Toshiaki Hino

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.

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.

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.

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.

Requirement of the immediate early gene vesl-1S/homer-1a for fear memory formation

BackgroundThe formation of long-term memory (LTM) and the late phase of long-term potentiation (L-LTP) depend on macromolecule synthesis, translation, and transcription in neurons. vesl-1S (V ASP/E na-related gene upregulated during s eizure and L TP, also known as homer-1a) is an LTP-induced immediate early gene. The short form of Vesl (Vesl-1S) is an alternatively spliced isoform of the vesl-1 gene, which also encodes the long form of the Vesl protein (Vesl-1L). Vesl-1L is a postsynaptic scaffolding protein that binds to and modulates the metabotropic glutamate receptor 1/5 (mGluR1/5), the IP3 receptor, and the ryanodine receptor. Vesl-1 null mutant mice show abnormal behavior, which includes anxiety- and depression-related behaviors, and an increase in cocaine-induced locomotion; however, the function of the short form of Vesl in behavior is poorly understood because of the lack of short-form-specific knockout mice.ResultsIn this study, we generated short-form-specific gene targeting (KO) mice by knocking in part of vesl-1L/homer-1c cDNA. Homozygous KO mice exhibited normal spine number and morphology. Using the contextual fear conditioning test, we demonstrated that memory acquisition and short-term memory were normal in homozygous KO mice. In contrast, these mice showed impairment in fear memory consolidation. Furthermore, the process from recent to remote memory was affected in homozygous KO mice. Interestingly, reactivation of previously consolidated fear memory attenuated the conditioning-induced freezing response in homozygous KO mice, which suggests that the short form plays a role in fear memory reconsolidation. General activity, emotional performance, and sensitivity to electrofootshock were normal in homozygous KO mice.ConclusionThese results indicate that the short form of the Vesl family of proteins plays a role in multiple steps of long-term, but not short-term, fear memory formation.

Activin plays a key role in the maintenance of long-term memory and late-LTP

A recent study has revealed that fear memory may be vulnerable following retrieval, and is then reconsolidated in a protein synthesis-dependent manner. However, little is known about the molecular mechanisms of these processes. Activin betaA, a member of the TGF-beta superfamily, is increased in activated neuronal circuits and regulates dendritic spine morphology. To clarify the role of activin in the synaptic plasticity of the adult brain, we examined the effect of inhibiting or enhancing activin function on hippocampal long-term potentiation (LTP). We found that follistatin, a specific inhibitor of activin, blocked the maintenance of late LTP (L-LTP) in the hippocampus. In contrast, administration of activin facilitated the maintenance of early LTP (E-LTP). We generated forebrain-specific activin- or follistatin-transgenic mice in which transgene expression is under the control of the Tet-OFF system. Maintenance of hippocampal L-LTP was blocked in the follistatin-transgenic mice. In the contextual fear-conditioning test, we found that follistatin blocked the formation of long-term memory (LTM) without affecting short-term memory (STM). Furthermore, consolidated memory was selectively weakened by the expression of follistatin during retrieval, but not during the maintenance phase. On the other hand, the maintenance of memory was also influenced by activin overexpression during the retrieval phase. Thus, the level of activin in the brain during the retrieval phase plays a key role in the maintenance of long-term memory.

Sirh7/Ldoc1 knockout mice exhibit placental P4 overproduction and delayed parturition

Sirh7/Ldoc1 [sushi-ichi retrotransposon homolog 7/leucine zipper, downregulated in cancer 1, also called mammalian retrotransposon-derived 7 (Mart7)] is one of the newly acquired genes from LTR retrotransposons in eutherian mammals. Interestingly, Sirh7/Ldoc1 knockout (KO) mice exhibited abnormal placental cell differentiation/maturation, leading to an overproduction of placental progesterone (P4) and placental lactogen 1 (PL1) from trophoblast giant cells (TGCs). The placenta is an organ that is essential for mammalian viviparity and plays a major endocrinological role during pregnancy in addition to providing nutrients and oxygen to the fetus. P4 is an essential hormone in the preparation and maintenance of pregnancy and the determination of the timing of parturition in mammals; however, the biological significance of placental P4 in rodents is not properly recognized. Here, we demonstrate that mouse placentas do produce P4 in mid-gestation, coincident with a temporal reduction in ovarian P4, suggesting that it plays a role in the protection of the conceptuses specifically in this period. Pregnant Sirh7/Ldoc1 knockout females also displayed delayed parturition associated with a low pup weaning rate. All these results suggest that Sirh7/Ldoc1 has undergone positive selection during eutherian evolution as a eutherian-specific acquired gene because it impacts reproductive fitness via the regulation of placental endocrine function.

Hippocampal function is not required for the precision of remote place memory.

BackgroundDuring permanent memory formation, recall of acquired place memories initially depends on the hippocampus and eventually become hippocampus-independent with time. It has been suggested that the quality of original place memories also transforms from a precise form to a less precise form with similar time course. The question arises of whether the quality of original place memories is determined by brain regions on which the memory depends.ResultsTo directly test this idea, we introduced a new procedure: a non-associative place recognition memory test in mice. Combined with genetic and pharmacological approaches, our analyses revealed that place memory is precisely maintained for 28 days, although the recall of place memory shifts from hippocampus-dependent to hippocampus-independent with time. Moreover, the inactivation of the hippocampal function does not inhibit the precision of remote place memory.ConclusionThese results indicate that the quality of place memories is not determined by brain regions on which the memory depends.

Motor discoordination of transgenic mice overexpressing a microtubule destabilizer, stathmin, specifically in Purkinje cells

The proper regulation of microtubule (MT) structure is important for dendritic and neural circuit development. However, the relationship between the regulation of the MTs in dendrites and the formation of neural function is still unclear. Stathmin is a MT destabilizer, and we have previously reported that the expression and the activity of stathmin is downregulated during cerebellar Purkinje cell (PC) development. In this study, we generated transgenic mice that specifically overexpress the constitutively active form of stathmin in the PCs. These mutant mice did not show any obvious morphological or excitatory transmission abnormalities in the cerebellum. In contrast, we observed a decline in the expression of MAP2 and KIF5 signal in the PC dendrites and a discoordination of motor function in the mutant mice, although they displayed normal general behavior. These data indicate that the overexpression of stathmin disrupts dendritic MT organization, motor protein distribution, and neural function in PCs.

PolyADP-Ribosylation Is Required for Pronuclear Fusion during Postfertilization in Mice

Background During fertilization, pronuclear envelope breakdown (PNEB) is followed by the mingling of male and female genomes. Dynamic chromatin and protein rearrangements require posttranslational modification (PTM) for the postfertilization development. Methodology/Principal Findings Inhibition of poly(ADP-ribose) polymerase activity (PARylation) by either PJ-34 or 5-AIQ resulted in developmental arrest of fertilized embryos at the PNEB. PARylation inhibition affects spindle bundle formation and phosphorylation of Erk molecules of metaphase II (MII) unfertilized oocytes. We found a frequent appearance of multiple pronuclei (PN) in the PARylation-inhibited embryos, suggesting defective polymerization of tubulins. Attenuated phosphorylation of lamin A/C by PARylation was detected in the PARylation-inhibited embryos at PNEB. This was associated with sustained localization of heterodomain protein 1 (HP1) at the PN of the one-cell embryos arrested by PARylation inhibition. Conclusions/Significance Our findings indicate that PARylation is required for pronuclear fusion during postfertilization processes. These data further suggest that PARylation regulates protein dynamics essential for the beginning of mouse zygotic development. PARylation and its involving signal-pathways may represent potential targets as contraceptives.