A humanized version of Foxp2 affects cortico-basal ganglia circuits in mice.

It has been proposed that two amino acid substitutions in the transcription factor FOXP2 have been positively selected during human evolution due to effects on aspects of speech and language. Here, we introduce these substitutions into the endogenous Foxp2 gene of mice. Although these mice are generally healthy, they have qualitatively different ultrasonic vocalizations, decreased exploratory behavior and decreased dopamine concentrations in the brain suggesting that the humanized Foxp2 allele affects basal ganglia. In the striatum, a part of the basal ganglia affected in humans with a speech deficit due to a nonfunctional FOXP2 allele, we find that medium spiny neurons have increased dendrite lengths and increased synaptic plasticity. Since mice carrying one nonfunctional Foxp2 allele show opposite effects, this suggests that alterations in cortico-basal ganglia circuits might have been important for the evolution of speech and language in humans.

Identification of ocular regulatory functions of core histone variant H3.2.

The posttranscriptional modifications (PTM) of the Histone H3 family play an important role in ocular system differentiation. However, there has been no study on the nature of specific Histone H3 subtype carrying these modifications. Fortuitously, we had previously identified a dominant small-eye mutant Aey69 mouse with a mutation in the H3.2 encoding Hist2h3c1 gene (Vetrivel et al., 2019). In continuation, in the present study, the role of Histone H3.2 with relation to the microphtalmic Aey69 has been elaborated. Foremost, a transgenic mouse line expressing the fusion protein H3.2-GFP was generated using Crispr/Cas9. The approach was intended to confer a unique tag to the Hist2h3c1 gene which is similar in sequence and encoded protein structure to other histones. The GFP tag was then used for ChIP Seq analysis of the genes regulated by H3.2. The approach revealed ocular specific H3.2 targets including Ephrin family genes. Altered enrichment of H3.2 was found in the mutant Aey69 mouse, specifically around the ligand Efna5 and the receptor Ephb2. The effect of this altered enrichment on Ephrin signaling was further analysed by QPCR and immunohistochemistry. This study identifies Hist2h3c1 encoded H3.2 as an important epigenetic player in ocular development. By binding to specific regions of ocular developmental factors Histone H3.2 facilitates the function of these genes for successful early ocular development.

Ionising radiation causes vision impairment in neonatal B6C3F1 mice.

Ionising radiation interacts with lenses and retinae differently. In human lenses, posterior subcapsular cataracts are the predominant observation, whereas retinae of adults are comparably resistant to even relatively high doses. In this study, we demonstrate the effects of 2 Gy of low linear energy transfer ionising radiation on eyes of B6C3F1 mice aged postnatal day 2. Optical coherence tomography and Scheimpflug imaging were utilised for the first time to monitor murine lenses and retinae in vivo. The visual acuity of the mice was determined and histological analysis was conducted. Our results demonstrated that visual acuity was reduced by as much as 50 % approximately 9 months after irradiation in irradiated mice. Vision impairment was caused by retinal atrophy and inner cortical cataracts. These results help to further our understanding of the risk of ionising radiation for human foeti (∼ 8 mo), which follow the same eye development stages as neonatal mice.

Laboratory mouse housing conditions can be improved using common environmental enrichment without compromising data.

Animal welfare requires the adequate housing of animals to ensure health and well-being. The application of environmental enrichment is a way to improve the well-being of laboratory animals. However, it is important to know whether these enrichment items can be incorporated in experimental mouse husbandry without creating a divide between past and future experimental results. Previous small-scale studies have been inconsistent throughout the literature, and it is not yet completely understood whether and how enrichment might endanger comparability of results of scientific experiments. Here, we measured the effect on means and variability of 164 physiological parameters in 3 conditions: with nesting material with or without a shelter, comparing these 2 conditions to a "barren" regime without any enrichments. We studied a total of 360 mice from each of 2 mouse strains (C57BL/6NTac and DBA/2NCrl) and both sexes for each of the 3 conditions. Our study indicates that enrichment affects the mean values of some of the 164 parameters with no consistent effects on variability. However, the influence of enrichment appears negligible compared to the effects of other influencing factors. Therefore, nesting material and shelters may be used to improve animal welfare without impairment of experimental outcome or loss of comparability to previous data collected under barren housing conditions.

CREB Signaling Mediates Dose-Dependent Radiation Response in the Murine Hippocampus Two Years after Total Body Exposure.

The impact of low-dose ionizing radiation (IR) on the human brain has recently attracted attention due to the increased use of IR for diagnostic purposes. The aim of this study was to investigate low-dose radiation response in the hippocampus. Female B6C3F1 mice were exposed to total body irradiation with 0 (control), 0.063, 0.125, or 0.5 Gy. Quantitative label-free proteomic analysis of the hippocampus was performed after 24 months. CREB signaling and CREB-associated pathways were affected at all doses. The lower doses (0.063 and 0.125 Gy) induced the CREB pathway, whereas the exposure to 0.5 Gy deactivated CREB. Similarly, the lowest dose (0.063 Gy) was anti-inflammatory, reducing the number of activated microglia. In contrast, induction of activated microglia and reactive astroglia was found at 0.5 Gy, suggesting increased inflammation and astrogliosis, respectively. The apoptotic markers BAX and cleaved CASP-3 and oxidative stress markers were increased only at the highest dose. Since the activated CREB pathway plays a central role in learning and memory, these data suggest neuroprotection at the lowest dose (0.063 Gy) but neurodegeneration at 0.5 Gy. The response to 0.5 Gy resembles alterations found in healthy aging and thus may represent radiation-induced accelerated aging of the brain.

A comprehensive and comparative phenotypic analysis of the collaborative founder strains identifies new and known phenotypes.

The collaborative cross (CC) is a large panel of mouse-inbred lines derived from eight founder strains (NOD/ShiLtJ, NZO/HILtJ, A/J, C57BL/6J, 129S1/SvImJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ). Here, we performed a comprehensive and comparative phenotyping screening to identify phenotypic differences and similarities between the eight founder strains. In total, more than 300 parameters including allergy, behavior, cardiovascular, clinical blood chemistry, dysmorphology, bone and cartilage, energy metabolism, eye and vision, immunology, lung function, neurology, nociception, and pathology were analyzed; in most traits from sixteen females and sixteen males. We identified over 270 parameters that were significantly different between strains. This study highlights the value of the founder and CC strains for phenotype-genotype associations of many genetic traits that are highly relevant to human diseases. All data described here are publicly available from the mouse phenome database for analyses and downloads.

Peroxidasin contributes to lung host defense by direct binding and killing of gram-negative bacteria.

Innate immune recognition is classically mediated by the interaction of host pattern-recognition receptors and pathogen-associated molecular patterns; this triggers a series of downstream signaling events that facilitate killing and elimination of invading pathogens. In this report, we provide the first evidence that peroxidasin (PXDN; also known as vascular peroxidase-1) directly binds to gram-negative bacteria and mediates bactericidal activity, thus, contributing to lung host defense. PXDN contains five leucine-rich repeats and four immunoglobulin domains, which allows for its interaction with lipopolysaccharide, a membrane component of gram-negative bacteria. Bactericidal activity of PXDN is mediated via its capacity to generate hypohalous acids. Deficiency of PXDN results in a failure to eradicate Pseudomonas aeruginosa and increased mortality in a murine model of Pseudomonas lung infection. These observations indicate that PXDN mediates previously unrecognized host defense functions against gram-negative bacterial pathogens.

Spectral domain - Optical coherence tomography (SD-OCT) as a monitoring tool for alterations in mouse lenses.

The eye lens displays a variety of phenotypes in the wake of genetic modifications or environmental influences. Therefore, a high-resolution in vivo imaging method for the lens is desirable. Optical coherence tomography (OCT) has become a powerful imaging tool in ophthalmology, especially for retinal imaging in small animal models such as mice. Here, we demonstrate an optimized approach specifically for anterior eye segment imaging with spectral domain OCT (SD-OCT) on several known murine lens cataract mutants. Scheimpflug and histological section images on the same eye were used in parallel to assess the observed pathologies. With SD-OCT images, we obtained detailed information about the different alterations from the anterior to the posterior pole of the lens. This capability makes OCT a valuable high-resolution imaging modality for the anterior eye segment in mouse.

Application of WES Towards Molecular Investigation of Congenital Cataracts: Identification of Novel Alleles and Genes in a Hospital-Based Cohort of South India.

Congenital cataracts are the prime cause for irreversible blindness in children. The global incidence of congenital cataract is 2.2-13.6 per 10,000 births, with the highest prevalence in Asia. Nearly half of the congenital cataracts are of familial nature, with a predominant autosomal dominant pattern of inheritance. Over 38 of the 45 mapped loci for isolated congenital or infantile cataracts have been associated with a mutation in a specific gene. The clinical and genetic heterogeneity of congenital cataracts makes the molecular diagnosis a bit of a complicated task. Hence, whole exome sequencing (WES) was utilized to concurrently screen all known cataract genes and to examine novel candidate factors for a disease-causing mutation in probands from 11 pedigrees affected with familial congenital cataracts. Analysis of the WES data for known cataract genes identified causative mutations in six pedigrees (55%) in PAX6, FYCO1 (two variants), EPHA2, P3H2,TDRD7 and an additional likely causative mutation in a novel gene NCOA6, which represents the first dominant mutation in this gene. This study identifies a novel cataract gene not yet linked to human disease. NCOA6 is a transcriptional coactivator that interacts with nuclear hormone receptors to enhance their transcriptional activator function.

Mouse models for microphthalmia, anophthalmia and cataracts.

Mouse mutants are a long-lasting, valuable tool to identify genes underlying eye diseases, because the absence of eyes, very small eyes and severely affected, cataractous eyes are easily to detect without major technical equipment. In mice, actually 145 genes or loci are known for anophthalmia, 269 for microphthalmia, and 180 for cataracts. Approximately, 25% of the loci are not yet characterized; however, some of the ancient lines are extinct and not available for future research. The phenotypes of the mutants represent a continuous spectrum either in anophthalmia and microphthalmia, or in microphthalmia and cataracts. On the other side, mouse models are still missing for some genes, which have been identified in human families to be causative for anophthalmia, microphthalmia, or cataracts. Finally, the mouse offers the possibility to genetically test the roles of modifiers and the role of SNPs; these aspects open new avenues for ophthalmogenetics in the mouse.

Mutation in the mouse histone gene Hist2h3c1 leads to degeneration of the lens vesicle and severe microphthalmia.

During an ENU (N-ethyl-N-nitrosourea) mutagenesis screen, we observed a dominant small-eye mutant mouse with viable homozygotes. A corresponding mutant line was established and referred to as Aey69 (abnormality of the eye #69). Comprehensive phenotyping of the homozygous Aey69 mutants in the German Mouse Clinic revealed only a subset of statistically significant alterations between wild types and homozygous mutants. The mutation causes microphthalmia without a lens but with retinal hyperproliferation. Linkage was demonstrated to mouse chromosome 3 between the markers D3Mit188 and D3Mit11. Sequencing revealed a 358 A-> C mutation (Ile120Leu) in the Hist2h3c1 gene and a 71 T-> C (Val24Ala) mutation in the Gja8 gene. Detailed analysis of eye development in the homozygous mutant mice documented a perturbed lens development starting from the lens vesicle stage including decreasing expression of crystallins as well as of lens-specific transcription factors like PITX3 and FOXE3. In contrast, we observed an early expression of retinal progenitor cells characterized by several markers including BRN3 (retinal ganglion cells) and OTX2 (cone photoreceptors). The changes in the retina at the early embryonic stages of E11.5-E15.5 happen in parallel with apoptotic processes in the lens at the respective stages. The excessive retinal hyperproliferation is characterized by an increased level of Ki67. The hyperproliferation, however, does not disrupt the differentiation and appearance of the principal retinal cell types at postnatal stages, even if the overgrowing retina covers finally the entire bulbus of the eye. Morpholino-mediated knock-down of the hist2h3ca1 gene in zebrafish leads to a specific perturbation of lens development. When injected into zebrafish zygotes, only the mutant mouse mRNA leads to severe malformations, ranging from cyclopia to severe microphthalmia. The wild-type Hist2h3c1 mRNA can rescue the morpholino-induced defects corroborating its specific function in lens development. Based upon these data, it is concluded that the ocular function of the Hist2h3c1 gene (encoding a canonical H3.2 variant) is conserved throughout evolution. Moreover, the data highlight also the importance of Hist2h3c1 in the coordinated formation of lens and retina during eye development.

Crybb2 associates with Tmsb4X and is crucial for dendrite morphogenesis.

Dendrite morphogenesis is a complex but well-orchestrated process. Various studies reported the involvement of alteration in dendrite morphology in different brain disorders, including neuropsychiatric disorders. Initially, ßB2-crystallin (gene symbol: Crybb2/CRYBB2) has been described as a structural protein of the ocular lens. Mutations of the corresponding gene, Crybb2, lead to cataract. Recent studies in mice suggested that mutations in Crybb2 cause alterations in hippocampal morphology and function, albeit its function in hippocampal neuron development remained elusive. In the current study, we found that Crybb2 contributes to dendritogenesis in vitro and in vivo. Furthermore, screening of previous data on differential expression-arrays, we found Tmsb4X up-regulated in Crybb2 mutants mouse brain. Additionally, Tmsb4X was co-expressed with Crybb2 at actin-enriched cell ruffles. Over-expression of Tmsb4X in cultured hippocampal neurons inhibited dendritogenesis, which phenocopied Crybb2 knock-down. The current study uncovers a new function of Crybb2 in brain development, especially in dendritogenesis, and the possible interplay partner Tmsb4X involved in this process.

Functional compensation among HMGN variants modulates the DNase I hypersensitive sites at enhancers.

DNase I hypersensitive sites (DHSs) are a hallmark of chromatin regions containing regulatory DNA such as enhancers and promoters; however, the factors affecting the establishment and maintenance of these sites are not fully understood. We now show that HMGN1 and HMGN2, nucleosome-binding proteins that are ubiquitously expressed in vertebrate cells, maintain the DHS landscape of mouse embryonic fibroblasts (MEFs) synergistically. Loss of one of these HMGN variants led to a compensatory increase of binding of the remaining variant. Genome-wide mapping of the DHSs in Hmgn1(-/-), Hmgn2(-/-), and Hmgn1(-/-)n2(-/-) MEFs reveals that loss of both, but not a single HMGN variant, leads to significant remodeling of the DHS landscape, especially at enhancer regions marked by H3K4me1 and H3K27ac. Loss of HMGN variants affects the induced expression of stress-responsive genes in MEFs, the transcription profiles of several mouse tissues, and leads to altered phenotypes that are not seen in mice lacking only one variant. We conclude that the compensatory binding of HMGN variants to chromatin maintains the DHS landscape, and the transcription fidelity and is necessary to retain wild-type phenotypes. Our study provides insight into mechanisms that maintain regulatory sites in chromatin and into functional compensation among nucleosome binding architectural proteins.

Meis1 coordinates a network of genes implicated in eye development and microphthalmia.

Microphthalmos is a rare congenital anomaly characterized by reduced eye size and visual deficits of variable degree. Sporadic and hereditary microphthalmos have been associated with heterozygous mutations in genes fundamental for eye development. Yet, many cases are idiopathic or await the identification of molecular causes. Here we show that haploinsufficiency of Meis1, which encodes a transcription factor with evolutionarily conserved expression in the embryonic trunk, brain and sensory organs, including the eye, causes microphthalmic traits and visual impairment in adult mice. By combining analysis of Meis1 loss-of-function and conditional Meis1 functional rescue with ChIP-seq and RNA-seq approaches we show that, in contrast to its preferential association with Hox-Pbx BSs in the trunk, Meis1 binds to Hox/Pbx-independent sites during optic cup development. In the eye primordium, Meis1 coordinates, in a dose-dependent manner, retinal proliferation and differentiation by regulating genes responsible for human microphthalmia and components of the Notch signaling pathway. In addition, Meis1 is required for eye patterning by controlling a set of eye territory-specific transcription factors, so that in Meis1(-/-) embryos boundaries among the different eye territories are shifted or blurred. We propose that Meis1 is at the core of a genetic network implicated in eye patterning/microphthalmia, and represents an additional candidate for syndromic cases of these ocular malformations.

Lifetime study in mice after acute low-dose ionizing radiation: a multifactorial study with special focus on cataract risk.

Because of the increasing application of ionizing radiation in medicine, quantitative data on effects of low-dose radiation are needed to optimize radiation protection, particularly with respect to cataract development. Using mice as mammalian animal model, we applied a single dose of 0, 0.063, 0.125 and 0.5 Gy at 10 weeks of age, determined lens opacities for up to 2 years and compared it with overall survival, cytogenetic alterations and cancer development. The highest dose was significantly associated with increased body weight and reduced survival rate. Chromosomal aberrations in bone marrow cells showed a dose-dependent increase 12 months after irradiation. Pathological screening indicated a dose-dependent risk for several types of tumors. Scheimpflug imaging of the lens revealed a significant dose-dependent effect of 1% of lens opacity. Comparison of different biological end points demonstrated long-term effects of low-dose irradiation for several biological end points.

From eyeless to neurological diseases.

Age-related cataracts are frequently associated with degenerative changes in the ocular lens including the aggregation of proteins - mainly crystallins, but also other proteins including amyloids (Aß) leading to the hypothesis that cataracts could be used as "biomarkers" for Alzheimer disease. Even if this hypothesis was rejected by David Beebe's last paper (Bei et al., Exp. Eye Res., 2015), it is a fascinating aspect to look for commonalities between eye diseases and neurological disorders. In this review, I discuss such commonalities between eye and brain mainly from a developmental point of view. The finding of the functional homology of the Drosophila eyeless gene with the mammalian Pax6 gene marks a first highlight in the developmental genetics of the eye - this result destroyed the "dogma" of the different evolutionary routes of eye development in flies and mammals. The second highlight was the finding that Pax6 is also involved in the development of the forebrain supporting the pleiotropic role of many genes. These findings opened a new avenue for research showing that a broad variety of transcription factors, but also structural proteins are involved both, in eye and brain development as well as into the maintenance of the functional integrity of the corresponding tissue(s). In this review recent findings are summarized demonstrating that genes whose mutations have been identified first to be causative for congenital or juvenile eye disorders are also involved in regenerative processes and neurogenesis (Pax6), but also in neurodegenerative diseases like Parkinson (e.g. Pitx3) or in neurological disorders like Schizophrenia (e.g. Crybb1, Crybb2).

Common eye diseases in older adults of southern Germany: results from the KORA-Age study.

Purpose: a population-based study in the region of Augsburg (Germany, KORA) was used to identify the prevalence of eye diseases and their risk factors in a sample of aged individuals. Methods: data originated from the KORA-Age study collected in 2012 and 822 participants (49.6% women, 50.4% men, aged 68-96 years) were asked standardised questions about eye diseases. Positive answers were validated and specified by treating ophthalmologists. Additional information came from laboratory data. Polymorphic markers were tested for candidate genes. Results: we received validations and specifications for 339 participants. The most frequent eye diseases were cataracts (299 cases, 36%), dry eyes (120 cases, 15%), glaucoma (72 cases, 9%) and age-related macular degeneration (AMD) (68 cases, 8%). Almost all participants suffering from glaucoma or from AMD also had cataracts. Cataract surgery was associated with diabetes (in men; OR = 2.24; 95% confidence interval [CI] 1.11-4.53; P = 0.025) and smoking (in women; OR = 6.77; CI 1.62-28.35; P = 0.009). In men, treatments in airway diseases was associated with cataracts (glucocorticoids: OR = 5.29, CI 1.20-23.37; P = 0.028; sympathomimetics: OR = 4.57, CI 1.39-15.00; P = 0.012). Polymorphisms in two genes were associated with AMD (ARMS2: OR = 2.28, CI 1.48-3.51; P = 0.005; CFH: OR = 2.03, CI 1.35-3.06; P = 0.010). Conclusion: combinations of eye diseases were frequent at old age. The importance of classical risk factors like diabetes, hypertension and airway diseases decreased either due to a survivor bias leaving healthier survivors in the older age group, or due to an increased influence of other up to now unknown risk factors.