Investigations into the roles of Wnt/beta-catenin pathway in retinal development and degeneration

Project Aim

The aim of this project was to develop techniques that would allow for the investigation of the role of the Wnt/beta-catenin signalling pathway in the developing and adult retina. The cell signalling pathway has been shown to exhibit changes in the presence of retinal degenerative disease. It is the goal of this study to better understand the Wnt/beta-catenin signalling pathway’s role in the development and function of the retina as well as how these changes occur and their significance in the context of retinal degeneration.

Project Summary and Impact

The Wnt/beta-catenin signalling pathway is present in both the developing and adult eye. Studies in other organs have shown this cell signalling pathway plays a crucial role in maintaining functional stem cell populations, particularly during embryonic development. Studies have shown that this signalling pathway exhibits changes in the presence of degenerative retinal disease. However, It is not yet understood whether these changes contribute to the cause of retinal degeneration, or if they are a consequence of the disease and reflect attempts by the cells of the retina to compensate for degenerative processes. Studies of this pathway in other organs indicate that it plays a crucial role in maintaining functional stem cell populations (the cells responsible for replacing damaged cells and have potential for future therapies), particularly during embryonic development.
An important way for us to understand how genes function is by manipulating the activity of genes in animals. A common way of doing this is by using a “gene knockout” approach in mice. The Cre-LoxP method allows a gene to be edited in a specific tissue or at a specific time by using a modular system, comprising of the Cre recombinase enzyme and its specific DNA binding site (LoxP sites). By manipulating where Cre recombinase is produced, we can mutate or delete a gene in the tissue of interest (the retina), without affecting the viability of the offspring.
The first aim of this project was to characterise the patterns of Cre activity in the two lines of genetically altered mice (MLR34 and MLR37) to see if these could be used to delete genes in the retina. The second aim was to investigate if Cre expressed in these mice could actually be used to delete a functional gene in the developing mouse retina. Beta-catenin is a central component of the Wnt signalling pathway and its deletion would prevent this pathway from functioning.
To address the first aim, we mated the MLR34 and MLR37 mice with “reporter” mice. The reporter mice have a special LoxP-flanked gene in their DNA that respond to the presence of Cre, by producing the enzyme beta-galactosidase, which can be stained blue with X-Gal. This technique was able to show where Cre is active in the developing retina.
To address the second aim, we investigated whether the levels of Cre in the retina were sufficient to actually delete a gene in the Cre mice (MLR34 and MLR37). By utilising polymerase chain reactions (PCR), we showed that the structure of the retinas in the mutant eyes was very similar to the normal eye. This could be due to the deletion of beta-catenin having minimal/subtle effects on retinal development, or that the levels of Cre in the retina were not sufficient to delete the gene from many of the target cells. To investigate this further we used antibodies to label the cells that have Cre red, and to label beta-catenin green. This showed that we had in fact deleted beta-catenin in some of the retinal cells, but the deletion was not very extensive. This means we can use these mice to study the area of affected cells compared to non-effected cells within the same retina.
Our future studies are aimed at increasing the efficiency of the Cre activity in the retina of our mutant mice through breeding experiments to hopefully generate more extensive gene deletions in the retina for us to study the effects of the beta-catenin gene in the developing eye.

This project was able to successfully use gene knockout technology in mice to investigate the role of the Wnt/beta-catenin pathway in the developing and adult retina. We were able to quantify the level of Cre in the mutant mice retinas as well as confirm the deletion of beta-catenin in some retinal cells, whilst others remained unaffected. This will allow us to compare the impact of the affected cells with non-affected cells within the same retina. Using this information, we are now undergoing further investigations into the role of the Wnt/beta-catenin pathway in the developing and adult retina, as well as its significance/role in the presence of retinal degeneration.

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Chief investigator:
Dr Robb de Iongh
University of Melbourne

Co-investigator/s:

Dr Helen E Abud, Ludwig Institute for Cancer Research

Grant awarded:
$35,000 (2005)