The Tibial1 Pioneer Pathway An in Vivo Model for Neuronal Outgrowth and Guidance

Jennifer Bonner, Kimberly A. Gerrow, and Timothy P. O'Connor

Program in Neuroscience Department of Anatomy University of British Columbia Vancouver, British Columbia V6T 1Z3 Canada

I. Introduction

II. Development of the Tibial-1 (Ti1) Pathway

A. Grasshopper Limb Development

B. Anatomy of Ti1 Neurons

C. The Path of Ti1 Pioneer Neurons: From the Periphery to the Central Nervous System

III. Guidance of Ti1 Axons

A. Mechanisms

B. Guidance Molecules

IV. Analysis of Axon Guidance Mechanisms in Grasshopper Procedures

A. Staging Grasshopper Embryos from 29 to 35% of Development

B. Culturing Grasshopper Embryos

C. Blocking the Function of Known Molecules (Antibodies and Peptides)

D. Ectopic Expression in the Developing Limb Bud

E. Microinjection and Labeling Ti1 Neurons V. Laboratory Protocols

A. Embryo Dissection

B. Embryo Cultures

C. Immunocytochemistry

D. Preparation of Glass Bottom Culture Dishes for Microinjection and Time-Lapse Imaging

E. Preparation of Poly-L-Lysine-Coated Coverslips

METHODS IN CELL BIOLOGY, VOL. 71 Copyright 2003, Elsevier Science (USA). All rights reserved. 0091-679X/03 $35.00

F. Filleting Grasshopper Limb Buds to Gain Access to the Ti1 Pathway

G. Labeling and Injecting Ti1 Neurons in Situ

H. Ectopic Overexpression in the Developing Limb Bud I. Blocking Protein Function


As neurons extend axons to their targets during development, growth cones must reorient their direction of migration in response to extracellular guidance cues. A variety of model systems have been employed in order to dissect the cellular and molecular mechanisms that underlie this complex process. One preparation, the developing grasshopper limb bud, has proved to offer a number of advantages in which to examine mechanisms of growth cone guidance and motility in vivo. First, the relatively large size of the embryonic nervous system allows for straightforward imaging of both fixed and live neurons in vivo. Second, the peripheral nerves generated in the limb bud are highly stereotyped. Third, intact embryos can be cultured for a period of days, allowing for fairly easy perturbations at precise developmental stages. Fourth, due to the ease of dissection, numerous cell biological and molecular techniques can be utilized in the limb bud. Finally, axon guidance molecules and mechanisms are conserved between grasshoppers and other organism, including vertebrates.

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