background, undergo complete XY gonadal sex reversal associated with failure to maintain expression of Sox9. Genetic crosses employing a null mutant of Fgfr2 suggest that Fgr2hob is a hypomorphic allele, affecting both the FGFR2b and FGFR2c splice isoforms of the receptor. We exploited the consistent phenotype of this constitutive mutant by analysing MAPK signalling at the sex-determining stage of gonad development, but no significant abnormalities in mutant embryos were detected.

Identification of a Z-band associated protein complex involving KY, FLNC and IGFN1

The KY protein underlies a form of muscular dystrophy in the mouse but its role in muscle remains elusive. Immunodetection of endogenous KY protein in C2C12-derived myotubes and expression of a recombinant form in neonatal cardiomyocytes indicated that KY is a Z-band associated protein. Moreover, characterization of a KY interacting protein fragment led to the identification of Igfn1 (Immunoglobulin-like and fibronectin type 3 domain containing 1). Igfn1 is a transcriptionally complex locus encoding many protein variants. A yeast two-hybrid screen identified the Z-band protein filamin C (FLNC) as an interacting partner. Consistent with this, expression of an IGFN1 recombinant fragment showed that the three N-terminal globular domains, common to at least five IGFN1 variants, are sufficient to provide Z-band targeting. Taken together, the yeast two-hybrid, biochemical and immunofluorescence data support the notion that KY, IGFN1 and FLNC are part of a Z-band associated protein complex likely to provide structural support to the skeletal muscle sarcomere.

Upregulation of PKD1L2 provokes a complex neuromuscular disease in the mouse

Following a screen for neuromuscular mouse mutants, we identified ostes, a novel N-ethyl N-nitrosourea-induced mouse mutant with muscle atrophy. Genetic and biochemical evidence shows that upregulation of the novel, uncharacterized transient receptor potential polycystic (TRPP) channel PKD1L2 (polycystic kidney disease gene 1-like 2) underlies this disease. Ostes mice suffer from chronic neuromuscular impairments including neuromuscular junction degeneration, polyneuronal innervation and myopathy. Ectopic expression of PKD1L2 in transgenic mice reproduced the ostes myopathic changes and, indeed, caused severe muscle atrophy in Tg(Pkd1l2)/Tg(Pkd1l2) mice. Moreover, double-heterozygous mice (ostes/+, Tg(Pkd1l2)/0) suffer from myopathic changes more profound than each heterozygote, indicating positive correlation between PKD1L2 levels and disease severity. We show that, in vivo, PKD1L2 primarily associates with endogenous fatty acid synthase in normal skeletal muscle, and these proteins co-localize to costameric regions of the muscle fibre. In diseased ostes/ostes muscle, both proteins are upregulated, and ostes/ostes mice show signs of abnormal lipid metabolism. This work shows the first role for a TRPP channel in neuromuscular integrity and disease.