ON-GOING PROJECTS IN THE MACAGNO LABORATORY AT UCSDPowerPoint PresentationSchematic of a segmental ganglion from its ventral aspectSlide 4Slide 5Slide 6Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15We are currently expressing GFP- and RFP-tagged actin monomers, MAPs and other molecules we have cloned in order to observe, using spinning disk confocal microscopy, the dynamics of the cytoskeleton.Slide 17Slide 18The PV and PD cells fully innervate the skin of the leechExtracellular Netrin Protein in the Leech EmbryoSlide 21Localized Knock-down of Netrin Expression Results in Abnormal Nerves in the Experimental RegionSlide 23Slide 24How is the gene expression profile of each neuron in a segmental ganglion, related to its physiological functions?Slide 26Slide 27ON-GOING PROJECTS IN THE MACAGNO LABORATORY AT UCSDHIRUDO MEDICINALIS GENOMICS, DEVELOPMENT, NEURAL STRUCTURE AND NEURAL FUNCTIONThe leech CNS is comprised of a head ganglion, 21 midbody segmental ganglia and a tail ganglion. Each midbody ganglion contains about 400 neurons and connects to adjacent ganglia and to the internal organs.Schematic of a segmental ganglion from its ventral aspect HEHE~185 bilateral pairs~30 unpairedEmbryos are large (6-8 mm) and easy to manipulateLiving Dissected Leech EmbryoHeadCNSTail??Embryonic sensory neuron extending through ventrum to innervate dorsumMACAGNO LABORATORY• Project 1: Functions of receptor phosphatases in development• Project 2: Dynamics of process outgrowth and the cytoskeleton• Project 3: Role of netrin in neuronal sensory tiling • Project 4: Expression and functions of Innexins• Project 5: Gene expression profiles of identified neuronsCOLLABORATIONS 1: Creation of Hirudo EST libraries and gene microarrays(Bento Soares, NU; Michel Salzet, ULille) 2: Creation and analysis of transcriptomic databases(Vineet Bafna, CSE, UCSD, and Terry Gaasterland, SIO) 3: Use of biolistics to modulate gene expression(Alex Groisman and Clare Simmonet, UCSD Physics)Structure of the Family of LAR-like Receptor Tyrosine Phosphatases LABORATORY Project 1: Functions of Receptor Phosphatases in DevelopmentCells 151 are candidates for the most prominent pairof neurons expressing HmLAR2 in the CNSComb Cells extend multiple parallel projections and express HmLAR2 in their Growth ConesAbnormal Morphology Following Injection of HmLAR2 dsRNALABORATORY Project 2: Dynamics of Process Outgrowth and the CytoskeletonWe are currently expressing GFP- and RFP-tagged actin monomers, MAPs and other molecules we have cloned in order to observe, using spinning disk confocal microscopy, the dynamics of the cytoskeleton.Actin - GFP MTAP - GFPHEHEP cellsLABORATORY Project 3: Role of netrin in neuronal sensory tilingThe PV and PD cells fully innervate the skin of the leechDORSAL DORSALVENTRALCNSLateralMidlinePV fieldPD fieldOne SegmentExtracellular Netrin Protein in the Leech EmbryoFigure 1, Aisemberg, Kuhn and Macagno, Dev Genes Evol 211:589 (2001)Pneumatic Capillary Gun for Biolistic Intracellular Delivery of Molecular ReagentsShefi et al., J. Neurosci. 26:6119 (2006)Localized Knock-down of Netrin Expression Results in Abnormal Nerves in the Experimental RegionTarget Area for siRNAControl AreaLABORATORY Project 4: Expression and Function of Innexins in DevelopmentWe are interested in the functions of gap junctions (innexins) in neural development. We have now cloned and have begun to characterize 11 different innexins - only five of these are expressed in the CNS, but we expect to find more. We are using RNAi and ectopic expression to test function.Inx-1 - 11day Inx-2 -11dayShot of INX2 siRNA-coated gold particles results in abnormal ganglia and nervesHow is the gene expression profile of each neuron in a segmental ganglion, related to its physiological functions?HEHEPEOPLECurrently In the Laboratory:Michael Baker - Research Scientist (PhD Dalhousie, CANADA)Alejandro Sanchez - PostDoc (PhD UABC, MEXICO)Orit Shefi - PostDoc (PhD Tel Aviv, ISRAEL)Li Bao - PostDoc (PhD UMiami, CHINA)Aloysius Phillips - PhD Student (Columbia)Sandra Peterson - Lab ManagerCollaborators:Gabriel Aisemberg - Lehman College, CUNYMichel Salzet - Universite de Lille, FRANCEBento Soares - University of Iowa, IOWAVineet Bafna – UCSDTerry Gaasterland – UCSDAlex Groisman – UCSDClare Simmonet -