Eduardo Macagno

Eduardo Macagno

Professor

Contact Information

NSB 6121
Office Tel: 822-5702
Lab Tel: 822-5700
Email: emacagno@ucsd.edu

Our studies focus on the cellular and molecular mechanisms underlying the specification of neuronal identity, growth cone motility and navigation, target selection and the generation of neuronal arbors. We work with identified neurons in the medicinal leech, Hirudo medicinalis, a system that has several advantages for our studies. First, we can study a particular neuron at all stages of development, in the animal; second, we can visualize many cells in the intact animal as they differentiate, using time-lapse 2-photon confocal imaging, and operate on them with a laser microbeam to ask very specific questions; and third, we can inject macromolecules into embryonic or adult neurons, which means that we can transform a single cell in a wild-type environment. In particular, we have adapted RNAi techniques to both single-cell and systemic uses, and have developed constructs for the ectopic expression of leech genes and mRNAs by micropressure injection into individual embryonic or adult leech cells. In current cellular studies, we are examining the formation of peripheral arbors by sensory and motor projections in the animal's bodywall and in specific target tissues, like the heart tubes, as well as the formation of arbors of efferent projections in the CNS, the latter in a collaboration with Prof. Birgit Zipser (MSU). Time-lapse observations in situ show that arbors are highly dynamic, with higher-order branches undergoing repeated cycles of extension and retraction. We have also identified several interactions (e.g., between homologous neurons, between pioneers and their followers, between axons and non-neuronal substrates, between different branches of the same neuron, between neurons and their targets) that play roles in defining the size and shape of the arbor. One experiment underway explores how a cell recognizes itself by examining what happens after a branch has been severed by laser photoablation - the result is that the cell stops recognizing the distal stump as self, suggesting that surface markers are not responsible for self recognition. To get some inkling of how back-branching might be induced by interactions with substrates or targets, in another set of experiments we are beginning to map the disposition and dynamics of cytoskeletal proteins at branch points forming behind growth cones. In a complementary set of molecular studies, we have begun to examine the functions of membrane-bound and secreted recognition/adhesion factors that are thought to be involved in the cell interactions we have documented to occur during arbor formation and target selection. We have recently cloned the leech homologues, HmLAR1 and HmLAR2, of the LAR family of receptor phosphatases and have determined that they are expressed by identified neurons (in processes and growth cones) as well as some muscle and other types of cells. Blocking function by injecting into embryos antibodies to the extracellular domain or recombinantly expressed extracellular domains, or using RNAi in tissues or individual cells to knock down expression of these receptors, shows that HmLAR1, which is expressed by the heart tubes, is necessary for the innervation of the heart, and that HmLAR2 is involved in the regulation of axonal pathfinding growth cone stability and process outgrowth. In addition, we have characterized a leech netrin, which is also expressed by specific central neurons as well as ventral (but not dorsal) longitudinal muscles. In a third area of work in molecular studies, we have initiated a major project to characterize the Hirudo medicinalis transcriptome using normalized EST libraries. In collaboration with Prof. Bento Soares (Northwestern University) and Prof. Michel Salzet (Universite de Lille), we generated two EST libraries, from complete embryos at several stages and from adult CNS. About 130,000 clones from these libraries have been sequenced (at the University of Iowa, the French genome agency Genoscope, and the Joint Genomics Institute at Berkeley), which have been clustered into 47,000 unique contigs and singletons with the help of Prof. Terry Gaasterland at UCSD. A major goal of this project is to spot transcript microarrays and query them with cDNA derived from individual identified cells, as well as cDNA derived from embryonic, adult and antigen-challenged CNS in order to identify and characterize genetic programs that confer unique properties to neurons as well as genetic programs involved in neurodevelopment, neuroregeneration and neuroimmunological responses. As part of this effort, we have also begun to create an international consortium of interested laboratories with W. Kristan (UCSD), M. Salzet (ULille, France) and V. Torre (SISSA, Italy) to exploit this new information. Finally, we have recently embarked in a developmental and physiological analysis of invertebrate gap junction proteins, the innexins. We have cloned and characterized 12 innexins in leech embryos and reported their patterns of expression, and are currently characterizing another group of 5-8 innexins obtained from the EST sequencing project described in the previous paragraph. We are also currently assaying the functions of those innexins expressed in the nervous system using RNAi to knock down their expression in embryos. In addition, using the frog oocyte expression system, we have characterized gap junctional channels comprised of leech inx1, inx2, inx3 and inx6. We have found that all of these innexins form hemichannels in single oocytes that can be opened by changing pH, increasing Ca and mechanically stretching the membrane. We are now checking whether these innexins also form hemichannels in the leech embryo, where they could play important signaling roles in neural development. Research in this laboratory has been or is currently supported by grants from the National Science Foundation and the National Institutes of Health.

RNAi of the Receptor Tyrosine Phosphatase HmLAR2 in a Single Cell of an Intact Leech Embryo Leads to Growth-Cone Collapse. M.W. Baker and E.R. Macagno. Curr. Biol. 10:1071-1074 (2000).

Possible Role of the Receptor Protein Tyrosine Phosphatase HmLAR2 in Interbranch Repulsion in a Leech Embryonic Cell. M.W. Baker, S.J. Rauth and E.R. Macagno. J. Neurobiol. 45:47-60 (2000).

The Role of a LAR-like Receptor Tyrosine Phosphatase in Growth Cone Collapse and Mutual-Avoidance by Sibling Processes. Michael W. Baker and E.R. Macagno. J. Neurobiol. 44:194-203 (2000).

Neuronal Growth and Target Recognition: Lessons from the Leech, Hirudo medicinalis. M.W. Baker and E.R. Macagno. Can. J. Zool. 79:204-217 (2001).

Netrin Signal is Produced in Leech Embryos by Segmentally Iterated Sets of Central Neurons and Longitudinal Muscle Cells. G. O. Aisemberg, J. Kuhn and E.R. Macagno. Devel. Genes Evol. 211:589-596 (2001).

Neuronal Growth and Target Recognition: Lessons from the Leech, Hirudo medicinalis. M.W. Baker and E.R. Macagno. Can. J. Zool. 79:204-217 (2001).

Association of LAR-like Receptor Protein Tyrosine Phosphatases with an Enabled homolog in Hirudo medicinalis. Subhas C. Biswas, Anindita Dutt, M. W. Baker and E. R. Macagno. Mol. Cell. Neurosci., 21:657-670 (2002).

In Vivo Dynamics of CNS Sensory Arbor Formation: A Time-Lapse Study in the Embryonic Leech. M. W. Baker, B. Kauffman, E. R. Macagno and B. Zipser. J. Neurobiol., 56:41-53 (2003).

Upregulation of Neurohemerythrin Expression in the Central Nervous System of the Medicinal Leech, Hirudo medicinalis, Following Septic Injury. D. Vergote, Pierre-Eric Sautiere, F. Vandenbulcke, D. Vieau, E. R. Macagno and M. Salzet. J. Biol. Chem. 279:43828-37 (2004).

Molecular Characterization and Embryonic Expression of Innexins in the Leech Hirudo medicinalis. Iain Dykes and E. R. Macagno. Devel Genes Evol 27:1-13 (2006).

Characterization of Hirudo medicinalis DNA Promoters for Targeted Gene Expression. Michael W. Baker and Eduardo R. Macagno. J. Neurosci. Meth. 156:145-153 (2006).

Microtargeted gene silencing and ectopic expression in live embryos using biolistic delivery with a pneumatic capillary gun. Orit Shefi, Claire Simonnet, Michael W. Baker, James R. Glass, Eduardo R. Macagno and Alex Groisman. J. Neurosci. 26:6119-6123 (2006).

Proteome modifications of the medicinal leech nervous system under bacterial challenge. David Vergote, Eduardo Macagno, Michel Salzet, and Pierre-Eric Sautière. Proteomics 6:4817-4825 (2006).

In Vivo Imaging of Growth Cone and Filopodial Dynamics: Evidence for Contact-Mediated Retraction of Filopodia Leading to the Tiling of Sibling Processes. M.W. Baker and E.R. Macagno. J.comp. Neurol. 500:850-862 (2007).

Innexins Form Two Types of Channels. L. Bao, S. Samuels, S. Locovei, E. R. Macagno, K. J. Muller, and G. Dahl. FEBS Letters 581:5703-5708 (2007).

Molecular MALDI Imaging: an Emerging Technology for Neuroscience Studies. I. Fournier, M. Wisztorski, D. Croix, E. Macagno and M. Salzet. Invited review, Special Issue on Dynamic Imaging, J. Glover, Guest Editor, Dev. Neurobiol. 68:845-858 (2008).

Microbial challenge promotes the regenerative process of the injured central nervous system of the medicinal leech by inducing the synthesis of antimicrobial peptides in neurons and microglia. D. Schikorski, V. Cuvillier Hot, M. Leippe, C. Wichlacz-Boidin, C. Slomianny, E. Macagno, M. Salzet and A. Tasiemski. J. Immunol. 181:1083-1095 (2008).

The Receptor Phosphatase HmLAR2 Sheds its Ectodomain and Collaborates with Focal Adhesion Proteins in Filopodial Tips to Control Growth Cone Morphology. M.W. Baker, S.M. Peterson and E.R. Macagno. Dev Biol, 320:215-225(2008).

MITICS (MALDI Imaging Team Imaging Computing System): a new open source mass spectrometry imaging software. Olivia Jardin-Mathé, David Bonnel, Julien Franck, Maxence Wisztorski, Eduardo Macagno, Isabelle Fournier and Michel Salzet. J. Proteomics 71 :332-345 (2008).