CD9 is an integral membrane protein associated with integrins and other membrane proteins. Mice lacking CD9 were produced by homologous recombination. Both male and female CD9-/- mice were born healthy and grew normally. However, the litter size from CD9-/- females was less than 2% of that of the wild type. In vitro fertilization experiments indicated that the cause of this infertility was due to the failure of sperm-egg fusion. When sperm were injected into oocytes with assisted microfertilization techniques, however, the fertilized eggs developed to term. These results indicate that CD9 has a crucial role in sperm-egg fusion.

Preeclampsia is a relatively common pregnancy-related disorder. Both maternal and fetal lives will be endangered if it proceeds unabated. Recently, the placenta-derived anti-angiogenic factors, such as soluble fms-like tyrosine kinase-1 (sFLT1) and soluble endoglin (sENG), have attracted attention in the progression of preeclampsia. Here, we established a unique experimental model to test the role of sFLT1 in preeclampsia using a lentiviral vector-mediated placenta-specific expression system. The model mice showed hypertension and proteinuria during pregnancy, and the symptoms regressed after parturition. Intrauterine growth restriction was also observed. We further showed that pravastatin induced the VEGF-like angiogenic factor placental growth factor (PGF) and ameliorated the symptoms. We conclude that our experimental preeclamptic murine model phenocopies the human case, and the model identifies low-dose statins and PGF as candidates for preeclampsia treatment.3-hydroxy-3-methyl-glutaryl-CoA | hemolytic anemia, elevated liver enzymes, and low platelet count | trophoblast P reeclampsia originates from placental insufficiency and is observed in ≈5% of pregnant females (1). Although preeclampsia is a major cause of maternal and infantile morbidity and mortality worldwide, the fundamental therapy available is to terminate the pregnancy. Therefore understanding the pathogenesis of preeclampsia is highly important and the generation of an appropriate animal model would be helpful to develop therapeutic drugs. In preeclamptic women, Maynard et al. found that increased soluble fms-like tyrosine kinase-1 (sFLT1) associated with decreased circulating levels of free VEGF and placental growth factor (PGF) (2). Because sFLT1 is a truncated form of VEGF receptor 1 and antagonizes VEGF and PGF function, impaired angiogenic signaling most likely results in endothelial dysfunction and preeclampsia (3).To establish an animal model of preeclampsia, placenta-specific gene manipulation is preferable because preeclampsia originates from a failure in placentation, and factors up-/ or down-regulated in the impaired placenta accentuate the symptoms. However, the lack of a facile and efficient method for placenta-specific gene manipulation has hampered the investigation of preeclampsia. The systemic administration of adenoviral vector (AdV-) expressing sFLT1 into pregnant rats resulted in classic signs of preeclampsia such as hypertension, proteinuria, and glomerular endotheliosis. However, unlike in patients, the sFLT1 expression in the rat model was transient and was mainly produced in the maternal liver, not in the placenta (2, 4). Previously, we and other groups have reported the placenta-specific transgenesis and expression by transducing blastocysts-stage embryos with HIV-I-based self-inactivating lentiviral vectors (5-7). The lentiviral vectors transduced the outermost layer of the blastocyst, the trophectoderm, that provides most of the main and functional components of the future placenta. By contrast, the vectors were not ab...

The heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family of growth factors that binds to and activates the EGF receptor (EGFR) and the related receptor tyrosine kinase, ErbB4. HB-EGF-null mice (HB del/del ) were generated to examine the role of HB-EGF in vivo. More than half of the HB del/del mice died in the first postnatal week. The survivors developed severe heart failure with grossly enlarged ventricular chambers. Echocardiographic examination showed that the ventricular chambers were dilated and that cardiac function was diminished. Moreover, HB del/del mice developed grossly enlarged cardiac valves. The cardiac valve and the ventricular chamber phenotypes resembled those displayed by mice lacking EGFR, a receptor for HB-EGF, and by mice conditionally lacking ErbB2, respectively. HB-EGF-ErbB interactions in the heart were examined in vivo by administering HB-EGF to WT mice. HB-EGF induced tyrosine phosphorylation of ErbB2 and ErbB4, and to a lesser degree, of EGFR in cardiac myocytes. In addition, constitutive tyrosine phosphorylation of both ErbB2 and ErbB4 was significantly reduced in HB del/del hearts. It was concluded that HB-EGF activation of receptor tyrosine kinases is essential for normal heart function. The ErbB family of receptor tyrosine kinases have fundamental roles in development, proliferation, and differentiation (1). There are four members of the receptor tyrosine kinase ErbB family, EGFR͞ErbB1͞HER1, ErbB2͞HER2͞neu, ErbB3͞HER3, and ErbB4͞HER4. Epidermal growth factor (EGF) family ligands bind to and activate their receptors by inducing the formation of homodimers and heterodimers, resulting in autophosphorylation of specific tyrosine residues within the cytoplasmic domain. The phosphorylated tyrosine residues bind adapter proteins, which are instrumental in mediating downstream signaling pathways that determine the biological activity of the ErbB family of ligands.In vertebrates, the EGF family of ligands bind to ErbB receptors with some degree of preference. EGF, transforming growth factor-␣, and amphiregulin bind to EGF receptor (EGFR); heparin-binding EGF-like growth factor (HB-EGF), epiregulin, and betacellulin bind to both EGFR and ErbB4; NRG-1 (neuregulin͞heregulin͞NDF) and NRG-2 bind to ErbB3 and ErbB4; and NRG-3 and NRG-4 bind to ErbB4 but not to ErbB3. Although no ligand for ErbB2 has yet been described, ErbB2 is active as a signaling receptor by forming heterodimers with other ErbB receptors (2).HB-EGF is synthesized as a type I transmembrane protein (proHB-EGF) composed of signal peptide, heparin-binding, EGF-like, juxtamembrane, transmembrane, and cytoplasmic domains (3, 4). The membrane-bound proHB-EGF is cleaved at the juxtamembrane domain, resulting in the shedding of soluble HB-EGF (5). The full-length proHB-EGF is biologically active as a juxtacrine growth factor that signals neighboring cells in a nondiffusible manner (6-8). ProHB-EGF forms complexes with CD9 (9) and integrin ␣3␤1 (10) on the cell membrane. ProHB-EGF is al...

Optogenetics has been enthusiastically pursued in recent neuroscience research, and the causal relationship between neural activity and behavior is becoming ever more accessible. Here, we established knockin-mediated enhanced gene expression by improved tetracycline-controlled gene induction (KENGE-tet) and succeeded in generating transgenic mice expressing a highly light-sensitive channelrhodopsin-2 mutant at levels sufficient to drive the activities of multiple cell types. This method requires two lines of mice: one that controls the pattern of expression and another that determines the protein to be produced. The generation of new lines of either type readily expands the repertoire to choose from. In addition to neurons, we were able to manipulate the activity of nonexcitable glial cells in vivo. This shows that our system is applicable not only to neuroscience but also to any biomedical study that requires understanding of how the activity of a selected population of cells propagates through the intricate organic systems.

Melanin-concentrating hormone (MCH) is a neuropeptide produced in neurons sparsely distributed in the lateral hypothalamic area.Recent studies have reported that MCH neurons are active during rapid eye movement (REM) sleep, but their physiological role in the regulation of sleep/wakefulness is not fully understood. To determine the physiological role of MCH neurons, newly developed transgenic mouse strains that enable manipulation of the activity and fate of MCH neurons in vivo were generated using the recently developed knockin-mediated enhanced gene expression by improved tetracycline-controlled gene induction system. The activity of these cells was controlled by optogenetics by expressing channelrhodopsin2 (E123T/T159C) or archaerhodopsin-T in MCH neurons. Acute optogenetic activation of MCH neurons at 10 Hz induced transitions from non-REM (NREM) to REM sleep and increased REM sleep time in conjunction with decreased NREM sleep. Activation of MCH neurons while mice were in NREM sleep induced REM sleep, but activation during wakefulness was ineffective. Acute optogenetic silencing of MCH neurons using archaerhodopsin-T had no effect on any vigilance states. Temporally controlled ablation of MCH neurons by cell-specific expression of diphtheria toxin A increased wakefulness and decreased NREM sleep duration without affecting REM sleep. Together, these results indicate that acute activation of MCH neurons is sufficient, but not necessary, to trigger the transition from NREM to REM sleep and that MCH neurons also play a role in the initiation and maintenance of NREM sleep.