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ON-GOING PROJECTS IN THE MACAGNO LABORATORY AT UCSD

ON-GOING PROJECTS IN THE MACAGNO LABORATORY AT UCSD. HIRUDO MEDICINALIS GENOMICS, DEVELOPMENT, NEURAL STRUCTURE AND NEURAL FUNCTION.

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ON-GOING PROJECTS IN THE MACAGNO LABORATORY AT UCSD

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  1. ON-GOING PROJECTS IN THE MACAGNO LABORATORY AT UCSD HIRUDO MEDICINALIS GENOMICS, DEVELOPMENT, NEURAL STRUCTURE AND NEURAL FUNCTION

  2. The 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.

  3. Schematic of a segmental ganglion from its ventral aspect HE HE ~185 bilateral pairs ~30 unpaired

  4. Embryos are large (6-8 mm) and easy to manipulate

  5. Head Living Dissected Leech Embryo CNS ? Tail

  6. Embryonic sensory neuron extending through ventrum to innervate dorsum

  7. MACAGNO 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 neurons • COLLABORATIONS • 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)

  8. LABORATORY Project 1: Functions of Receptor Phosphatases in Development Structure of the Family of LAR-like Receptor Tyrosine Phosphatases

  9. Cells 151 are candidates for the most prominent pair of neurons expressing HmLAR2 in the CNS

  10. Comb Cells extend multiple parallel projections and express HmLAR2 in their Growth Cones

  11. Abnormal Morphology Following Injection of HmLAR2 dsRNA

  12. We 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. LABORATORY Project 2: Dynamics of Process Outgrowth and the Cytoskeleton Actin - GFP MTAP - GFP

  13. LABORATORY Project 3: Role of netrin in neuronal sensory tiling HE HE P cells

  14. ThePVandPDcells fully innervate the skin of the leech CNS Lateral Midline One Segment PV field PD field VENTRAL DORSAL DORSAL

  15. Extracellular Netrin Protein in the Leech Embryo Figure 1, Aisemberg, Kuhn and Macagno, Dev Genes Evol 211:589 (2001)

  16. Pneumatic Capillary Gun for Biolistic IntracellularDelivery of Molecular Reagents Shefi et al., J. Neurosci. 26:6119 (2006)

  17. Localized Knock-down of Netrin Expression Results in Abnormal Nerves in the Experimental Region Target Area for siRNA Control Area

  18. LABORATORY Project 4: Expression and Function of Innexins in Development We 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 -11day

  19. Shot of INX2 siRNA-coated gold particles results in abnormal ganglia and nerves

  20. How is the gene expression profile of each neuron in a segmental ganglion, related to its physiological functions? HE HE

  21. PEOPLE Currently 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 Manager Collaborators: Gabriel Aisemberg - Lehman College, CUNY Michel Salzet - Universite de Lille, FRANCE Bento Soares - University of Iowa, IOWA Vineet Bafna – UCSD Terry Gaasterland – UCSD Alex Groisman – UCSD Clare Simmonet - UCSD

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