Munekazu Komada

Elevated Plus Maze for Mice

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given gene on a specific behavior can be determined by behavioral analysis of mutant mice. If a target gene is expressed in the brain, behavioral phenotype of the mutant mice could elucidate the genetic mechanism of normal behaviors. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior. The test is based on the natural aversion of mice for open and elevated areas, as well as on their natural spontaneous exploratory behavior in novel environments. The apparatus consists of open arms and closed arms, crossed in the middle perpendicularly to each other, and a center area. Mice are given access to all of the arms and are allowed to move freely between them. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced anxiety in mice. Unfortunately, the procedural differences that exist between laboratories make it difficult to duplicate and compare results among laboratories. Here, we present a detailed movie demonstrating our protocol for the elevated plus maze test. In our laboratory, we have assessed more than 90 strains of mutant mice using the protocol shown in the movie. These data will be disclosed as a part of a public database that we are now constructing. Visualization of the protocol will promote better understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used in different laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

Hedgehog signaling is involved in development of the neocortex

Sonic hedgehog (Shh) function is essential for patterning and cell fate specification, particularly in ventral regions of the central nervous system. It is also a crucial mitogen for cerebellar granule neuron precursors and is important in maintenance of the stem cell niche in the postnatal telencephalon. Although it has been reported that Shh is expressed in the developing dorsal telencephalon, functions of Shh in this region are unclear, and detailed characterization of Shh mRNA transcripts in situ has not been demonstrated. To clarify the roles of Shh signaling in dorsal pallium (neocortex primordium) development, we have knocked out the Shh and Smo genes specifically in the early developing dorsal telencephalon by using Emx1cre mice. The mutants showed a smaller dorsal telencephalon at E18.5, which was caused by cell cycle kinetics defects of the neural progenitor/stem cells. The cell cycle length of the progenitor/stem cells was prolonged, and the number of cycle-exiting cells and neurogenesis were decreased. Birth-date analysis revealed abnormal positioning of neurons in the mutants. The characteristics of the subventricular zone, ventricular zone and subplate cells were also affected. Weak immunoreactivity of Shh was detected in the dorsal telencephalon of wild types. Reduced Shh immunoreactivity in mutant dorsal telencephalons supports the above phenotypes. Our data indicate that Shh signaling plays an important role in development of the neocortex.

Mice with Altered Myelin Proteolipid Protein Gene Expression Display Cognitive Deficits Accompanied by Abnormal Neuron–Glia Interactions and Decreased Conduction Velocities

Conduction velocity (CV) of myelinated axons has been shown to be regulated by oligodendrocytes even after myelination has been completed. However, how myelinating oligodendrocytes regulate CV, and what the significance of this regulation is for normal brain function remain unknown. To address these questions, we analyzed a transgenic mouse line harboring extra copies of the myelin proteolipid protein 1 (plp1) gene (plp1 tg/− mice) at 2 months of age. At this stage, the plp1 tg/− mice have an unaffected myelin structure with a normally appearing ion channel distribution, but the CV in all axonal tracts tested in the CNS is greatly reduced. We also found decreased axonal diameters and slightly abnormal paranodal structures, both of which can be a cause for the reduced CV. Interestingly the plp1 tg/− mice showed altered anxiety-like behaviors, reduced prepulse inhibitions, spatial learning deficits and working memory deficit, all of which are schizophrenia-related behaviors. Our results implicate that abnormalities in the neuron-glia interactions at the paranodal junctions can result in reduced CV in the CNS, which then induces behavioral abnormalities related to schizophrenia.

Maternal bisphenol A oral dosing relates to the acceleration of neurogenesis in the developing neocortex of mouse fetuses.

Bisphenol A (BPA), an endocrine-disruptor, is widely used in the production of plastics and resins. Human perinatal exposure to this chemical has been proposed to be a potential risk to public health. Animal studies indicate that postnatal exposure to BPA may affect neocortex development in embryos by accelerated neurogenesis and causing neuronal migration defects. The detailed phenotypes and pathogenetic mechanisms, especially with regard to the proliferation and differentiation of neural stem/progenitor cells, however, have not been clarified. C57BL/6J pregnant mice were orally administered BPA at 200μg/kg from embryonic day (E) 8.5 to 13.5, and the fetuses were observed histologically at E14.5. To clarify the histological changes, especially in terms of neurogenesis, proliferation and cell cycle, we performed histological analysis using specific markers of neurons/neural stem cells and cell cycle-specific labeling experiments using thymidine-analog substances. Cortical plate was hyperplastic and the number of neural stem/progenitor cells was decreased after the exposure to BPA. In particular, the maternal BPA oral dosing related to the effects on intermediate progenitor cells (IPCs, neural progenitor cells) in the subventricular zone (SVZ) of dorsal telencephalon. Exposure to BPA associated the promotion of the cell cycle exit in radial glial cells (RGCs, neural stem cells) and IPCs, and decreased the proliferation resulting from the prolong cell cycle length of IPCs in the SVZ. Our data show that maternal oral exposure to BPA related to the disruption of the cell cycle in IPCs and the effects of neurogenesis in the developing neocortex.

The cyst-branch difference in developing chick lung results from a different morphogen diffusion coefficient.

The developing avian lung is formed mainly by branching morphogenesis, but there is also a unique cystic structure, the air sac, in the ventral region. It has been shown that mesenchymal tissue is responsible for the differential development of a cystic or branched structure, and that the transcription factor Hoxb may be involved in determining this regional difference. We have previously developed two scenarios for branch-cyst transition, both experimentally and theoretically: increased production or increased diffusion of FGF. The aim of the present study was to discover whether one of these scenarios actually operates in the ventral region of the chick lung. We found that the FGF10 level was lower while the diffusion of FGF10 was more rapid in the ventral lung, indicating that the second scenario is more plausible. There are two possibilities as to why the diffusion of FGF10 differs between the two regions: (1) diffusion is facilitated by the looser tissue organisation of the ventral lung mesenchyme; (2) stronger expression of heparan sulphate proteoglycan (HSPG) in the dorsal lung traps FGF and decreases the effective diffusion coefficient. Mathematical analysis showed that the dorsal-ventral difference in the amount of HSPG is not sufficient to generate the observed difference in pattern, indicating that both extracellular matrix and tissue architecture play a role in this system. These results suggest that the regional cystic-branched difference within the developing chick lung results from a difference in the rate of diffusion of morphogen between the ventral and dorsal regions due to differential levels of HSPG and a different mesenchymal structure.

Newborn mice exposed prenatally to bisphenol A show hyperactivity and defective neocortical development

The central nervous system is especially susceptible to toxic insults during development. Prenatal administration of bisphenol A (BPA) induces histologic anomalies in the dorsal telencephalon of the embryo. Whether these anomalies affect the morphogenesis and maturation of neuronal function of the newborn neocortex, however, is unknown. To evaluate the neurodevelopmental and behavioral effects of prenatal BPA exposure at 20 and 200μg/kg/day in newborn mice, we performed a detailed histologic analysis of the neocortex and tested for the presence of behavioral abnormalities in newborn mice prenatally exposed to BPA using our newly developed behavioral test. Observations of newborn mice prenatally exposed to BPA revealed abnormal neuronal distribution and layer formation, hypoplasia of layer 6b, and abnormal dopaminergic neuronal projections in the neocortex. Further, the newborn mice exhibited hyperactivity. These findings suggest that prenatal BPA exposure induces neurobehavioral toxicity associated with abnormal dopaminergic neuronal projections, and abnormal corticogenesis and lamination. Histologic and behavioral analyses of newborn mice are considered useful for assessing the neurodevelopmental and behavioral toxicity of chemicals.

Expression of the mouse Fgf15 gene is directly initiated by sonic hedgehog signaling in the diencephalon and midbrain

Sonic hedgehog (Shh) is a secreted molecule that is thought to regulate tissue growth and patterning in vertebrate embryos. Although it has been reported that Gli transcription factors mediate Shh signaling to the nucleus, little is known about developmental target genes of Gli. In the previous genetic study, we showed that Shh is required for Fgf15 expression in the diencephalon and midbrain. Here, we examined whether Fgf15 is a direct target of Shh signaling through Gli. Shh was expressed in the midline cells and Fgf15 in the medial region of the diencephalon/midbrain by the seven‐somite stage. The Fgf15 expression domain coincided with that of Gli1 and overlapped with that of Gli2 at this stage. Fgf15 expression in the diencephalon/midbrain was greatly reduced in the seven‐somite Shh mutant embryos. Transgenic analysis showed that the 3.6‐kb 5′‐flanking region of the Fgf15 gene is sufficient for induction of Fgf15 in the medial/ventral diencephalon/midbrain. Luciferase assay showed that the 3.6‐kb Fgf15 enhancer/promoter was activated by Gli2. A Gli‐binding site was located 1 kb upstream of the transcription start site and was required for expression in the medial/ventral diencephalon/midbrain in transgenic embryos and for activation in luciferase assay. These findings indicate that Fgf15 is directly regulated by Shh signaling through Gli proteins. Developmental Dynamics 232:282–292, 2005.

Sonic hedgehog signaling coordinates the proliferation and differentiation of neural stem/progenitor cells by regulating cell cycle kinetics during development of the neocortex

Sonic hedgehog (Shh) acts as a morphogen in normal development of various vertebrate tissues and organs. Shh signaling is essential for patterning and cell‐fate specification, particularly in the central nervous system. Shh signaling plays different roles depending on its concentration, area, and timing of exposure. During the development of the neocortex, a low level of Shh is expressed in the neural stem/progenitor cells as well as in mature neurons in the dorsal telencephalon. Shh signaling in neocortex development has been shown to regulate cell cycle kinetics of radial glial cells and intermediate progenitor cells, thereby maintaining the proliferation, survival and differentiation of neurons in the neocortex. During the development of the telencephalon, endogenous Shh signaling is involved in the transition of slow‐cycling neural stem cells to fast‐cycling neural progenitor cells. It seems that high‐level Shh signaling in the ventral telencephalon is essential for ventral specification, while low‐level Shh signaling in the dorsal telencephalon plays important roles in the fine‐tuning of cell cycle kinetics. The Shh levels and multiple functions of Shh signaling are important for proper corticogenesis in the developing brain. The present paper discusses the roles of Shh signaling in the proliferation and differentiation of neural stem/progenitor cells.

Embryogenesis of holoprosencephaly.

Holoprosencephaly (HPE) is a malformation of the human brain caused primarily by incomplete division of the prosencephalon into two halves and is often associated with various facial anomalies. Although HPE is rather rare in newborns (1/10,000–15,000 births), it is frequently encountered in therapeutic abortuses (>1/250). To date, nine gene mutations responsible for human HPE have been identified, but the pathogenetic mechanisms of the craniofacial anomalies in HPE have just begun to be understood. Here, we summarize our studies on human embryos with HPE and discuss the embryogenesis and the underlying molecular mechanisms of HPE malformations under the following headings: pathology, pathogenesis, and critical period of development.

Smoothened controls cyclinD2 expression and regulates the generation of intermediate progenitors in the developing cortex

Translocation of the Smoothened to the cell membrane is critical for sonic hedgehog activity. However, the biological importance of Smoothened itself has not been fully studied. To address this issue, we disabled Smoothened specifically in the dorsal telencephalon. Birth-date analysis and layer marker expression patterns revealed the slightly impaired development of the superficial layer neurons in the embryos of Emx1-Cre; Smoothened(fl/-) conditional knockout mice. Further analysis of the mutant embryos revealed a decrease in the number of intermediate progenitor cells. In the knockout mice, the expression of cyclin D2, but not cyclin D1 or cyclin E, was reduced in the dorsal telencephalon. In addition, the projections of dopaminergic neurons were affected during development, and the number of activated astrocytes was increased in the neocortex of the mutant mice. Our data suggest that Smoothened signaling, acting through cyclin D2, is critical for the proper development and maturation of the neocortex.