UNIVERSITY OF CALIFORNIA, SAN DIEGO (UCSD). Julie Sylvia BACKGROUND @UCSD.
ON-GOING PROJECTS IN THE MACAGNO LABORATORY AT UCSD
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Transcript of 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
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
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~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
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 pairof 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 - GFP MTAP - GFP
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P cells
LABORATORY Project 3: Role of netrin in neuronal sensory tiling
The PV and PD cells fully innervate the skin of the leech
DORSAL DORSALVENTRAL
CNS LateralMidline
PV field
PD 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-1 - 11day Inx-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?
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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