ON-GOING PROJECTS IN THE MACAGNO LABORATORY AT UCSD HIRUDO MEDICINALIS GENOMICS, DEVELOPMENT, NEURAL STRUCTURE AND NEURAL FUNCTION
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.
Schematic of a segmental ganglion from its ventral aspect HE ~185 bilateral pairs ~30 unpaired HE
Embryos are large (6 -8 mm) and easy to manipulate
Living Dissected Leech Embryo Head CNS ? Tail
Embryonic sensory neuron extending through ventrum to innervate dorsum
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)
LABORATORY Project 1: Functions of Receptor Phosphatases in Development Structure of the Family of LAR-like Receptor Tyrosine Phosphatases
Cells 151 are candidates for the most prominent pair of neurons expressing Hm. LAR 2 in the CNS
Comb Cells extend multiple parallel projections and express Hm. LAR 2 in their Growth Cones
Abnormal Morphology Following Injection of Hm. LAR 2 ds. RNA
LABORATORY Project 2: Dynamics of Process Outgrowth and the Cytoskeleton 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. Actin - GFP MTAP - GFP
LABORATORY Project 3: Role of netrin in neuronal sensory tiling HE P cells HE
The PV and PD cells fully innervate the skin of the leech CNS Lateral Midline One Segment PV field PD field DORSAL VENTRAL DORSAL
Extracellular Netrin Protein in the Leech Embryo Figure 1, Aisemberg, Kuhn and Macagno, Dev Genes Evol 211: 589 (2001)
Pneumatic Capillary Gun for Biolistic Intracellular Delivery of Molecular Reagents Shefi et al. , J. Neurosci. 26: 6119 (2006)
Localized Knock-down of Netrin Expression Results in Abnormal Nerves in the Experimental Region Control Area Target Area for si. RNA
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 - 11 day Inx-2 -11 day
Shot of INX 2 si. RNA-coated gold particles results in abnormal ganglia and nerves
How is the gene expression profile of each neuron in a segmental ganglion, related to its physiological functions? HE HE
PEOPLE Currently In the Laboratory: Michael Baker - Research Scientist (Ph. D Dalhousie, CANADA) Alejandro Sanchez - Post. Doc (Ph. D UABC, MEXICO) Orit Shefi - Post. Doc (Ph. D Tel Aviv, ISRAEL) Li Bao - Post. Doc (Ph. D UMiami, CHINA) Aloysius Phillips - Ph. D 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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