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| Nerve Cell Projections: using a molecular marker to study brain development in the medaka fish |
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Author:
Alexander Picker
Description:
In this activity, we will show how a protein can be visualised in an embryo and how it can be used as a molecular marker.
Molecular markers are molecules that can outline or ëpaintí biological structures by their restricted expression and localisation in a particular tissue. Thus, they can be used to study the anatomy of biological systems as they define tissue territories.
We will visualize the localisation of acetylated a-tubulin, a molecular marker for early neuronal differentiation and will thereby highlight the anatomical structure of the early brain: a complex scaffold of differentiating neurons and their axonal projections.
Acetylated a-tubulin is a particular variant of tubulin, the major protein component of microtubules. Microtubules are an integral part of the cytoskeleton of eukaryotic cells and are involved in a variety of cellular functions, including movement, cell division and sub-cellular protein transport (Please, refer to Practical Activity 2).
In differentiating neurons, microtubules are very important in providing structural support and in ensuring axonal extensions. In this context, a-tubulin on microtubules is highly acetylated and this acetylation stabilizes the microtubules.
The visualization of tubulin will be carried out using immunocytochemistry, a biochemical technique involving the use of an antibody recognising tubulin, and we will compare the localization of tubulin in the brain of wild type and mutant medaka embryos.
Our exercise is intended as an introduction to immunocytochemical methods and their importance in the study of the nervous system.
Objective:
During this activity, students will be exposed to:
The concept of molecular markers
Basic principles of early neural development
The principles of immunocytochemistry
Biological Model:
Oryzias latipes (Medaka fish)
Material:
Protocol: Whole-mount antibody staining of neurons in larvae of the medaka fish [PDF]
Tutorial
1: Principles of Immunocytochemistry [PDF]
Tutorial
2: Basic principles of neurulation [PDF]
Results [PDF]
Glossary [PDF] |
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