Here we propose a new protocol for whole-mount bone staining, which allows the rapid preparation of highly cleared and nondestructive specimens. It only takes 3 days to complete whole procedure for small vertebrates, such as medaka, zebrafish, and Xenopus frogs. In this procedure, we used a newly developed fixative containing formalin, Triton X-100, and potassium hydroxide, which allows the fixation, decolorization, and transparentization of specimens at the same time. A bone staining solution containing alizarin red S with ethylene glycol and a clearing solution containing Tween 20 and potassium hydroxide also contributed the specificity and swiftness of this new system. As expected, although details of the skeletal system could be observed in specimens with high transparency, it was noteworthy that high-resolution fluorescence images acquired using zoom microscopes clearly delineated the shape of each bone. This new procedure would be expected to be widely used as a standard procedure for bone staining in the testing the developmental toxicity of chemicals and in the screening test of knockout or mutant animals.

Recent studies in rodents suggest that maternal immune activation (MIA) by viral infection is associated with schizophrenia and autism in offspring. Although maternal IL-6 is though t to be a possible mediator relating MIA induced these neuropsychiatric disorders, the mechanism remains to be elucidated. Previously, we reported that the maternal leukemia inhibitory factor (LIF)–placental ACTH–fetal LIF signaling relay pathway (maternal–fetal LIF signal relay) promotes neurogenesis of fetal cerebrum in rats. Here we report that the maternal–fetal LIF signal relay in mice is suppressed by injection of polyriboinosinic-polyribocytidylic acid into dams, which induces MIA at 12.5 days post-coitum. Maternal IL-6 levels and gene expression of placental suppressor of cytokine signaling 3 (Socs3) increased according to the severity of MIA and gene expression of placental Socs3 correlated with maternal IL-6 levels. Furthermore, we show that MIA causes reduction of LIF level in the fetal cerebrospinal fluid, resulting in the decreased neurogenesis in the cerebrum. These findings suggest that maternal IL-6 interferes the maternal–fetal LIF signal relay by inducing SOCS3 in the placenta and leads to decreased neurogenesis.

The rapid rise in the prevalence of autism spectrum disorders (ASD) and other psychiatric disorders displaying similar traits has increased the need to elucidate their molecular mechanisms.Epidemiological studies have shown that maternal infection during mid-pregnancy is associated with increased risk of neurodevelopmental disorders such as ASD in offspring. Using maternal infection models, researchers have gathered evidence relevant to such disorders. A comprehensive summary of the changes in the brain structure, function, and behavior in offspring induced by maternal immune activation (MIA) has been reported. However, the molecular mechanisms underlying the association between MIA and improper brain development, which ultimately lead to neurodevelopmental disorders, have not been fully reviewed. This paper summarizes the currently known molecular mechanisms associated with the MIA model, with a special focus on the role of the placenta in fetal brain development.K E Y W O R D S fetal brain development, interleukin-6 (IL-6) family signaling pathway, maternal immune activation (MIA), neurodevelopmental disorders, placenta

Maternal immune activation (MIA) in midpregnancy is a risk factor for neurodevelopmental disorders. Improper brain development may cause malformations of the brain; maldevelopment induced by MIA may lead to a pathology-related phenotype. In this study, a single intraperitoneal injection of 20 mg/kg polyriboinosinic–polyribocytidylic acid [poly(I:C)] was administered to C57BL/6J mice on embryonic day (E) 12.5 to mimic maternal viral infection. Histopathological analysis of neurogenesis was performed using markers for Pax6, Tbr2, and Tbr1. In these fetuses, significant increases were observed in the proportion of Pax6-positive neural progenitor cells and Pax6/Tbr2 double-positive cells 24 h after poly(I:C) injection. There were no differences in the proportion of Tbr1-positive postmitotic neurons 48 h after poly(I:C) injection. At E18.5, there were more Pax6-positive and Tbr2-positive neural progenitor cells in the poly(I:C)-injected group than in the saline-injected group. Gene ontology enrichment analysis of poly(I:C)-induced differentially expressed genes in the fetal brain at E12.5 demonstrated that these genes were enriched in terms including response to cytokine, response to decreased oxygen levels in the category of biological process. At E13.5, activating transcription factor 4 (Atf4), which is an effector of integrated stress response, was significantly upregulated in the fetal brain. Our results show that poly(I:C)-induced MIA at E12.5 leads to dysregulated neurogenesis and upregulates Atf4 in the fetal brain. These findings provide a new insight in the mechanism of MIA causing improper brain development and subsequent neurodevelopmental disorders.

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