Axon Guidance Module

Axon Guidance Module

Instructor:

Focus:

I chose this module because the story begins with the first description of chemical guidance during axon growth in the central nervous system. It includes some of the seminal papers in the axon guidance field.
The Tessier-Lavigne lab has generated a large body of work describing axon guidance and these are many of the seminal papers from his laboratory. The work is rigorous and includes a wide variety of techniques ranging from complex biochemical analysis to ex vivo culture of primary tissue.
In addition to being one of the first groups to characterize chemotropic guidance in the nervous system, this lab has identified and characterized a large number of novel axon guidance cues and their receptors so it is good for students to have the opportunity to discuss the process through which an initial observation can lead to the identification of novel signaling pathways, molecules and receptors.
Tessier-Lavigne has also held a variety of positions in both academia and industry (which has been followed in the popular press) and students thus have the opportunity to discuss alternative options for scientific careers.

Overview:

The papers I have chosen cover approximately eight years of research beginning with the original identification of a chemical cue that guides growing axons in the central nervous system. This is followed by the identification of the floor plate as the source of one of these chemical cues, the identification of netrin as a floor plate secreted axon guidance cue and the identification of one of the netrin receptors. The first two papers are similar and relatively simple, so they give a chance for the students to get practice reading papers (two different styles) before diving into more complex and diverse methods.
Several of these papers are quite complicated with many figures depicting different chemical approaches, so I would design this course as an upper level seminar/capstone course for students who have taken mid-level Molecular or Cell Biology courses.

Applicable for Courses:

Educational Level:

Upper-level

Roadmap Objectives:

    • Article: Chemotropic guidance of developing axons in the mammalian central nervous system. Tessier-Lavigne M, Placzek M, Lumsden AGS, Dodd J, Jessell TM. (1988). Nature 336, 775-778.
    • Content area/major concepts: This paper establishes the presence of a molecular cue secreted from rat spinal cord floor plates that regulates both growth and the orientation of growth of spinal cord axons.

      Chemical gradients, diffusion, nervous system organization/development (basic), antibody/antigen
    • Methods or technology used to obtain data: ex vivo 3D tissue culture, immunocytochemistry, nuclear staining (Hoechst).
    • How the CREATE strategy was used:
    • Biggest teaching challenge: The experiments in this paper are relatively limited and seem quite basic to students used to learning about the latest technology/scientific tools. It is most challenging to grasp how such little “data” can lead to such an important conclusion about how axons grow and know where to go. Because it is a Nature Letter, there is a lack of methods described (they are in the figure legends).
    • Article: Orientation of commissural axons in vitro in response to a floor plate-derived chemoattractant. Placzek M, Tessier-Lavigne M, Jessell TM, Dodd J. (1990). Development 110: 19-30.
    • Content area/major concepts: In this paper, they extend their previous study to quantitatively evaluate the effectiveness of the secreted floor-plate derived factor in orienting axon growth. They show the factor can diffuse a significant distance and that this effect is independent of neuronal survival.

      Chemical gradients and diffusion, cell surface vs. secreted molecules, cell adhesion molecules, extracellular matrix, directed cell migration, cell polarity.
    • Methods or technology used to obtain data: ex vivo 3D tissue culture, cell culture, immunocytochemistry, Di-I labeling (identification of specific neuronal populations.
    • How the CREATE strategy was used:
    • Biggest teaching challenge: Relatively straight forward (complements the first one pretty well……similar challenges but better methods)
    • Article: The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6. (1994). Serafini T, Kennedy TE, Galko MJ, Mirzayan C, Jessell TM, Tessier-Lavigne M. Cell 78: 409-424
    • Content area/major concepts: In this paper, the authors identified two proteins from rat embryonic brain that promote commissural axon growth, netrin 1 and netrin 2. They also show that netrin 1 and 2 are homologs of the C. elegans protein UNC-6.

      Chemical gradients/diffusion, chemoattraction/repulsion, CNS development, protein structure/amino acid sequence, RNA vs. DNA vs. protein, transcription/translation,
    • Methods or technology used to obtain data: ex vivo 3D tissue culture, immunohistochemistry, outgrowth activity assay (cell extraction of soluble and application of serial dilutions to tissue explants), protein purification (subcellular fractionation, salt extraction, affinity chromatography etc.), immunoblotting, protein sequencing, RNA isolation, PCR, cloning, RACE, DNA sequencing, southern blot, recombinant protein production, transfection,
    • How the CREATE strategy was used:
    • Biggest teaching challenge: This paper is challenging because for the first time in the series, we now bring the brain into the picture and students have to make the connection that the diffusible cue identified in the spinal cord may be the same molecule that was identified in the brain. The methods are also incredibly extensive and complicated. I predict that this paper will take a lot of time because there are a number of figures and very complex methods.
    • Article: Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord. Kennedy TE, Serafini T, de la Torre JR, Tessier-Lavigne M. (1994). Cell 78: 425-435
    • Content area/major concepts: This paper was published back to back with the previous paper that identified netrin 1 and 2 from brain extracts. Here, they show where netrin 1 and 2 are expressed in the spinal cord and investigate whether soluble netrin is capable of influencing commissural axon growth.

      Cell signaling, protein homology, chemical gradients, anatomy of the embryonic CNS (brain and spinal cord images shown in figures and labeled), transcription/translation,
    • Methods or technology used to obtain data: Northern blot, In situ hybridization, ex vivo tissue culture, expression of recombinant proteins in cell culture, immunoblotting, immunohistochemistry
    • How the CREATE strategy was used:
    • Biggest teaching challenge: In this paper, they argue that it is the combination of netrin 1 and 2 gradients that provide the long-range guidance. This is kind of tricky as the two proteins localize to different regions of the spinal cord. All papers so far have focused so much on the role of the floor plate……but only netrin 1 is found there. This is a bit confusing.
    • Article: Deleted in colorectal cancer (DCC) encodes a netrin receptor. (1996). Keino-Masu K, Masu M, Hinck L, Lenoardo ED, Chan SSY, Culotti JG, Tessier-Lavigne M. Cell 87: 175-785
    • Content area/major concepts: In this paper, the authors identify DCC, the mammalian homolog of UNC-6 (C. elegans) as the netrin-1 receptor that mediates commissural axon growth.

      DNA/RNA/protein (central dogma), sequence homology, phylogenetic conservation, genetic mutations, protein structure (receptors: immunoglobulin , fibronectin domains), protein specificity
    • Methods or technology used to obtain data: degenerate PCR/subcloning to identify receptor, in situ hybridization, immunohistochemistry, recombinant protein expression and purification, binding assays (including radiolabeling for equilibrium binding assays , ex vivo 3D tissue culture
    • How the CREATE strategy was used:
    • Biggest teaching challenge: The identification and cloning of UNC-40 homologs in the rat through the use degenerate primers to amplify sequences by PCR and the subsequent identification of the products by BLAST searching. The methods to identify the protein are similar to those used in paper 3, but in this paper they are a more prominent part of the story and it is necessary to understand it thoroughly to get through the first few figures (in paper 3 it gets overshadowed by the sheer number of other techniques that are used, so the actual identification of netrin and how it is done is not as prominent.)

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