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
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
Section of Chick Spinal Cord from Ramon y Cajal (circa 1890)
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)
Structure of the Family of LAR-like Receptor Tyrosine Phosphatases LABORATORY Project 1: Functions of Receptor Phosphatases in Development
Cells 151 are candidates for the most prominent pair of neurons expressing HmLAR2 in the CNS
Comb Cells extend multiple parallel projections and express HmLAR2 in their Growth Cones
Abnormal Morphology Following Injection of HmLAR2 dsRNA
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 - GFPMTAP - GFP
HE P cells LABORATORY Project 3: Role of netrin in neuronal sensory tiling
The P V and P D cells fully innervate the skin of the leech DORSAL VENTRAL CNS Lateral Midline P V field P D field One Segment
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 Target Area for siRNA Control Area
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 dayInx-2 -11day
Shot of INX2 siRNA-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
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