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Author:
Alexander Manaia, Ana Cyrklaff, Anne Ephrussi
Description:
During the development of many organisms, the main body axes (A-P: anterior-posterior; D-V: dorsal-ventral) are established at very early stages.
In the fruit fly Drosophila melanogaster, the polarity of these axes is already determined in the oocyte, the cell that will later develop into the fly egg. In the oocyte, messenger mRNAs are produced and are placed in precise regions, acting as position determinants and drawing the map of the body as the fly develops.
Specific sets of mRNAs are positioned anteriorly, others posteriorly and others at the extremities of the oocyte, guiding the formation of the anterior-posterior axes of the body. The localization of the maternal mRNAs in the egg depends upon binding to specific proteins present in the cytoplasm of the oocyte, one of them is the Staufen protein.
The Staufen protein is responsible for the establishment of the A-P axis and it must be located at the posterior pole of the oocyte. It can hold to this particular position through binding to specific components of the oocyte cytoskeleton, the microtubules. These are dynamic and polarized structures, possessing a fast growing end (plus end), oriented to the posterior pole of the egg and a slow growing end (minus end), oriented towards the anterior pole of the egg. Staufen is exclusively able to bind to the plus pole of microtubules.
Similarly as in Practical Activity 2, we will see how mutations in the DNA sequence result in modifications in the usual characteristics of organisms, making them different from the normal population (wildtype). Drosophila melanogaster is a very powerful experimental model for genetic studies.
We will use mutant flies that have a default at the microtubule polarisation level. Instead of being oriented towards the posterior pole of the oocyte, the plus ends of the microtubules are oriented towards the centre. As a consequence, the staufen protein is mislocalised to the centre of the oocyte and the A-P axis is disrupted.
During this hands-on activity you will be able to identify mutant flies by observing their striking external morphological aspects. We will investigate the mutation at the molecular level by comparing the localisation of staufen in wild type and mutant flies by immunocytochemistry.
Objective:
Screen mutants both at the organism and molecular levels.Learn how to visualize the global distribution of a protein in the context of an organism at a particular phase of its development.
Biological Model:
The fruit fly Drosophila
Keywords:
Drosophila, oocyte, embryo, body plan, body axes, microtubules, adult fly.
Material:
Tutorial
1: Drosophila as a model organism [PDF]
Tutorial
2: Principles of immunocytochemistry [PDF]
Protocol
Day 1: Sorting adult flies [PDF]
Protocol
Day 2: When a protein is out of place [PDF]
Results
[PDF]
Glossary
[PDF]
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