The molecular mechanism that underlies neuronal growth cone guidance
	Elke Stein, Ph.D. Assistant Professor of Molecular, Cellular & Developmental Biology and Cell Biology Yale University, KBT 232 PO Box 208103, 266 Whitney Ave New Haven, CT 06520 Phone (203) 432 8402; (203) 432 7651 Email: elke.stein@yale.edu Ph.D. Vanderbilt University 1996

Turning responses of Xenopus spinal neurons after exposure to gradients of netrin-1 and Slit-2. Shown here spinal neurons derived from Stage 22 are attracted to a netrin-1 gradient, whereas netrin-1 mediated attraction is silenced in the presence of Slit-2 (Stage 22). Xenopus spinal neurons gain responsiveness to repellent Slit-2 with development (Stage 28). Spinal neurons were stained for F-actin. Similar to axons guidance events occurring at the vertebrate midline.

One of the earliest steps in neuronal development is the growth of axons from their cell bodies of origin to their appropriate targets, to form a precise pattern of neuronal connections. The growth of axons is highly directed, as axons are guided to their targets by specific guidance cues displayed in the extracellular environment. These cues can be attractive, steering axons towards particular sources of the cues, or repulsive, steering axons away from inappropriate regions. We are interested to elucidate the molecular mechanism how attractive and repulsive guidance cues and their respective receptors participate during neuronal development, elucidate their precise functions in guiding axons in vivo, and determining how axons respond to these cues with directed growth. Recent progress in the field lead to the identification of several families of attractants and repellents, including the netrin, semaphorin and slit protein families, as well as receptors involved in mediating the responses of these factors, however only little is known how these guidance cues function in vivo.

Our central focus is to understand the molecular mechanism that underlies neuronal growth cone guidance. We are currently focusing on several guidance systems, including the netrin receptors DCC and UNC-5, slit receptors of the Robo family and the DSCAM (Down syndrome cell adhesion molecule) family of orphan receptors. Our current efforts are aimed at

1. is identifying the signal transduction mechanisms through which these receptors trigger axonal steering, branching of axons and dendrites and
2. elucidating their functions and signaling mechanism in vivo using gene targeting and transgenic approaches. To address this we are using a series of biochemical, cell biological and molecular techniques, in combination with functional approaches, including axon turning assays utilizing Xenopus spinal and neuronal rodent cultures, in vitro explant and slice cultures, and in vivo assays using the developing chicken as a model.

Selected Publications

Hong K., Hinck L. Nishiyama M., Poo M-M., Tessier-Lavigne M. and Stein E. (1999) A ligand-gated association between cytoplasmic domains of UNC5 and DCC family receptors converts netrin-induced growth cone attraction to repulsion. Cell 97: 927-941

Stein E. and Tesier-Lavigne M. (2001) Hierarchical organization of guidance receptors: Silencing of netrin attraction by slit through a Robo/DCC receptor complex. Science 291: 1928-1938

Stein E., Zou Y., Poo .M-M. and Tessier-Lavigne M. (2001) Binding of DCC by netrin-1 to mediate axon guidance independent of Adenosine A2B receptor activation. Science 291: 1976-1982

Forcet C., Stein E., Pays L., Llambi F., Corset V., Tessier-Lavigne M. and Mehlen P. (2002) DCC-dependent MAPK activation is required for netrin-1 mediated axon outgrowth. Nature 417: 443-447.

Charron F., Stein E., Jeong J., McMahon A.P. and Tessier-Lavigne M. (2003) The morphogen Sonic hedgehog is an axonal chemoattractant that collaborates with netrin-1 in midline axon guidance. Cell 113, 11-23.

			Netrin-1 mediates axonal outgrowth of commissural axons in vitro. E13 dorsal spinal cord explants are cultured in 3-D collagen bed in the absence or presence of purified netrin-1 protein.

			Matured primary cortical neurons derived from rat E16 rat embryos. Stained with MAP1b (red) and F-actin (green).

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