Novel gene function revealed by mouse mutagenesis screens for models of age-related disease

Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss

Functional analysis of a novel ENU-induced PHD finger 11 (Phf11) mouse mutant

Previously, human genetic studies have shown association between polymorphisms within the gene encoding plant homeodomain zinc finger protein 11 (PHF11) and asthma-related phenotypes. Initial functional studies have suggested that PHF11 may be involved in the immune response through regulation of T cell activities. In order to study further the gene’s functions, we have investigated the mouse Phf11 locus. We have established and characterised a mouse line harbouring a point mutation in the PHD domain of Phf11. Full-length mouse cDNA for Phf11 was obtained by applying rapid amplification of cDNA ends (RACE). All five exons encoding the PHD domain of Phf11 were directly sequenced in 3840 mouse DNA samples from the UK MRC Harwell ENU (N-ethyl-N-nitrosourea)-mutagenised DNA archive. Mice harbouring a valine to alanine substitution, predicted to have a significant functional impact on the PHD zinc finger domain, were re-derived. These Phf11 mutant mice were outcrossed to C3H mice and then backcrossed for ten generations in order to establish a congenic line harbouring the single point mutation in Phf11. Macroscopic examination, haematology and histological examination of lung structure revealed no significant differences between mutant and wild-type mice. After administration of lipopolysaccharide, the level of expression of Il2, NF-kB and Setdb2 were significantly increased in Phf11 mutant homozygous lungs compared to control littermates. Our results provide evidence that Phf11 can operate as a Th1 cell regulator in immune responses. Moreover, our data indicate that these mice may provide a useful model for future studies on Phf11. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00335-014-9535-x) contains supplementary material, which is available to authorised users

Helios is a key transcriptional regulator of outer hair cell maturation

The sensory cells that are responsible for hearing include the cochlear inner hair cells (IHCs) and outer hair cells (OHCs), with the OHCs being necessary for sound sensitivity and tuning1. Both cell types are thought to arise from common progenitors; however, our understanding of the factors that control the fate of IHCs and OHCs remains limited. Here we identify Ikzf2 (which encodes Helios) as an essential transcription factor in mice that is required for OHC functional maturation and hearing. Helios is expressed in postnatal mouse OHCs, and in the cello mouse model a point mutation in Ikzf2 causes early-onset sensorineural hearing loss. Ikzf2cello/cello OHCs have greatly reduced prestin-dependent electromotile activity, a hallmark of OHC functional maturation, and show reduced levels of crucial OHC-expressed genes such as Slc26a5 (which encodes prestin) and Ocm. Moreover, we show that ectopic expression of Ikzf2 in IHCs: induces the expression of OHC-specific genes; reduces the expression of canonical IHC genes; and confers electromotility to IHCs, demonstrating that Ikzf2 can partially shift the IHC transcriptome towards an OHC-like identity

Characterisation of cello: an ENU-induced mouse model of early-onset hearing loss

cello is a recessive mouse model of early-onset, sensorineural hearing loss that was identified from a large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis screen undertaken at MRC Harwell. Linkage analysis and whole-genome sequencing identified that cello mice have a non-synonymous point mutation in Ikaros family zinc finger 2 (Ikzf2), which encodes a zinc finger transcription factor called helios. To investigate the role of helios in the auditory system, cello mice underwent comprehensive molecular, phenotypic and functional characterisation. The cello mutation was found to disrupt a highly conserved, zinc-coordinating residue in the final C-terminal zinc finger domain of helios. In vitro analyses demonstrated that mutant helios can still localise to the nucleus, but has an impaired ability to homodimerise – a prerequisite for DNA binding. In both Ikzf2+/+ and Ikzf2cello/cello cochleae, helios is expressed in the nuclei of the outer hair cells (OHCs) from the first postnatal week, coinciding with a critical period of OHC functional maturation. Although Ikzf2cello/cello exhibit severely impaired hearing at postnatal day (P) 16, cochlear morphology appears grossly normal up to one month of age, after which the OHCs and inner hair cells begin to progressively degenerate. Transcriptome profiling was subsequently undertaken to identify potential target genes of helios in the auditory system. Results suggested that loss of helios function leads to dysregulation of over 450 genes in Ikzf2cello/cello cochleae at P8. This included the OHC-specific genes Slc26a5 (which encodes the OHC motor protein, prestin) and Ocm (oncomodulin), both of which were significantly decreased in Ikzf2cello/cello mutants compared to Ikzf2+/+ controls. Electrophysiological studies demonstrated that Ikzf2cello/cello OHCs fail to acquire full electromotility, a property that is fundamental to their function, but otherwise mature normally. Using luciferase reporter assays, wild-type helios was found to induce transactivation of the Slc26a5 promoter, whilst this ability was lost in the mutant protein. Additional in silico network analyses identified helios as a putative direct regulator of 40 genes that are enriched in the OHCs and upregulated during development, which suggests that helios has a key role in regulating the maturing OHC transcriptome. Overall, the work presented in this thesis identifies Ikzf2 as a novel hearing loss-causing gene and establishes that the helios transcription factor is essential for OHC functional maturation in the mammalian auditory system.</p

Universal Southern blot protocol with cold or radioactive probes for the validation of alleles obtained by homologous recombination

International audienc

Manipulation of dipeptidylpeptidase 10 in mouse and human and models indicates a protective role in asthma.

We previously identified dipeptidylpeptidase 10 () on chromosome 2 as a human asthma susceptibility gene, through positional cloning. Initial association results were confirmed in many subsequent association studies but the functional role of DPP10 in asthma remains unclear. Using the MRC Harwell N-ethyl-N-nitrosourea (ENU) DNA archive, we identified a point mutation in that caused an amino acid change from valine to aspartic acid in the β-propeller region of the protein. Mice carrying this point mutation were recovered and a congenic line was established ( ). Macroscopic examination and lung histology revealed no significant differences between wild-type and mice. However, after house dust mite (HDM) treatment, mutant mice showed significantly increased airway resistance in response to 100 mg/ml methacholine. Total serum IgE levels and bronchoalveolar lavage (BAL) eosinophil counts were significantly higher in homozygotes than in control mice after HDM treatment. DPP10 protein is present in airway epithelial cells and altered expression is observed in both tissue from asthmatic patients and in mice following HDM challenge. Moreover, knockdown of in human airway epithelial cells results in altered cytokine responses. These results show that a point mutation leads to increased airway responsiveness following allergen challenge and provide biological evidence to support previous findings from human genetic studies. This article has an associated First Person interview with the first author of the paper

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

AbstractDuring 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

The epilepsy-associated protein TBC1D24 is required for normal development, survival and vesicle trafficking in mammalian neurons

Mutations in the Tre2/Bub2/Cdc16 (TBC)1 domain family member 24 (TBC1D24) gene are associated with a range of inherited neurological disorders, from drug-refractory lethal epileptic encephalopathy and DOORS syndrome (deafness, onychodystrophy, osteodystrophy, mental retardation, seizures) to non-syndromic hearing loss. TBC1D24 has been implicated in neuronal transmission and maturation, although the molecular function of the gene and the cause of the apparently complex disease spectrum remain unclear. Importantly, heterozygous TBC1D24 mutation carriers have also been reported with seizures, suggesting that haploinsufficiency for TBC1D24 is significant clinically. Here we have systematically investigated an allelic series of disease-associated mutations in neurons alongside a new mouse model to investigate the consequences of TBC1D24 haploinsufficiency to mammalian neurodevelopment and synaptic physiology. The cellular studies reveal that disease-causing mutations that disrupt either of the conserved protein domains in TBC1D24 are implicated in neuronal development and survival and are likely acting as loss-of-function alleles. We then further investigated TBC1D24 haploinsufficiency in vivo and demonstrate that TBC1D24 is also crucial for normal presynaptic function: genetic disruption of Tbc1d24 expression in the mouse leads to an impairment of endocytosis and an enlarged endosomal compartment in neurons with a decrease in spontaneous neurotransmission. These data reveal the essential role for TBC1D24 at the mammalian synapse and help to define common synaptic mechanisms that could underlie the varied effects of TBC1D24 mutations in neurological disease