Improving laboratory animal genetic reporting: LAG-R guidelines.

The biomedical research community addresses reproducibility challenges in animal studies through standardized nomenclature, improved experimental design, transparent reporting, data sharing, and centralized repositories. The ARRIVE guidelines outline documentation standards for laboratory animals in experiments, but genetic information is often incomplete. To remedy this, we propose the Laboratory Animal Genetic Reporting (LAG-R) framework. LAG-R aims to document animals' genetic makeup in scientific publications, providing essential details for replication and appropriate model use. While verifying complete genetic compositions may be impractical, better reporting and validation efforts enhance reliability of research. LAG-R standardization will bolster reproducibility, peer review, and overall scientific rigor.

An Interactive Database for the Assessment of Histone Antibody Specificity.

Access to high-quality antibodies is a necessity for the study of histones and their posttranslational modifications (PTMs). Here we debut the Histone Antibody Specificity Database (http://www.histoneantibodies.com), an online and expanding resource cataloging the behavior of widely used, commercially available histone antibodies by peptide microarray. This interactive web portal provides a critical resource to the biological research community that routinely uses these antibodies as detection reagents for a wide range of applications.

Epigenetic basis for aberrant upregulation of autoantigen genes in humans with ANCA vasculitis.

Antineutrophil cytoplasmic autoantibody (ANCA) causes vascular injury that leads to small-vessel vasculitis. Patients with ANCA aberrantly express neutrophil granule-encoding genes, including 2 that encode autoantigens: proteinase 3 (PR3) and myeloperoxidase (MPO). To uncover a potential transcriptional regulatory mechanism for PR3 and MPO disrupted in patients with ANCA vasculitis, we examined the PR3 and MPO loci in neutrophils from ANCA patients and healthy control individuals for epigenetic modifications associated with gene silencing. We found that levels of the chromatin modification H3K27me3, which is associated with gene silencing, were depleted at PR3 and MPO loci in ANCA patients compared with healthy controls. Interestingly, in both patients and controls, DNA was unmethylated at a CpG island in PR3, whereas in healthy controls, DNA was methylated at a CpG island in MPO. Consistent with decreased levels of H3K27me3, JMJD3, the demethylase specific for H3K27me3, was preferentially expressed in ANCA patients versus healthy controls. In addition, we describe a mechanism for recruiting the H3K27 methyltransferase enhancer of zeste homolog 2 (EZH2) to PR3 and MPO loci mediated by RUNX3. RUNX3 message was decreased in patients compared with healthy controls, and may also be under epigenetic control. DNA methylation was increased at the RUNX3 promoter in ANCA patients. These data indicate that epigenetic modifications associated with gene silencing are perturbed at ANCA autoantigen-encoding genes, potentially contributing to inappropriate expression of PR3 and MPO in ANCA patients.

Social approach and repetitive behavior in eleven inbred mouse strains.

Core symptoms of autism include deficits in social interaction, impaired communication, and restricted, repetitive behaviors. The repetitive behavior domain encompasses abnormal motoric stereotypy, an inflexible insistence on sameness, and resistance to change. In recent years, many genetic mouse models of autism and related disorders have been developed, based on candidate genes for disease susceptibility. The present studies are part of an ongoing initiative to develop appropriate behavioral tasks for the evaluation of mouse models relevant to autism. We have previously reported profiles for sociability, preference for social novelty, and resistance to changes in a learned pattern of behavior, as well as other functional domains, for 10 inbred mouse strains of divergent genetic backgrounds. The present studies extend this multi-component behavioral characterization to several additional strains: C58/J, NOD/LtJ, NZB/B1NJ, PL/J, SJL/J, SWR/J, and the wild-derived PERA/EiJ. C58/J, NOD/LtJ, NZB/B1NJ, SJL/J, and PERA/EiJ demonstrated low sociability, measured by time spent in proximity to an unfamiliar conspecific, with 30-60% of mice from these strains showing social avoidance. In the Morris water maze, NZB/B1NJ had a persistent bias for the quadrant where the hidden platform was located during acquisition, even after 9 days of reversal training. A particularly interesting profile was found for C58/J, which had low social preference, poor performance in the T-maze, and overt motoric stereotypy. Overall, this set of tasks and observational methods provides a strategy for evaluating novel mouse models in behavioral domains relevant to the autism phenotype.

Mouse behavioral tasks relevant to autism: phenotypes of 10 inbred strains.

Three defining clinical symptoms of autism are aberrant reciprocal social interactions, deficits in social communication, and repetitive behaviors, including motor stereotypies and insistence on sameness. We developed a set of behavioral tasks designed to model components of these core symptoms in mice. Male mice from 10 inbred strains were characterized in assays for sociability, preference for social novelty, and reversal of the spatial location of the reinforcer in T-maze and Morris water maze tasks. Six strains, C57BL/6J, C57L/J, DBA/2J, FVB/NJ, C3H/HeJ, and AKR/J, showed significant levels of sociability, while A/J, BALB/cByJ, BTBR T(+)tf/J, and 129S1/SvImJ mice did not. C57BL/6J, C57L/J, DBA/2J, FVB/NJ, BALB/cByJ, and BTBR T(+)tf/J showed significant preference for social novelty, while C3H/HeJ, AKR/J, A/J, and 129S1/SvImJ did not. Normal scores on relevant control measures confirmed general health and physical abilities in all strains, ruling out artifactual explanations for social deficits. Elevated plus maze scores confirmed high anxiety-like behaviors in A/J, BALB/cByJ, and 129S1/SvImJ, which could underlie components of their low social approach. Strains that showed high levels of performance on acquisition of a T-maze task were also able to reach criterion for reversal learning. On the Morris water maze task, DBA/2J, AKR/J, BTBR T(+)tf/J, and 129S1/SvImJ failed to show significant quadrant preference during the reversal probe trial. These results highlight a dissociation between social task performance and reversal learning. BTBR T(+)tf/J is a particularly interesting strain, displaying both low social approach and resistance to change in routine on the water maze, consistent with an autism-like phenotype. Our multitask strategy for modeling symptoms of autism will be useful for investigating targeted and random gene mutations, QTLs, and microarray analyses.

Large-scale gene expression differences across brain regions and inbred strains correlate with a behavioral phenotype.

Behaviors are often highly heritable, polygenic traits. To investigate molecular mediators of behavior, we analyzed gene expression patterns across seven brain regions (amygdala, basal ganglia, cerebellum, frontal cortex, hippocampus, cingulate cortex, and olfactory bulb) of 10 different inbred mouse strains (129S1/SvImJ, A/J, AKR/J, BALB/cByJ, BTBR T+ tf/J, C3H/HeJ, C57BL/6J, C57L/J, DBA/2J, and FVB/NJ). Extensive variation was observed across both strain and brain region. These data provide potential transcriptional intermediates linking polygenic variation to differences in behavior. For example, mice from different strains had variable performance on the rotarod task, which correlated with the expression of >2000 transcripts in the cerebellum. Correlation with this task was also found in the amygdala and hippocampus, but not in other regions examined, indicating the potential complexity of motor coordination. Thus we can begin to identify expression profiles contributing to behavioral phenotypes through variation in gene expression.

Reduced maternal expression of adrenomedullin disrupts fertility, placentation, and fetal growth in mice.

Adrenomedullin (AM) is a multifunctional peptide vasodilator that is essential for life. Plasma AM expression dramatically increases during pregnancy, and alterations in its levels are associated with complications of pregnancy including fetal growth restriction (FGR) and preeclampsia. Using AM+/- female mice with genetically reduced AM expression, we demonstrate that fetal growth and placental development are seriously compromised by this modest decrease in expression. AM+/- female mice had reduced fertility characterized by FGR. The incidence of FGR was also influenced by the genotype of the embryo, since AM-/- embryos were more often affected than either AM+/- or AM+/+ embryos. We demonstrate that fetal trophoblast cells and the maternal uterine wall have coordinated and localized increases in AM gene expression at the time of implantation. Placentas from growth-restricted embryos showed defects in trophoblast cell invasion, similar to defects that underlie human preeclampsia and placenta accreta. Our data provide a genetic in vivo model to implicate both maternal and, to a lesser extent, embryonic levels of AM in the processes of implantation, placentation, and subsequent fetal growth. This study provides the first genetic evidence to our knowledge to suggest that a modest reduction in human AM expression during pregnancy may have an unfavorable impact on reproduction.

Mouse models of autism spectrum disorders: the challenge for behavioral genetics.

Autism is a severe neurodevelopmental disorder, which typically emerges early in childhood. The core symptoms of autism include deficits in social interaction, impaired communication, and aberrant repetitive behavior, including self-injury. Despite the strong genetic component for the disease, most cases of autism have not been linked to mutations in a specific gene, and the etiology of the disorder has yet to be established. At the present time, there is no generally accepted therapeutic strategy to treat the core symptoms of autism, and there remains a critical need for appropriate animal models and relevant behavioral assays to promote the understanding and treatment of the clinical syndrome. Challenges for the development of valid mouse models include complex genetic interactions underlying the high heritability of the disease in humans, diagnosis based on deficits in social interaction and communication, and the lack of confirmatory neuropathological markers to provide validation for genetic models of the disorder. Research focusing on genes that mediate social behavior in mice may help identify neural circuitry essential for normal social interaction, and lead to novel genetic animal models of the autism behavioral phenotype.

Defects in yolk sac vasculogenesis, chorioallantoic fusion, and embryonic axis elongation in mice with targeted disruption of Yap65.

YAP is a multifunctional adapter protein and transcriptional coactivator with several binding partners well described in vitro and in cell culture. To explore in vivo requirements for YAP, we generated mice carrying a targeted disruption of the Yap gene. Homozygosity for the Yap(tm1Smil) allele (Yap-/-) caused developmental arrest around E8.5. Phenotypic characterization revealed a requirement for YAP in yolk sac vasculogenesis. Yolk sac endothelial and erythrocyte precursors were specified as shown by histology, PECAM1 immunostaining, and alpha globin expression. Nonetheless, development of an organized yolk sac vascular plexus failed in Yap-/- embryos. In striking contrast, vasculogenesis proceeded in both the allantois and the embryo proper. Mutant embryos showed patterned gene expression domains along the anteroposterior neuraxis, midline, and streak/tailbud. Despite this evidence of proper patterning and tissue specification, Yap-/- embryos showed developmental perturbations that included a notably shortened body axis, convoluted anterior neuroepithelium, caudal dysgenesis, and failure of chorioallantoic fusion. These results reveal a vital requirement for YAP in the developmental processes of yolk sac vasculogenesis, chorioallantoic attachment, and embryonic axis elongation.

H1 linker histones are essential for mouse development and affect nucleosome spacing in vivo.

Most eukaryotic cells contain nearly equimolar amounts of nucleosomes and H1 linker histones. Despite their abundance and the potential functional specialization of H1 subtypes in multicellular organisms, gene inactivation studies have failed to reveal essential functions for linker histones in vivo. Moreover, in vitro studies suggest that H1 subtypes may not be absolutely required for assembly of chromosomes or nuclei. By sequentially inactivating the genes for three mouse H1 subtypes (H1c, H1d, and H1e), we showed that linker histones are essential for mammalian development. Embryos lacking the three H1 subtypes die by mid-gestation with a broad range of defects. Triple-H1-null embryos have about 50% of the normal ratio of H1 to nucleosomes. Mice null for five of these six H1 alleles are viable but are underrepresented in litters and are much smaller than their littermates. Marked reductions in H1 content were found in certain tissues of these mice and in another compound H1 mutant. These results demonstrate that the total amount of H1 is crucial for proper embryonic development. Extensive reduction of H1 in certain tissues did not lead to changes in nuclear size, but it did result in global shortening of the spacing between nucleosomes.