A single amino acid mutation in SNAP-25 induces anxiety-related behavior in mouse.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a presynaptic protein essential for neurotransmitter release. Previously, we demonstrate that protein kinase C (PKC) phosphorylates Ser(187) of SNAP-25, and enhances neurotransmitter release by recruiting secretory vesicles near to the plasma membrane. As PKC is abundant in the brain and SNAP-25 is essential for synaptic transmission, SNAP-25 phosphorylation is likely to play a crucial role in the central nervous system. We therefore generated a mutant mouse, substituting Ser(187) of SNAP-25 with Ala using "knock-in" technology. The most striking effect of the mutation was observed in their behavior. The homozygous mutant mice froze readily in response to environmental change, and showed strong anxiety-related behavior in general activity and light and dark preference tests. In addition, the mutant mice sometimes exhibited spontaneously occurring convulsive seizures. Microdialysis measurements revealed that serotonin and dopamine release were markedly reduced in amygdala. These results clearly indicate that PKC-dependent SNAP-25 phosphorylation plays a critical role in the regulation of emotional behavior as well as the suppression of epileptic seizures, and the lack of enhancement of monoamine release is one of the possible mechanisms underlying these defects.

Coordinated regulation of differentiation and proliferation of embryonic cardiomyocytes by a jumonji (Jarid2)-cyclin D1 pathway.

In general, cell proliferation and differentiation show an inverse relationship, and are regulated in a coordinated manner during development. Embryonic cardiomyocytes must support embryonic life by functional differentiation such as beating, and proliferate actively to increase the size of the heart. Therefore, progression of both proliferation and differentiation is indispensable. It remains unknown whether proliferation and differentiation are related in these embryonic cardiomyocytes. We focused on abnormal phenotypes, such as hyperproliferation, inhibition of differentiation and enhanced expression of cyclin D1 in cardiomyocytes of mice with mutant jumonji (Jmj, Jarid2), which encodes the repressor of cyclin D1. Analysis of Jmj/cyclin D1 double mutant mice showed that Jmj was required for normal differentiation and normal expression of GATA4 protein through cyclin D1. Analysis of transgenic mice revealed that enhanced expression of cyclin D1 decreased GATA4 protein expression and inhibited the differentiation of cardiomyocytes in a CDK4/6-dependent manner, and that exogenous expression of GATA4 rescued the abnormal differentiation. Finally, CDK4 phosphorylated GATA4 directly, which promoted the degradation of GATA4 in cultured cells. These results suggest that CDK4 activated by cyclin D1 inhibits differentiation of cardiomyocytes by degradation of GATA4, and that initiation of Jmj expression unleashes the inhibition by repression of cyclin D1 expression and allows progression of differentiation, as well as repression of proliferation. Thus, a Jmj-cyclin D1 pathway coordinately regulates proliferation and differentiation of cardiomyocytes.

Three human ARX mutations cause the lissencephaly-like and mental retardation with epilepsy-like pleiotropic phenotypes in mice.

ARX (the aristaless-related homeobox gene) is a transcription factor that participates in the development of GABAergic and cholinergic neurons in the forebrain. Many ARX mutations have been identified in X-linked lissencephaly and mental retardation with epilepsy, and thus ARX is considered to be a causal gene for the two syndromes although the neurobiological functions of each mutation remain unclear. We attempted to elucidate the causal relationships between individual ARX mutations and disease phenotypes by generating a series of mutant mice. We generated three types of mice with knocked-in ARX mutations associated with X-linked lissencephaly (P353R) and mental retardation [P353L and 333ins(GCG)7]. Mice with the P355R mutation (equivalent to the human 353 position) that died after birth were significantly different in Arx transcript/protein amounts, GABAergic and cholinergic neuronal development, brain morphology and lifespan from mice with P355L and 330ins(GCG)7 but considerably similar to Arx-deficient mice with truncated ARX mutation in lissencephaly. Mice with the 330ins(GCG)7 mutation showed severe seizures and impaired learning performance, whereas mice with the P355L mutation exhibited mild seizures and only slightly impaired learning performance. Both types of mutant mice exhibited the mutation-specific lesser presence of GABAergic and cholinergic neurons in the striatum, medial septum and ventral forebrain nuclei when compared with wild-type mice. Present findings that reveal a causal relationship between ARX mutations and the pleiotropic phenotype in mice, suggest that the ARX-related syndrome, including lissencephaly or mental retardation, is caused by only the concerned ARX mutations without the involvement of other genetic factors.

Marked improvement of fertility of cryopreserved C57BL/6J mouse sperm by depletion of Ca2+ in medium.

Cryopreservation of mouse sperm is useful for maintaining various strains. However, fertility generally decreases after freezing. In particular, the fertility of cryopreserved C57BL/6J sperm is very low. To improve the fertility of frozen sperm, we examined the efficiencies of various media used for sperm preincubation (SP) and in vitro fertilization (IVF) in frozen C57BL/6J sperm. In this study, SP medium was examined for efficiency of fertility with respect to content, especially calcium (Ca(2+)), phosphate (PO(4)(3-)) and lactate. In all media containing no Ca(2+), including medium lacking Ca(2+), lacking Ca(2+) and PO(4)(3-), lacking Ca(2+) and lactate and lacking Ca(2+), PO(4)(3-) and lactate, high IVF rates were obtained (79, 69, 76 and 71%, respectively). On the other hand, the rates for media containing Ca(2+) were significantly lower (30-38%, P<0.05). After transfer, 41-50% of newborns were obtained in all media containing no Ca(2+). In conclusion, preincubation of thawed sperm in medium containing no Ca(2+) markedly improved the fertility of cryopreserved C57BL/6J sperm. These results indicate that the present method of IVF using medium with no Ca(2+) is practical for use in cryopreserved C57BL/6J sperm.

Paternal deletion of Meg1/Grb10 DMR causes maternalization of the Meg1/Grb10 cluster in mouse proximal Chromosome 11 leading to severe pre- and postnatal growth retardation.

Mice with maternal duplication of proximal Chromosome 11 (MatDp(prox11)), where Meg1/Grb10 is located, exhibit pre- and postnatal growth retardation. To elucidate the responsible imprinted gene for the growth abnormality, we examined the precise structure and regulatory mechanism of this imprinted region and generated novel model mice mimicking the pattern of imprinted gene expression observed in the MatDp(prox11) by deleting differentially methylated region of Meg1/Grb10 (Meg1-DMR). It was found that Cobl and Ddc, the neighboring genes of Meg1/Grb10, also comprise the imprinted region. We also found that the mouse-specific repeat sequence consisting of several CTCF-binding motifs in the Meg1-DMR functions as a silencer, suggesting that the Meg1/Grb10 imprinted region adopted a different regulatory mechanism from the H19/Igf2 region. Paternal deletion of the Meg1-DMR (+/DeltaDMR) caused both upregulation of the maternally expressed Meg1/Grb10 Type I in the whole body and Cobl in the yolk sac and loss of paternally expressed Meg1/Grb10 Type II and Ddc in the neonatal brain and heart, respectively, demonstrating maternalization of the entire Meg1/Grb10 imprinted region. We confirmed that the +/DeltaDMR mice exhibited the same growth abnormalities as the MatDp(prox11) mice. Fetal and neonatal growth was very sensitive to the expression level of Meg1/Grb10 Type I, indicating that the 2-fold increment of the Meg1/Grb10 Type I is one of the major causes of the growth retardation observed in the MatDp(prox11) and +/DeltaDMR mice. This suggests that the corresponding human GRB10 Type I plays an important role in the etiology of Silver-Russell syndrome caused by partial trisomy of 7p11-p13.

Role of retrotransposon-derived imprinted gene, Rtl1, in the feto-maternal interface of mouse placenta.

Eutherian placenta, an organ that emerged in the course of mammalian evolution, provides essential architecture, the so-called feto-maternal interface, for fetal development by exchanging nutrition, gas and waste between fetal and maternal blood. Functional defects of the placenta cause several developmental disorders, such as intrauterine growth retardation in humans and mice. A series of new inventions and/or adaptations must have been necessary to form and maintain eutherian chorioallantoic placenta, which consists of capillary endothelial cells and a surrounding trophoblast cell layer(s). Although many placental genes have been identified, it remains unknown how the feto-maternal interface is formed and maintained during development, and how this novel design evolved. Here we demonstrate that retrotransposon-derived Rtl1 (retrotransposon-like 1), also known as Peg11 (paternally expressed 11), is essential for maintenance of the fetal capillaries, and that both its loss and its overproduction cause late-fetal and/or neonatal lethality in mice.

Functions of a jumonji-cyclin D1 pathway in the coordination of cell cycle exit and migration during neurogenesis in the mouse hindbrain.

During development of the mouse central nervous system (CNS), most neural progenitor cells proliferate in the ventricular zone (VZ). In many regions of the CNS, neural progenitor cells give rise to postmitotic neurons that initiate neuronal differentiation and migrate out of the VZ to the mantle zone (MZ). Thereafter, they remain in a quiescent state. Here, we found many ectopic mitotic cells and cell clusters expressing neural progenitor or proneural marker genes in the MZ of the hindbrain of jumonji (jmj) mutant embryos. When we examined the expression of cyclin D1, which is repressed by jmj in the repression of cardiac myocyte proliferation, we found many ectopic clusters expressing both cyclin D1 and Musashi 1 in the MZ of mutant embryos. jmj is mainly expressed in the cyclin D1 negative region in the hindbrain, and cyclin D1 expression in the VZ was upregulated in jmj mutant mice. In jmj and cyclin D1 double mutant mice, the ectopic mitosis and formation of the abnormal clusters in the MZ were rescued. These results suggest that a jmj-cyclin D1 pathway is required for the precise coordination of cell cycle exit and migration during neurogenesis in the mouse hindbrain.

Versatile roles of R-Ras GAP in neurite formation of PC12 cells and embryonic vascular development.

Ras GTPase-activating proteins (GAP) are negative regulators of Ras that convert active Ras-GTP to inactive Ras-GDP. R-Ras GAP is a membrane-associated molecule with stronger GAP activity for R-Ras, an activator of integrin, than H-Ras. We found that R-Ras GAP is down-regulated during neurite formation in rat pheochromocytoma PC12 cells by nerve growth factor (NGF), which is blocked by the transient expression of R-Ras gap or dominant negative R-ras cDNA. By establishing a PC12 subclone that stably expresses exogenous R-Ras GAP, it was found that NGF reduced endogenous R-Ras GAP but not exogenous R-Ras GAP, suggesting that down-regulation of R-Ras GAP occurs at the transcription level. To clarify the physiological role of R-Ras GAP, we generated mice that express mutant Ras GAP with knocked down activity. While heterozygotes are normal, homozygous mice die at E12.5-13.5 of massive subcutaneous and intraparenchymal bleeding, probably due to underdeveloped adherens junctions between capillary endothelial cells. These results show essential roles of R-Ras GAP in development and differentiation: its expression is needed for embryonic development of blood vessel barriers, whereas its down-regulation facilitates NGF-induced neurite formation of PC12 cells via maintaining activated R-Ras.

Cryoprotective effects of various saccharides on cryopreserved mouse sperm from various strains.

Aim: Cryopreservation of mouse sperm commonly uses raffinose, which is a trisaccharide, plus 3% skim milk. Because of the present lack of knowledge of the effectiveness of any other saccharides, we examined the cryoprotective effects of various saccharides on the viability of mouse sperm from various strains to determine which saccharides are the best cryoprotectants for mouse sperm. Methods: Sperm from the caudae epididymides of mature C57BL/6J mice were frozen with monosaccharides (fructose, glucose, rhamnose, xylose), disaccharides (lactose, maltose, sucrose, trehalose) or trisaccharides (melezitose, raffinose) in a range of concentrations (4-33%). After thawing, the optimal concentration was determined to be the concentration in which there was the highest proportion of motile sperm. In addition, sperm of inbred and hybrid mice were frozen with the saccharides at the optimal concentrations and used for in vitro fertilization. Results: The optimal concentration was 12% for the disaccharides and 18% for the trisaccharides. The fertility of all strains, except C57BL/6J, showed the best cryoprotective effects with maltose, melezitose and raffinose when compared with fresh sperm. Conclusion: Maltose, melezitose and raffinose have the best effects when used as a protectant for cryopreservation of mouse sperm. (Reprod Med Biol 2007; 6: 229-233).

Successful pregnancies after transplantation of frozen-thawed mouse ovaries into chimeric mice that received lethal-dose radiation.

OBJECTIVE: To study whether fecundity was recovered in mice into which umbilical cord blood cells (UCBCs) were transfused after lethal-dose radiation, followed by transplantation of frozen-thawed ovaries. DESIGN: Prospective basic research study. SETTING: Academic research laboratory. ANIMAL(S): Female C57BL/6 mice as recipients of UCBCs and ovaries, male B6C3F1 mice for mating, and green fluorescent protein (GFP)-transgenic mice: 18.5-day-old fetuses (-/+) for UCBCs and adult GFP mice (+/+) for ovarian tissues. INTERVENTION(S): The UCBCs were transfused into each irradiated mouse, with GFP+ ovaries transplanted 4 weeks later. The chimeric mice were mated 3 weeks after ovarian transplantation and were examined 14 to 16 weeks after the transfusion of UCBCs. MAIN OUTCOME MEASURE(S): Percentage of chimerism, number of GFP+ pups. RESULT(S): The percentage of chimerism in these mice tends to increase with the radiation dose. The recovery of fecundity was observed in the chimeric mice that were transplanted with fresh and previously vitrified ovaries after exposure to radiation. CONCLUSION(S): Even when the exposure dose of radiation administered as pretreatment is lethal, the fecundity of recipients can be maintained if their ovaries are cryopreserved before they are exposed to radiation.

Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice.

Autophagy is an intracellular bulk degradation process through which a portion of the cytoplasm is delivered to lysosomes to be degraded. Although the primary role of autophagy in many organisms is in adaptation to starvation, autophagy is also thought to be important for normal turnover of cytoplasmic contents, particularly in quiescent cells such as neurons. Autophagy may have a protective role against the development of a number of neurodegenerative diseases. Here we report that loss of autophagy causes neurodegeneration even in the absence of any disease-associated mutant proteins. Mice deficient for Atg5 (autophagy-related 5) specifically in neural cells develop progressive deficits in motor function that are accompanied by the accumulation of cytoplasmic inclusion bodies in neurons. In Atg5-/- cells, diffuse, abnormal intracellular proteins accumulate, and then form aggregates and inclusions. These results suggest that the continuous clearance of diffuse cytosolic proteins through basal autophagy is important for preventing the accumulation of abnormal proteins, which can disrupt neural function and ultimately lead to neurodegeneration.

Deletion of Peg10, an imprinted gene acquired from a retrotransposon, causes early embryonic lethality.

By comparing mammalian genomes, we and others have identified actively transcribed Ty3/gypsy retrotransposon-derived genes with highly conserved DNA sequences and insertion sites. To elucidate the functions of evolutionarily conserved retrotransposon-derived genes in mammalian development, we produced mice that lack one of these genes, Peg10 (paternally expressed 10), which is a paternally expressed imprinted gene on mouse proximal chromosome 6. The Peg10 knockout mice showed early embryonic lethality owing to defects in the placenta. This indicates that Peg10 is critical for mouse parthenogenetic development and provides the first direct evidence of an essential role of an evolutionarily conserved retrotransposon-derived gene in mammalian development.

Cardiac abnormalities cause early lethality of jumonji mutant mice.

jumonji (jmj) mutant mice, obtained by a gene trap strategy, showed several morphological abnormalities including neural tube and cardiac defects, and died in utero around embryonic day 11.5 (E11.5). It is unknown what causes the embryonic lethality. Here, we demonstrate that exogenous expression of jmj gene in the heart of jmj mutant mice rescued the morphological phenotypes in the heart, and these embryos survived until E13.5. These results suggest that there are at least two lethal periods in jmj mutant mice, and that cardiac abnormalities may cause the earlier lethality. In addition, the rescue of the cardiac abnormalities by the jmj transgene provided solid evidence that the cardiac abnormalities resulted from mutation of the jmj gene.

Laminar patterning in the developing neocortex by temporally coordinated fibroblast growth factor signaling.

Laminar organization, a fundamental neural architecture in the CNS, is a prominent feature of the neocortex, where the cortical neurons in spatially distinct layers are generated from the common progenitors in a temporally distinct manner during development. Despite many advances in the characterization of the molecular mechanisms of the radial migration of cortical neurons, the way in which the early-late temporal sequence of cortical neuron generation is linked with the deep-superficial spatial sequence of cell body positioning remains obscure. Using in vivo electroporation-mediated gene transfer, we show here that the activities mediated by fibroblast growth factor receptors (FGFRs) in cortical progenitors are critical for conferring proper migratory properties on nascent neuronal progeny. Furthermore, we provide supportive evidence that Pea3 subfamily members of Ets (Pea3-Ets) transcription factors mediate the activities of FGFR at the mid to late phase of neocortical development. In addition, using FGF18 knock-out mice, we demonstrate that FGF18 expressed by early-generated cortical neurons in the cortical plate is critical for the expression of Pea3-Ets transcription factors and that FGF18 is sufficient to induce their expressions. Our results thus imply that a feedback mechanism mediated by FGF signaling is involved in setting up the proper laminar positioning of cortical neurons; FGF18 derived from early-generated cortical neurons acts on the cortical progenitors expressing FGFRs and induces the expression of Pea3-Ets transcription factors that, in turn, confer proper migratory behaviors on nascent cortical progeny during the mid to late stages of neocortical development.

Generation of reelin-positive marginal zone cells from the caudomedial wall of telencephalic vesicles.

An early and fundamental step of the laminar organization of developing neocortex is controlled by the developmental programs that critically depend on the activities of reelin-positive cells in the marginal zone. However, the ontogeny of reelin-positive cells remained elusive. To gain insights into the spatial and temporal regulation of reelin-positive marginal zone cell development, we used a transgenic mouse line in which we defined the green fluorescent protein (GFP) transgene as a novel reliable molecular marker of reelin-positive marginal zone cells from the early stages of their development. We further used exo utero electroporation-mediated gene transfer that allows us to mark progenitor cells and monitor the descendants in the telencephalon in vivo. We show here the generation of reelin-positive marginal zone cells from the caudomedial wall of telencephalic vesicles, including the cortical hem, where the prominent expression of GFP is initially detected. These neurons tangentially migrate at the cortical marginal zone and are distributed throughout the entire neocortex in a caudomedial-high to rostrolateral-low gradient during the dynamic developmental period of corticogenesis. Therefore, our findings on reelin-positive marginal zone cells, in addition to the cortical interneurons, add to the emerging view that the neocortex consists of neuronal subtypes that originate from a focal source extrinsic to the neocortex, migrate tangentially into the neocortex, and thereby underlie neural organization of the neocortex.

jumonji downregulates cardiac cell proliferation by repressing cyclin D1 expression.

Spatiotemporal regulation of cell proliferation is necessary for normal tissue development. The molecular mechanisms, especially the signaling pathways controlling the cell cycle machinery, remain largely unknown. Here, we demonstrate a negative relationship between the spatiotemporal patterns of jumonji (jmj) expression and cardiac myocyte proliferation. cyclin D1 expression and cell proliferation are enhanced in the cardiac myocytes of jmj-deficient mutant embryos. In contrast, jmj overexpression represses cyclin D1 expression in cardiac cells, and Jmj protein binds to cyclin D1 promoter in vivo and represses its transcriptional activity. cyclin D1 overexpression causes hyperproliferation in the cardiac myocytes, but the absence of cyclin D1 in jmj mutant embryos rescues the hyperproliferation. Therefore, Jmj might control cardiac myocyte proliferation and consequently cardiac morphogenesis by repressing cyclin D1 expression.

Successful cryopreservation of mouse ovaries by vitrification.

We developed a new method of cryopreservation of whole ovaries by vitrification using DAP213 (2 M dimethyl sulfoxide, 1 M acetamide, and M propylene glycol) as a cryoprotectant. Four-week-old C57BL/6 mice that underwent partial ovariectomy were orthotopically transplanted with cryopreserved or fresh ovaries (experimental or control group) isolated from 10-day-old green fluorescent protein (GFP)-transgenic mice (+/+). GFP-positive pups were similarly obtained from both groups by natural mating or in vitro fertilization (IVF) followed by embryo transfer, indicating that the cryopreserved ovaries by vitrification retain their fecundity. However, a statistically significant difference (P < 0.05) was found between both groups with respect to the following parameters: the number of GFP-positive pups born by natural mating/grafted ovary (0.8 +/- 0.3 for the experimental group versus 2.0 +/- 0.7 for the control group, mean +/- SEM), the number of collected oocytes by superovulation per mouse (7.0 +/- 1.7 for the experimental group versus 22.7 +/- 3.2 for the control group), the percentage of two-cell embryos obtained from GFP-positive oocytes by IVF (38.5% for the experimental group versus 90.0% for the control group). Histologically, normal development of follicles and formation of corpora lutea were observed in frozen-thawed grafts. However, estimated number of follicles decreased in frozen-thawed ovaries compared with fresh ovaries. Taken together, cryopreservation of the ovary by vitrification seems a promising method to preserve ovarian function, but further studies are required to overcome the possible inhibitory effects of this method on the growth of the ovarian graft.