Browsing by Subject "Neuropeptides"
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Item Analysis of Circadian Rhythm Using a Novel SCN-Specific Cre Transgenic Mouse Line(2010-07-12) Chang, Alexander S.; Yanagisawa, MasashiThe neurons that make up the suprachiasmatic nucleus (SCN) temporally organize behavior into circadian cycles of activity and rest. When dissociated, these neurons individually oscillate with various period, phase, and amplitude. These conflicting results can be reconciled if inter-neuronal networking in the SCN is required for a consolidated behavioral circadian rhythm. To test this hypothesis, a novel SCN-specific Cre transgenic mouse line, named NMS-Cre, was developed by inserting a bicistronic Cre expression cassette at the 3’-untranslated region of the Neuromedin S (NMS) gene. By crossing NMS-Cre line to a lox-STOP-lox diphtheria toxin receptor line, behavioral circadian rhythm was disrupted upon intraperitoneal injection of diphtheria toxin. A histological examination showed that diphtheria toxin injection eliminated ~85% of NMS-Cre containing neurons at the SCN. Next, I generated NMS-Cre mediated Bmal1 conditional knockout animals to study behavioral rhythm output when most of the SCN neurons are without a molecular oscillator. The NMS-Cre(+);Bmal1flox/flox animals have essentially normal circadian rhythm of locomotor activity. Then, I generated NMS-Cre mediated Vesicular GABA Transporter (VGAT) conditional knockout animals because GABA has long been suspected to play a role in behavioral circadian rhythm. The NMS-Cre;VGATflox/flox animals performed normally in behavioral circadian rhythm parameter such as free-running period, robustness, and phase response curve. These in vivo data demonstrated a model that intra-SCN neuronal network is required for behavioral circadian rhythm, and can be a conduit that mediates molecular clock outputs from a small number of SCN neurons. Despite the fact that virtually all SCN neurons are GABAergic, GABA is an unlikely transmitter for this intra-SCN networking. Finally, NMS knockout animals have a well-consolidated behavioral circadian rhythm. However, when subjected to photic phase advancement, NMS knockout animals shifted their activity onset time quicker than wild type control animals. In situ hybridization results ruled out that an altered response to light stimuli or dampened molecular clock oscillation in the SCN as the cause for the rapid phase shift. NMS knockout animals switching from constant illumination to constant dark environment are unable to return to the typical less than twenty-four hour free-running period. Therefore, NMS is involved in a circadian pacemaker function.Item Cellular Basis of Behavioral Circadian Rhythms in Mammals: The Role of Neuromedin S (Nms)-Producing Cells in the Suprachiasmatic Nucleus(2013-08-07) Lee, Ivan T.; Elmquist, Joel; Russell, David W.; Greene, Robert W.; Yanagisawa, Masashi; Takahashi, JosephBehavioral circadian rhythms in mammals are controlled by highly heterogeneous populations of neurons located in the suprachiasmatic nucleus (SCN). Lesion and transplantation studies have established that the SCN is both necessary and sufficient for the generation of daily rhythms in locomotion. It remains uncertain, however, whether this pacemaking property of the SCN is limited to certain subsets of cells or intrinsic to all neurons within the SCN. To dissect out the cellular properties of circadian rhythms, we utilized a BAC transgenic mouse line in which Cre recombinase (iCre) is driven by the promoter of neuromedin S (Nms), a neuropeptide that has restricted expression in ~40% of cells within the SCN. Using this cell-type specific driver, we genetically altered the molecular oscillation of Nms-positive cells by overexpressing the ClockΔ19 or the Period2 transgene. ClockΔ19 is a semi-dominant mutation that leads to lengthened behavioral circadian periods when expressed in the majority of SCN cells. Likewise, Period2, when overexpressed in all or almost all of the SCN neurons, lead to the loss of behavioral circadian rhythms. We found that, intriguingly, the transgenic expression of ClockΔ19 only in Nms-positive neurons leads to a lengthened period in circadian rhythms while the overexpression of Per2 in Nms-expressing neurons causes the loss of daily rhythms altogether, suggesting that behavioral rhythms can be controlled by the molecular oscillation of Nms-positive cells. Next, to ascertain whether Nms-expressing neurons are required for normal behavioral circadian rhythms, we utilized a tetanus toxin-based technology that permits the inducible and reversible inhibition of neurotransmission. Surprisingly, this genetic manipulation revealed that synaptic neurotransmission from Nms neurons is essential for the generation of behavioral circadian rhythms. Taken together, these results indicate that Nms marks a specialized subgroup of neurons that is both necessary and sufficient for the production of circadian rhythms in behavior.Item Deconstructing Collective Cell Death in a Genetic Model(2015-07-27) Garcia Hughes, Gianella; Rothenfluh, Adrian; Abrams, John M.; Kraus, W. Lee; Krämer, HelmutElimination of cells and tissues by apoptosis is a highly conserved process. In Drosophila, the entire wing epithelium is completely removed shortly after eclosion. The cells that make up this epithelium are collectively eliminated through a highly synchronized form of apoptotic cell death, involving canonical apoptosome genes. Here I present evidence that transcription of the IAP antagonist, head involution defective (hid), is acutely induced in wing epithelial cells prior to this process. hid mRNAs accumulate to levels that exceed a component of the ribosome and likewise, Hid protein becomes highly abundant in these same cells. hid function is required for collective cell death, since loss of function mutants show persisting wing epithelial cells and, furthermore, silencing of the hormone bursicon in the CNS produced collective cell death defective phenotypes manifested in the wing epithelium. Taken together, these observations suggest that acute induction of Hid primes wing epithelial cells for collective cell death and that Bursicon is a strong candidate to trigger this process, possibly by activating the abundant pool of Hid already present. This model of collective cell death in the wing is predictable, easy to observe, and experimentally tractable. Previous studies have shown that mutants in the canonical apoptotic pathways share a late-onset blemishing and persisting cells phenotypes and here I present data of two other possible cell death gene candidates. First, I show that a compound deletion, 33B(del), which removes vps33B and part of fur1, generates late-onset blemishes and persisting cells in the wing blade. These phenotypes cannot be rescued by a vps33B genomic rescue, and the possibility exists that the phenotypes are due to fur1. Finally, I show that silencing of CTCF, a protein involved in chromosomal looping and 3D genomic organization, also generates late-onset blemishes and persisting cells in the wing blade. These results, together with the binding sites annotation in the Reaper region, suggest that CTCF might be coordinating the expression of the IAP antagonists, hid in particular, by chromosomal looping. Taken together, these studies contribute to the characterization of collective cell death in Drosophila.Item Lessons from Sleepy Mice: Narcolepsy and the Orexin Neuropeptide System(2005-04-29) Willie, Jon Timothy; Yanagisawa, MasashiThe hypothalamic orexin neuropeptide system is a neuronal pathway regulating behavioral vigilance states and metabolic functions. Orexins activate the orexin receptors type 1 and type 2 (OX2R). Melanin-concentrating hormone (MCH) is an anatomically-related peptide that may have complementary functions. Orexin null (orexin-/-) mice exhibit a behavioral and electroencephalographic phenotype similar to narcolepsy-cataplexy, a neurological disorder caused by orexin deficiency. Narcolepsy-cataplexy consists of inability to maintain wakefulness, abnormal intrusions of rapid-eye-movement (REM) sleep and related phenomena (i.e. cataplexy) into wakefulness, and poorly characterized metabolic abnormalities. I demonstrate that both OX2R-/- and orexin-/- mice are unable to maintain wakefulness normally. In contrast, OX2R-/- mice are only mildly affected with abnormalities of REM sleep, whereas orexin-/- mice are severely affected. Thus, the profound dysregulation of REM sleep control unique to the narcolepsy-cataplexy syndrome emerges from loss of signaling through both OX2R-dependent and OX2R-independent pathways. Transgenic mice, in which orexin neurons are ablated, fail to respond normally to fasting with increased wakefulness and activity indicating that orexin neurons provide a crucial link between energy balance and arousal. Orexin-/- and MCH-/- mice have increased and decreased adiposity and susceptibility to diet-induced obesity, respectively. While orexin-/- mice exhibit sleepiness and cataplexy, MCH-/- mice are more wakeful than wild-type mice. Orexin-/-;MCH-/- exhibit exacerbation of the narcolepsy phenotype, indicating that orexin and MCH complementarily regulate behavioral stabilty. Mice that ectopically overexpress orexin in the brain exhibit reduced body weight and resistance to diet-induced obesity, inability to maintain sleep, and reduced REM sleep with abnormal myoclonic activity relative to wild-type controls, providing further evidence that orexin alters homeostatic set-points of both energy metabolism and sleep/wakefulnuss. Modafinil, a stimulant used to treat narcoleptics, effectively increases wakefulness but does not suppress cataplexy in orexin-/- mice, indicating that orexin is not required for the wake-promoting action of the drug. By contrast, expression of an orexin transgene in the brain completely prevented cataplectic arrests and other abnormalities of REM sleep in the absence of endogenous orexin neurons. Thus, orexin neuron-ablated mice retain the ability to respond to orexin neuropeptides and orexin receptor agonists would likely contribute to treating human narcolepsy.Item Liver Receptor Homolog-1 Regulates Kisspeptin Expression in the Arcuate Nucleus to Promote Reproductive Axis Function(2014-04-08) Atkin, Stan Dean; Elmquist, Joel; Mangelsdorf, David J.; Kliewer, Steven A.; Takahashi, Joseph; Mendelson, Carole R.The timing of ovulation in mammals is set by a complex hypothalamic pituitary neuroendocrine axis. Kisspeptin neurons in the arcuate nucleus (Arc) are thought to secrete kisspeptin (Kiss1) to stimulate the release of follicle stimulating hormone (FSH) from the pituitary. However, mechanisms that drive Arc KISS1 output and maintain adequate FSH secretion required for folliculogenesis are not well understood. Here, we report that the nuclear receptor, liver receptor homolog-1 (LRH-1), is expressed in kisspeptin neurons of the Arc. Kiss1 is found to be a direct target gene of LRH-1, whereby LRH-1 sets a basal tone of kiss1 expression in the Arc. Deletion of Lrh-1 from kisspeptin neurons causes decreased Arc Kiss1 expression. This leads to reduced plasma FSH, prolongation of the estrous cycle, and decreased follicle maturation and ovulation. These defects ultimately compromise fertility. Overexpression of Lrh-1 in a kisspeptin neuron-specific transgenic mouse model increases Arc Kiss1 expression and plasma FSH. Chromatin immunoprecipitation and luciferase-based promoter assays demonstrate that LRH-1 binds to a putative LRH-1 response element in the Kiss1 promoter to stimulate activity. In conclusion, LRH-1 is expressed in the Arc to set the basal tone of Kiss1 output needed to promote FSH secretion for folliculogenesis prior to ovulation.Item Rasip1 Regulates Vascular Tubulogenesis(2011-12-15) Xu, Ke; Cleaver, OndineCardiovascular function depends on patent blood vessel formation by endothelial cells (ECs). However very little is known about the mechanisms underlying vascular ‘tubulogenesis’. This study identifies Rasip1 as a unique, endothelial-specific regulator of Rho GTPase signaling, which is essential for endothelial lumen morphogenesis. We found that Rasip1 is strongly expressed in vascular endothelial cells throughout development across species. Similar to the well-characterized vascular markers VEGFR2 and PECAM, Rasip1 is specifically expressed in angioblasts prior to vessel formation, in the initial embryonic vascular plexus, in the growing blood vessels during angiogenesis and in the endothelium of mature blood vessels into the postnatal period. Rasip1 expression is undetectable in VEGFR2 null embryos, which lack endothelial cells, suggesting that Rasip1 is endothelial-specific. Ablation of Rasip1 both in vitro and in vivo strongly affects vascular integrity. Specifically, siRNA-mediated reduction of Rasip1 severely impairs angiogenesis in endothelial cell cultures, and morpholino knockdown experiments demonstrate that Rasip1 is required for embryonic vessel formation in frog embryos. Mice lacking Rasip1 fail to form patent lumens in all blood vessels, including the early endocardial tube. Rasipl null angioblasts fail to properly localize the polarity determinant Par3 and display defective cell polarity, resulting in mislocalized junctional complexes and loss of adhesion to extracellular matrix (ECM). Depletion of either Rasip1 or its binding partner RhoGAP Arhgap29 in cultured ECs blocks in vitro lumen formation, fundamentally alters the cytoskeleton and reduces integrin-dependent adhesion to ECM. These defects result from increased RhoA/ROCK/myosin II activity and blockade of Cdc42 and Rac1 signaling. Together, our work identify Rasip1 as a novel endothelial factor that plays an essential role in vascular tubulogenesis.Item The Small GTPase Rheb Is Required for Spermatogenesis but not Oogenesis(2013-10-01) Baker, Michael David; Lum, Lawrence; Castrillon, Diego H.; Brekken, Rolf A.; Amatruda, James F.The process of germ cell development is under the tight control of various signaling pathways among which the PI3K-PKB-mTOR pathway is of critical importance. Previous studies have demonstrated sex-specific roles for several components of this pathway. In the current study I aimed to evaluate the role of Rheb, a member of the small GTPase superfamily and a critical component for mTORC1 activation, in male and female gametogenesis. The function of Rheb in development and the nervous system has been extensively studied, but little was known about its role in the germline. I have exploited genetic approaches in the mouse to study the role of Rheb in the germline and have identified an essential role in spermatogenesis. Conditional knockout (cKO) of Rheb in the male germline resulted in severe oligoasthenoteratozoospermia and male sterility. More detailed phenotypic analyses uncovered an age-dependent meiotic progression defect combined with subsequent abnormalities in spermiogenesis as evidenced by abnormal sperm morphology. In the female, however, germ-cell specific inactivation of Rheb was not associated with any discernible abnormality; these cKO mice were fertile with morphologically unremarkable ovaries, normal primordial follicle formation, and subsequent follicle maturation. The absence of an abnormal ovarian phenotype is striking given previous studies demonstrating a critical role for the mTORC1 pathway in the maintenance of primordial follicle pool. In conclusion, our findings demonstrate an essential role of Rheb in diverse aspects of spermatogenesis but suggest the existence of functionally-redundant factors that can compensate for Rheb deficiency within oocytes.Item [Southwestern News](1998-02-20) Steeves, Susan A.