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