參考說明session306段佳麗

1、Molecular mechanism of CO2 sensing in breathing regulation in DrosophilaJiali DuanYulong Li labSep 23rd, 201812Why breathing?O2CO23Eleanor Lutz, 2014 Animals evolve diverse respiratory systems and breathing behaviors4higher CO2 level in the bodylower O2 level in the environmentBreathing is dynamical

2、ly regulated by environments5CO2 regulates breathing behavior via negative feedbackGerard Tortora et al., Principles of Anatomy and Physiology, 2012High CO2 promotes breathing by opening spiracles in insectsHigh CO2 sensing responseHoward Schneiderman, The Biological Bulletin, 19606Spiracle (closed

3、state)How is CO2 sensed during breathing regulation?7Using Drosophila as the model to study the CO2 sensing in breathing regulationDrosophila has a similar breathing behavior and responses to high CO2 (to mammals)Powerful genetic tools8Experiment setup and protocol1st2nd3rd9th50 m20 m200 mmHypercapn

4、ia treatment92ndVolker Hartenstein, Atlas of Drosophila Development, 1993Images and cartoons from Ao Dong and Rongbo Sun, Li Lab, unpublished Gas flow inGas flow outCamera10How is CO2 sensed during breathing?11Where?the CO2 receptorWhat?There are many sensory related receptors on legs, wings and hea

5、d12Ana Depetris-Chauvin et al. Front. Ecol. Evol., 2015 intactlegs removedwings & legsremovedhead & legs & wingsremovedCentral brain, legs and wings are not necessary in the CO2-induced breathing regulation.13Guided by Ao Dong, Li LabHow is CO2 sensed during breathing?14Where?the CO2 receptorWhat?Wa

6、lton Jones et al. Nature, 2007Jae Young Kwon et al. PNAS, 2007Ji Hu et al. Science, 2007M. A. Ruda et al. Science, 2000Cartoons form Ao Dong, Li Lab15Identified receptors for CO2 in animals and fungiCA IIDrosophilaORNCO2AversionGr21a, Gr63aGq, G30A?Two GrsTAX-2, TAX-4MouseOlfactory epiCO2HCO3-GTPcGM

7、PCNGGC-DC. elegansBAG neuronsCO2HCO3-AversionGTPcGMP?GCY-9Guanylyl cyclasesEwan St. John Smith et al. Journal of Biological Chemistry, 2013Cawas Engineer et al. Trends in plant science, 2016Cartoons form Ao Dong, Li Lab16Identified receptors for CO2 in animals and fungiMammalSpermHCO3-HCO3-Sperm act

8、ivationATPcAMPsACAdenylyl cyclasesFungiCO2NCE103HCO3-CDC35FilammtationATPcAMP?CO2 receptor in breathing regulation in Drosophila? GeneFamilyStock #StrategyLethality under elav-Gal4Spiracular response to 10% CO2 Gr21aGustatory receptorTHU5648RNAiViableNormalGr63aGustatory receptorBL9941MutantNormaldR

9、GCA3rGCTHU3054RNAiViableNormaldRGCA3rGCTH04281.NRNAiLethalN.A.dRGCA2rGCTHU3860RNAiLethalN.A.dRGCA2rGCTH04360.NRNAiViableNormaldRGCA9rGCTHU3514RNAiLethalN.A.dRGCA1rGCBL56937RNAiLethalN.A.dRGCA1rGCBL58224RNAiViable“Fluttering”dRGCA1rGCBL28604RNAiViableNormaldRGCA4rGCBL28524RNAiViableNormaldRGCA5rGCBL5

10、4528MutantNormalGeneFamilyStock #StrategyLethality under elav-Gal4Spiracular response to 10% CO2 dASGCA6asGCTHU0446RNAiViableNormaldASGCA7asGCTHU0528RNAiViableNormaldASGCA8asGCTHU0504RNAiViableNormaldACA1rACTH04485.NRNAiViableNormaldACA2rACTH04486.NRNAiViableNormaldACA7rACTH04487.NRNAiViableOngoingd

11、ACA3rACTH04488.NRNAiViableNormaldACA4rACTH04489.NRNAiViableNormaldACA8rACTH04490.NRNAiLethalN.A.dACA9rACTH04492.NRNAiLethalN.A.dACA5rACTH02059.NRNAiViableNormaldACA10rACTH04773.NRNAiLethalN.A.dACA6rACTHU2421RNAiViableNormaldACA11rACTHU3027RNAiLethalN.A.elav-Gal4 (pan-neural driver) x UAS-shRNALoss-o

12、f-function mutantsScreening of CO2 receptors17Two GrsGuanylyl cyclasesAdenylyl cyclasesGuided and collaborated by Ao Dong, Li LabelavUAS-shRNAdRGCA1Wild typeIndex calculation0 1ClosedOpenKnockdown of dRGCA1 in neurons leads to impaired spiracle movements induced by 10% CO218Each genotype: N = 6-10 i

13、ndividualsKnockdown of dRGCA1 in neurons leads to impaired spiracle movements induced by 10% CO219Guided by Ao Dong, Li LabGCCGCUUAUACAAAAGUAGUUGAUAGCGGCGAAUAUGUUUUCAUCAAUAUGATUCGGGUGAGUACGUCUUCAUAAAUAU S G E Y V F I N S G E Y V F I N dRGCA1 mRNA dRGCA1 ProteindRGCA1 shRNAdRGCA1R mRNAdRGCA1R protein

14、Rescue of dRGCA1 in neurons restores the phenotype20elav-Gal4 ; UAS-shRNAdRGCA1/UAS-dRGCA1Relav-Gal4 ; UAS-shRNAdRGCA1/CyOKnockdown of dRGCA1 is specific.Systematic RNAi screeningIdentification of candidate genesMolecular levelEnzymatic activity21What is the CO2 receptor regulating breathing?RFP-dea

15、ddRGCA1Generation of stable cell lines expressing dRGCA1 and deaddRGCA122RFP-deaddRGCA1RFP-dRGCA1RFP-dRGCA1Extracellular domainPseudo-kinase domainGuanylyl cyclase domaindRGCA1 can be activated by proton in vitro23dRGCA1deaddRGCA1: undetectableSystematic RNAi screeningIdentification of candidate gen

16、esMolecular levelCircuit levelEnzymatic activityAnatomy and function24What is the CO2 receptor regulating breathing?Coding SequencedRGCA1 KnockoutHomologous armattP site3xP3-RFPsgRNA targeting sitesexon8exon1exon1Cas9Guanylyl cyclase domaindRGCA1 KO/dRGCA1 KO flies are lethal.Generation of the dRGCA

17、1 knockout and Gal4 driver lines25attB siteT2A-Gal4dRGCA1 KnockoutdRGCA1-Gal4exon1exon1exon8exon826Characterizing dRGCA1 expressing neurons in vivo using dRGCA1-Gal4 driver linesMorphology:Membrane GFP, synapse marker, dendrite marker, et al.Function:CO2 sensitive or not calcium imaging using GCaMP.

18、Manipulation:Optogenetic activation/inhibition CsChrimson/GTACR.Summary27Extracellular domainPseudo-kinase domainGuanylyl cyclase domaindRGCA1dRGCA1deaddRGCA1: undetectableThank School of Life Sciences and UHPB for support.Thank Dr. Yulong Li for patient and instructive guidance. Thank Ao Dong for t

19、eaching me all of the experiments.Thank all Li lab members for generous help all the time.Any questions and suggestions are ed.Acknowledgements28Future plan1. Identifying the signaling pathway in dRGCA1 expressing neurons.29DrosophiladRGCA1 neuronsCO2Breathing regulationGTPcGMPCNGdRGCA1CAHCO3-H+PKGP

20、DEProposed model?Future plan2. Characterizing dRGCA1 expressing neurons in vivo.30Morphology:Membrane GFP, synapse marker, dendrite marker, et al.Function:CO2 sensitive or not calcium imaging using GCaMP.Manipulation:Optogenetic activation/inhibition CsChrimson/GTACR.Intersectional approaches.Future

21、 plan3. Mapping the CO2 sensory circuit.31Mustafa Talay et al. Neuron, 2017trans-Tango technologyDosage dependent curve of spiracular response to CO2Case, James F. Carbon dioxide and oxygen effects on the spiracles of flies.Physiological Zoology29.2 (1956): 163-171.3334FlyAtlas Anatomical Expression

22、 Data (Microarray)Expression pattern of dRGCA1 in Drosophila. 3536dRGCA1GC-DIntracellular cGMP level measured by ELISA pH dependence3738Sun, Liming, et al. Guanylyl cyclase-D in the olfactory CO2 neurons is activated by bicarbonate.Proceedings of the National Academy of Sciences106.6 (2009): 2041-20

23、46.39Discontinuous respiration in insectsLevy, R. I., and H. A. Schneiderman. Discontinuous respiration in insects. II. The direct measurement and significance of changes in tracheal gas composition during the respiratory cycle of silkworm pupae. Journal of Insect Physiology 12.1(1966):83.4041Imaging the cGMP signals in HEK cells +cGMPcng channelCa2+GTPdRGCA1GEXH+Coexpression of GEX and rCNGA2300 uM SNAPSNAP: NO donorData from Ao Dong42Imaging the cGMP signals in HEK cells n=14n=10dRGCA1TDGEXmScarlet