Effects of Aberrant Pax6 Gene Dosage on Mouse Corneal Pathophysiology and Corneal Epithelial Homeostasis

Background: Altered dosage of the transcription factor PAX6 causes multiple human eye pathophysiologies. PAX6(+/-) heterozygotes suffer from aniridia and aniridia-related keratopathy (ARK), a corneal deterioration that probably involves a limbal epithelial stem cell (LESC) deficiency. Heterozygous Pax6(+/Sey-Neu) (Pax6(+/-)) mice recapitulate the human disease and are a good model of ARK. Corneal pathologies also occur in other mouse Pax6 mutants and in PAX77(Tg/-) transgenics, which over-express Pax6 and model human PAX6 duplication. Methodology/Principal Findings: We used electron microscopy to investigate ocular defects in Pax6(+/-) heterozygotes (low Pax6 levels) and PAX77(Tg/-) transgenics (high Pax6 levels). As well as the well-documented epithelial defects, aberrant Pax6 dosage had profound effects on the corneal stroma and endothelium in both genotypes, including cellular vacuolation, similar to that reported for human macular corneal dystrophy. We used mosaic expression of an X-linked LacZ transgene in X-inactivation mosaic female (XLacZ(Tg/-)) mice to investigate corneal epithelial maintenance by LESC clones in Pax6(+/-) and PAX77(Tg/-) mosaic mice. PAX77(Tg/-) mosaics, over-expressing Pax6, produced normal corneal epithelial radial striped patterns (despite other corneal defects), suggesting that centripetal cell movement was unaffected. Moderately disrupted patterns in Pax6(+/-) mosaics were corrected by introducing the PAX77 transgene (in Pax6(+/-), PAX77(Tg/-) mosaics). Pax6(Leca4/+), XLacZ(Tg/-) mosaic mice (heterozygous for the Pax6(Leca4) missense mutation) showed more severely disrupted mosaic patterns. Corrected corneal epithelial stripe numbers (an indirect estimate of active LESC clone numbers) declined with age (between 15 and 30 weeks) in wild-type XLacZ(Tg/-) mosaics. In contrast, corrected stripe numbers were already low at 15 weeks in Pax6(+/-) and PAX77(Tg/-) mosaic corneas, suggesting Pax6 under-and over-expression both affect LESC clones. Conclusions/Significance: Pax6(+/-) and PAX77(Tg/-) genotypes have only relatively minor effects on LESC clone numbers but cause more severe corneal endothelial and stromal defects. This should prompt further investigations of the pathophysiology underlying human aniridia and ARK

Normal X-inactivation mosaicism in corneas of heterozygous FlnaDilp2/+ female mice--a model of human Filamin A (FLNA) diseases

<p>Abstract</p> <p>Background</p> <p>Some abnormalities of mouse corneal epithelial maintenance can be identified by the atypical mosaic patterns they produce in X-chromosome inactivation mosaics and chimeras. Human <it>FLNA</it>/+ females, heterozygous for X-linked, filamin A gene (<it>FLNA</it>) mutations, display a range of disorders and X-inactivation mosaicism is sometimes quantitatively unbalanced. <it>Flna</it>^{<it>Dilp2/+ </it>}mice, heterozygous for an X-linked filamin A (<it>Flna</it>) nonsense mutation have variable eye, skeletal and other abnormalities, but X-inactivation mosaicism has not been investigated. The aim of this study was to determine whether X-inactivation mosaicism in the corneal epithelia of <it>Flna</it>^{<it>Dilp2/+ </it>}mice was affected in any way that might predict abnormal corneal epithelial maintenance.</p> <p>Results</p> <p>X-chromosome inactivation mosaicism was studied in the corneal epithelium and a control tissue (liver) of <it>Flna</it>^{<it>Dilp2/+ </it>}and wild-type (WT) female X-inactivation mosaics, hemizygous for the X-linked, <it>LacZ </it>reporter H253 transgene, using β-galactosidase histochemical staining. The corneal epithelia of <it>Flna</it>^{<it>Dilp2/+ </it>}and WT X-inactivation mosaics showed similar radial, striped patterns, implying epithelial cell movement was not disrupted in <it>Flna</it>^{<it>Dilp2/+ </it>}corneas. Corrected stripe numbers declined with age overall (but not significantly for either genotype individually), consistent with previous reports suggesting an age-related reduction in stem cell function. Corrected stripe numbers were not reduced in <it>Flna</it>^{<it>Dilp2/+ </it>}compared with WT X-inactivation mosaics and mosaicism was not significantly more unbalanced in the corneal epithelia or livers of <it>Flna</it>^{<it>Dilp2/+ </it>}than wild-type <it>Flna^+/+</it>X-inactivation mosaics.</p> <p>Conclusions</p> <p>Mosaic analysis identified no major effect of the mouse <it>Flna^Dilp2</it>mutation on corneal epithelial maintenance or the balance of X-inactivation mosaicism in the corneal epithelium or liver.</p

Haploinsufficiency of the NOTCH1 Receptor as a Cause of Adams-Oliver Syndrome With Variable Cardiac Anomalies.

BACKGROUND: Adams-Oliver syndrome (AOS) is a rare disorder characterized by congenital limb defects and scalp cutis aplasia. In a proportion of cases, notable cardiac involvement is also apparent. Despite recent advances in the understanding of the genetic basis of AOS, for the majority of affected subjects, the underlying molecular defect remains unresolved. This study aimed to identify novel genetic determinants of AOS. METHODS AND RESULTS: Whole-exome sequencing was performed for 12 probands, each with a clinical diagnosis of AOS. Analyses led to the identification of novel heterozygous truncating NOTCH1 mutations (c.1649dupA and c.6049_6050delTC) in 2 kindreds in which AOS was segregating as an autosomal dominant trait. Screening a cohort of 52 unrelated AOS subjects, we detected 8 additional unique NOTCH1 mutations, including 3 de novo amino acid substitutions, all within the ligand-binding domain. Congenital heart anomalies were noted in 47% (8/17) of NOTCH1-positive probands and affected family members. In leukocyte-derived RNA from subjects harboring NOTCH1 extracellular domain mutations, we observed significant reduction of NOTCH1 expression, suggesting instability and degradation of mutant mRNA transcripts by the cellular machinery. Transient transfection of mutagenized NOTCH1 missense constructs also revealed significant reduction in gene expression. Mutant NOTCH1 expression was associated with downregulation of the Notch target genes HEY1 and HES1, indicating that NOTCH1-related AOS arises through dysregulation of the Notch signaling pathway. CONCLUSIONS: These findings highlight a key role for NOTCH1 across a range of developmental anomalies that include cardiac defects and implicate NOTCH1 haploinsufficiency as a likely molecular mechanism for this group of disorders

Three Dimensional Visualization and Fractal Analysis of Mosaic Patches in Rat Chimeras: Cell Assortment in Liver, Adrenal Cortex and Cornea

The production of organ parenchyma in a rapid and reproducible manner is critical to normal development. In chimeras produced by the combination of genetically distinguishable tissues, mosaic patterns of cells derived from the combined genotypes can be visualized. These patterns comprise patches of contiguously similar genotypes and are different in different organs but similar in a given organ from individual to individual. Thus, the processes that produce the patterns are regulated and conserved. We have previously established that mosaic patches in multiple tissues are fractal, consistent with an iterative, recursive growth model with simple stereotypical division rules. Fractal dimensions of various tissues are consistent with algorithmic models in which changing a single variable (e.g. daughter cell placement after division) switches the mosaic pattern from islands to stripes of cells. Here we show that the spiral pattern previously observed in mouse cornea can also be visualized in rat chimeras. While it is generally held that the pattern is induced by stem cell division dynamics, there is an unexplained discrepancy in the speed of cellular migration and the emergence of the pattern. We demonstrate in chimeric rat corneas both island and striped patterns exist depending on the age of the animal. The patches that comprise the pattern are fractal, and the fractal dimension changes with the age of the animal and indicates the constraint in patch complexity as the spiral pattern emerges. The spiral patterns are consistent with a loxodrome. Such data are likely to be relevant to growth and cell division in organ systems and will help in understanding how organ parenchyma are generated and maintained from multipotent stem cell populations located in specific topographical locations within the organ. Ultimately, understanding algorithmic growth is likely to be essential in achieving organ regeneration in vivo or in vitro from stem cell populations

Minor Physical Anomalies in Patients with Schizophrenia, Unaffected First-Degree Relatives, and Healthy Controls: A Meta-Analysis

Background: Minor physical anomalies (MPAs) have been found to be more prevalent in schizophrenia than control participants in numerous studies and may index a potential endophenotype for schizophrenia.</p

Analysis of compound heterozygotes reveals that the mouse floxed Pax6 tm1Ued allele produces abnormal eye phenotypes

Analysis of abnormal phenotypes produced by different types of mutations has been crucial for our understanding of gene function. Some floxed alleles that retain a neomycin-resistance selection cassette (neo cassette) are not equivalent to wild-type alleles and provide useful experimental resources. Pax6 is an important developmental gene and the aim of this study was to determine whether the floxed Pax6(tm1Ued) (Pax6(fl)) allele, which has a retained neo cassette, produced any abnormal eye phenotypes that would imply that it differs from the wild-type allele. Homozygous Pax6(fl/fl) and heterozygous Pax6(fl/+) mice had no overt qualitative eye abnormalities but morphometric analysis showed that Pax6(fl/fl) corneas tended be thicker and smaller in diameter. To aid identification of weak effects, we produced compound heterozygotes with the Pax6(Sey-Neu) (Pax6(−)) null allele. Pax6(fl/−) compound heterozygotes had more severe eye abnormalities than Pax6(+/−) heterozygotes, implying that Pax6(fl) differs from the wild-type Pax6(+) allele. Immunohistochemistry showed that the Pax6(fl/−) corneal epithelium was positive for keratin 19 and negative for keratin 12, indicating that it was abnormally differentiated. This Pax6(fl) allele provides a useful addition to the existing Pax6 allelic series and this study demonstrates the utility of using compound heterozygotes with null alleles to unmask cryptic effects of floxed alleles. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11248-016-9962-4) contains supplementary material, which is available to authorized users

Overexpression of Pax6 results in microphthalmia, retinal dysplasia and defective retinal ganglion cell axon guidance

Background: The transcription factor Pax6 is expressed by many cell types in the developing eye. Eyes do not form in homozygous loss-of-function mouse mutants (Pax6(Sey/Sey)) and are abnormally small in Pax6(Sey/+) mutants. Eyes are also abnormally small in PAX77 mice expressing multiple copies of human PAX6 in addition to endogenous Pax6; protein sequences are identical in the two species. The developmental events that lead to microphthalmia in PAX77 mice are not well-characterised, so it is not clear whether over- and under-expression of Pax6/PAX6 cause microphthalmia through similar mechanisms. Here, we examined the consequences of over-expression for the eye and its axonal connections. Results: Eyes form in PAX77(+/+) embryos but subsequently degenerate. At E12.5, we found no abnormalities in ocular morphology, retinal cell cycle parameters and the incidence of retinal cell death. From E14.5 on, we observed malformations of the optic disc. From E16.5 into postnatal life there is progressively more severe retinal dysplasia and microphthalmia. Analyses of patterns of gene expression indicated that PAX77(+/+) retinae produce a normal range of cell types, including retinal ganglion cells (RGCs). At E14.5 and E16.5, quantitative RT-PCR with probes for a range of molecules associated with retinal development showed only one significant change: a slight reduction in levels of mRNA encoding the secreted morphogen Shh at E16.5. At E16.5, tract-tracing with carbocyanine dyes in PAX77(+/+) embryos revealed errors in intraretinal navigation by RGC axons, a decrease in the number of RGC axons reaching the thalamus and an increase in the proportion of ipsilateral projections among those RGC axons that do reach the thalamus. A survey of embryos with different Pax6/PAX6 gene dosage (Pax6(Sey/+), Pax6(+/+), PAX77(+) and PAX77(+/+)) showed that (1) the total number of RGC axons projected by the retina and (2) the proportions that are sorted into the ipsilateral and contralateral optic tracts at the optic chiasm vary differently with gene dosage. Increasing dosage increases the proportion projecting ipsilaterally regardless of the size of the total projection. Conclusion: Pax6 overexpression does not obviously impair the initial formation of the eye and its major cell-types but prevents normal development of the retina from about E14.5, leading eventually to severe retinal degeneration in postnatal life. This sequence is different to that underlying microphthalmia in Pax6(+/-) heterozygotes, which is due primarily to defects in the initial stages of lens formation. Before the onset of severe retinal dysplasia, Pax6 overexpression causes defects of retinal axons, preventing their normal growth and navigation through the optic chiasm

Effectiveness of an implementation optimisation intervention aimed at increasing parent engagement in HENRY, a childhood obesity prevention programme - the Optimising Family Engagement in HENRY (OFTEN) trial: study protocol for a randomised controlled trial

Background: Family-based interventions to prevent childhood obesity depend upon parents’ taking action to improve diet and other lifestyle behaviours in their families. Programmes that attract and retain high numbers of parents provide an enhanced opportunity to improve public health and are also likely to be more cost-effective than those that do not. We have developed a theory-informed optimisation intervention to promote parent engagement within an existing childhood obesity prevention group programme, HENRY (Health Exercise Nutrition for the Really Young). Here, we describe a proposal to evaluate the effectiveness of this optimisation intervention in regard to the engagement of parents and cost-effectiveness. Methods/design: The Optimising Family Engagement in HENRY (OFTEN) trial is a cluster randomised controlled trial being conducted across 24 local authorities (approximately 144 children’s centres) which currently deliver HENRY programmes. The primary outcome will be parental enrolment and attendance at the HENRY programme, assessed using routinely collected process data. Cost-effectiveness will be presented in terms of primary outcomes using acceptability curves and through eliciting the willingness to pay for the optimisation from HENRY commissioners. Secondary outcomes include the longitudinal impact of the optimisation, parent-reported infant intake of fruits and vegetables (as a proxy to compliance) and other parent-reported family habits and lifestyle. Discussion: This innovative trial will provide evidence on the implementation of a theory-informed optimisation intervention to promote parent engagement in HENRY, a community-based childhood obesity prevention programme. The findings will be generalisable to other interventions delivered to parents in other community-based environments. This research meets the expressed needs of commissioners, children’s centres and parents to optimise the potential impact that HENRY has on obesity prevention. A subsequent cluster randomised controlled pilot trial is planned to determine the practicality of undertaking a definitive trial to robustly evaluate the effectiveness and cost-effectiveness of the optimised intervention on childhood obesity prevention

Functional Amyloids Composed of Phenol Soluble Modulins Stabilize Staphylococcus aureus Biofilms

Staphylococcus aureus is an opportunistic pathogen that colonizes the skin and mucosal surfaces of mammals. Persistent staphylococcal infections often involve surface-associated communities called biofilms. Here we report the discovery of a novel extracellular fibril structure that promotes S. aureus biofilm integrity. Biochemical and genetic analysis has revealed that these fibers have amyloid-like properties and consist of small peptides called phenol soluble modulins (PSMs). Mutants unable to produce PSMs were susceptible to biofilm disassembly by matrix degrading enzymes and mechanical stress. Previous work has associated PSMs with biofilm disassembly, and we present data showing that soluble PSM peptides disperse biofilms while polymerized peptides do not. This work suggests the PSMs' aggregation into amyloid fibers modulates their biological activity and role in biofilms