Autism-Like Behavior and Epigenetic Changes Associated with Autism as Consequences of In Utero Exposure to Environmental Pollutants in a Mouse Model

We tested the hypothesis that in utero exposure to heavy metals increases autism-like behavioral phenotypes in adult animals and induces epigenetic changes in genes that have roles in the etiology of autism. Mouse dams were treated with cadmium, lead, arsenate, manganese, and mercury via drinking water from gestational days (E) 1–10. Valproic acid (VPA) injected intraperitoneally once on (E) 8.5 served as a positive control. Young male offspring were tested for behavioral deficits using four standardized behavioral assays. In this study, in utero exposure to heavy metals resulted in multiple behavioral abnormalities that persisted into adulthood. VPA and manganese induced changes in perseverative/impulsive behavior and social dominance behavior, arsenic caused changes only in perseverative/impulsive behavior, and lead induced abnormalities in social interaction in comparison to the control animals. Brain samples from Mn, Pb, and VPA treated and control animals were evaluated for changes in CpG island methylation in promoter regions and associated changes in gene expression. The Chd7 gene, essential for neural crest cell migration and patterning, was found to be hypomethylated in each experimental animal tested compared to water-treated controls. Furthermore, distinct patterns of CpG island methylation yielded novel candidate genes for further investigation

Postimplantation Whole Embryo Culture Assay for Hamsters: An Alternative to Rat and Mouse

Postimplantation whole embryo culture (WEC) assay for rats and mice has been well established and introduced to many laboratories. Recently WEC technique for rabbits has been developed; however, information on culture of other species is very limited. Knowing the usefulness of hamsters in classical embryotoxicology, we reasoned that hamster WEC could be an alternative model for the most frequently used rat and mouse WEC. Previously we have optimized culture conditions for postimplantation hamster embryos. The aim of this study was to test the susceptibility of hamster embryos cultured in vitro to embryotoxic compounds and to compare our results with those reported by others on rat or mouse embryo culture. For that purpose we choose three known embryotoxic compounds�valproic acid, cadmium chloride, and diethylstilbestrol�and tested them using a postimplantation hamster whole embryo culture assay. Hamster embryos were cultured from 7.5 days gestation for 24 h in a medium consisting of 35% hamster serum and 65% synthetic culture medium (Iscove�s or McCoy 5A). At the end of the culture period, the embryos were examined morphologically, measured with the aid of a computer image analysis system, and total protein content was assessed. All three compounds exhibited dose-related embryotoxic and teratogenic effects in hamster embryos. The malformations observed were similar to those reported on rat and mouse embryos. Comparison of the results with data reported by other authors indicates that hamster embryos cultured in vitromight be more susceptible to embryotoxic stimuli than rat and mouse embryos

Autism-Like Behavior and Epigenetic Changes Associated with Autism as Consequences of In Utero

We tested the hypothesis that in utero exposure to heavy metals increases autism-like behavioral phenotypes in adult animals and induces epigenetic changes in genes that have roles in the etiology of autism. Mouse dams were treated with cadmium, lead, arsenate, manganese, and mercury via drinking water from gestational days (E) 1–10. Valproic acid (VPA) injected intraperitoneally once on (E) 8.5 served as a positive control. Young male offspring were tested for behavioral deficits using four standardized behavioral assays. In this study, in utero exposure to heavy metals resulted in multiple behavioral abnormalities that persisted into adulthood. VPA and manganese induced changes in perseverative/impulsive behavior and social dominance behavior, arsenic caused changes only in perseverative/impulsive behavior, and lead induced abnormalities in social interaction in comparison to the control animals. Brain samples from Mn, Pb, and VPA treated and control animals were evaluated for changes in CpG island methylation in promoter regions and associated changes in gene expression. The Chd7 gene, essential for neural crest cell migration and patterning, was found to be hypomethylated in each experimental animal tested compared to water-treated controls. Furthermore, distinct patterns of CpG island methylation yielded novel candidate genes for further investigation

CIC de novo loss of function variants contribute to cerebral folate deficiency by downregulating FOLR1 expression

Background Cerebral folate deficiency (CFD) syndrome is characterised by a low concentration of 5-methyltetrahydrofolate in cerebrospinal fluid, while folate levels in plasma and red blood cells are in the low normal range. Mutations in several folate pathway genes, including FOLR1 (folate receptor alpha, FRα), DHFR (dihydrofolate reductase) and PCFT (proton coupled folate transporter) have been previously identified in patients with CFD.Methods In an effort to identify causal mutations for CFD, we performed whole exome sequencing analysis on eight CFD trios and identified eight de novo mutations in seven trios.Results Notably, we found a de novo stop gain mutation in the capicua (CIC) gene. Using 48 sporadic CFD samples as a validation cohort, we identified three additional rare variants in CIC that are putatively deleterious mutations. Functional analysis indicates that CIC binds to an octameric sequence in the promoter regions of folate transport genes: FOLR1, PCFT and reduced folate carrier (Slc19A1; RFC1). The CIC nonsense variant (p.R353X) downregulated FOLR1 expression in HeLa cells as well as in the induced pluripotent stem cell (iPSCs) derived from the original CFD proband. Folate binding assay demonstrated that the p.R353X variant decreased cellular binding of folic acid in cells.Conclusion This study indicates that CIC loss of function variants can contribute to the genetic aetiology of CFD through regulating FOLR1 expression. Our study described the first mutations in a non-folate pathway gene that can contribute to the aetiology of CFD

A tentative model linking <i>Fuz</i> to Shh, Wnt and PCP signal pathways.

<p><b>A</b>) Fuz acts downstream of Frizzled and Dishevelled to regulate PCP signaling. Non-canonical Wnt signaling represses Wnt/β-catenin signaling and Fuz is involved in a negative feedback loop of Wnt/β-catenin signal regulation. Fuz also regulates assembly of the apical actin cytoskeleton, which is critical for ciliogenesis. Cilia formation can be directly linked to Hedgehog signaling and Gli transcription factor activation. Activated Gli factors then regulate Hedgehog target genes. <b>B</b>) Loss of <i>Fuz</i> leads to impaired PCP signaling and up-regulation of Wnt/β-catenin signaling. In addition, it results in impaired actin cap and cilia, which inhibits the activation of Gli transcription factors and weakens Hedgehog signaling. Loss of <i>Fuz</i> also results in the up-regulation of Sox9.</p

Wnt/β-catenin signaling is increased in the <i>Fuz</i> null mice.

<p>Immunofluorescence on sagittal sections of E14.5 embryos. <b>A</b>) β-catenin expression was increased in the oral epithelium and mesenchyme in mutant (<i>Fuz</i>^−/−) embryos. The bottom panels are the <i>Fuz^−/−</i> embryos. <b>B</b>) β-catenin expression was also increased in the <i>Fuz</i> null Meckel's cartilage (bottom panel), compared to the heterozygote (<i>Fuz</i>^+/−, top panel) samples. <b>C</b>) Lef-1 expression in the dental epithelium, oral and dental mesenchyme at E14.5. Lef-1 expression increased in the <i>Fuz</i> null oral mesenchyme (bottom panel). <b>D</b>) These are higher magnification of the boxed areas in C. Lef-1 expression was expanded in the <i>Fuz^−/−</i> mice oral mesenchyme. <b>E</b>) The expression of <i>Tcf4</i> (<i>Tcf7l2</i>) was increased in <i>Fuz</i> mutant Meckel's cartilage (bottom panels) at E14.5 compared with wild type samples (top panels). <b>F</b>) Real-time PCR with mRNA from dissected E14.5 Meckel's cartilage and surrounding mesenchyme. Canonical Wnt target gene expression was increased whereas non-canonical Wnt pathway gene expression was decreased. β-actin served as the reference gene. Experiments were repeated three to five times each from multiple samples. <b>G</b>) Topflash reporter activity was repressed by co-transfection of <i>Fuz</i> in HEK 293FT and CHO cells. The activities are shown as mean fold activation compared to reporter activation co-transfected with pcDNA3.1 empty vector and normalized to SV-40 β-galactosidase activity. Error bars indicate S.E. *: <i>p</i>-values<0.05; **: <i>p</i>-values<0.01.</p

The cilium defect in the <i>Fuz</i> null mouse.

<p><b>A</b>) The primary cilia are shown by immunofluorescence with an Arl13b antibody in mandible mesenchyme at E14.5. <b>B</b>) The amount of cilia in the sagittal sections of <i>Fuz^−/−</i> mandible mesenchyme was quantitated and compared to wild type mandible mesenchyme. Error bars indicate S.E., n = 8, <i>p</i><0.01.</p