This web page was produced as an assignment for Genetics 677, an undergraduate course at UW-Madison.
Conclusions
Because Crouzon Syndrome is not well studied, and there is little known about how it manifests in humans, there are many possible directions to take further research about the disease and about Fgfr2. In reading the literature that has been published about Crouzon Syndrome and related diseases, it seemed there were many possible routes from which the variety of phenotypes could develop. These possibilities include poor cell migration, incorrect cell differentiation, dysfunctional cell proliferation, and countless others. I chose to look into cell migration for this project.
Hypothesis 1: a disruption of cell migration which causes the trachea phenotype of Crouzon Syndrome
This website exemplifies how beneficial bioinformatics databases can be to science today. I used those bioinformatic tools to propose a hypothesis about the Fgfr2 gene and its role in Crouzon Syndrome. As mentioned, there are countless paths one could take to begin researching this disease, and I chose to focus on one particular phenotype. Since the skull development phenotypes are best studied, I wanted to take my search in a different direction. I chose to look into the improper tracheal development that occurs in some Crouzon patients.
I chose to look at this phenotype in flies because, as a model organism, they best fit my ideas for research, and the Fgfr2 gene is well conserved in them. Other model organisms were not an option simply because they do not have a tracheal system, and others, like mice, are too expensive and time consuming to maintain. Also, in flies, the phenotype seems to be fairly consistant. In mammals, the phenotypes vary so much that is difficult to draw solid conclusions or repeat results. As mentioned, flies have a well conserved Fgfr2 homolog, Breathless (Btl), and I began by looking at the STRING network for Btl. The proteins in this network had a number of different term associations when I used AmiGO to search the biological processes in which they are involved, but only one protein, Stumps, was associated with cell migration in the tracheal system. Doing a simple literature search of Btl and Stumps, I found the two genes, when mutated seperately, produce very similar phenotypes. Also, I found the two interact with one another in a yeast-two-hybrid assay. Therefore, it is likely the two genes are working in the same pathway. To further make sure Stumps was a good candidate, I did an alignment in T-Coffee with it and its homologs humans, mice, and mosquitoes. Using Prosite, I found that certain areas of the alignment corresponded to a protein domain known as the DBB domain. This domain is only found in Stumps and its homologs. Prosite also provided information about the domain's binding abilities, and according to that, the DBB domain is capable of binding to a tyrosine phosphatase site. These sites are involved in phosphate transferring, and there is a domain within the Fgfr2 protein, a tyrosine kinase domain, that is responsible for transferring phosphate groups. This provides a link between Btl and Stumps and how they might be interacting. Knowing this information, I revisited my hypothesis to narrow it and focus on future experiments that might lead to more insight.
Hypothesis 2: Disruption of the Btl/Stumps interaction causes a disruption of cell migration which causes the trachea phenotype of Crouzon Syndrome
With this more directed hypothesis, I would perform a number of experiments to test its validity. First, I propose an experiment to test whether or not migration is the culprit of the poor tracheal development. Using flies with a mutated Btl gene which display the phenotype seen here, it would be simple to take transverse sections (with a one cell thickness) of the tracheal system and stain the tissue for epithelial cells. This could be compared to stained tissue sections of wild-type flies, and a cell count could be performed. If the same number of cells exists just in different formations between the control and mutant tissue, it would indicate migration is a likely cause for the truncated phenotype. Further migration experiments would be done using a Boyden Chamber Assay (1). Using the Boyden Chamber method, I could test the ability of the cells which express Btl and/or Stumps to migrate in response to different stimuli. This could be compared with cells where Btl and/or Stumps is knocked down to see if their migration patterns differ. If the migration patters differ.
Next, I would propose would be an immunofluorescence test to see if Btl and Stumps are colocalizing in tracheal tissue. By using an antibody of one color to detect the presence of Btl and an antibody of another color to detect Stumps I could compare the expression of the two proteins in wild-type and mutant tissue to see if the interaction between them differs. A decrease in colocalization in the mutant would suggest the disruption of this interaction is involved in the mutant phenotype.
In summary, my goal is to determine the role of cell migration in the Breathless mutant tracheal phenotype and to understand how Stumps is working in the Breathless pathway.
Hypothesis 1: a disruption of cell migration which causes the trachea phenotype of Crouzon Syndrome
This website exemplifies how beneficial bioinformatics databases can be to science today. I used those bioinformatic tools to propose a hypothesis about the Fgfr2 gene and its role in Crouzon Syndrome. As mentioned, there are countless paths one could take to begin researching this disease, and I chose to focus on one particular phenotype. Since the skull development phenotypes are best studied, I wanted to take my search in a different direction. I chose to look into the improper tracheal development that occurs in some Crouzon patients.
I chose to look at this phenotype in flies because, as a model organism, they best fit my ideas for research, and the Fgfr2 gene is well conserved in them. Other model organisms were not an option simply because they do not have a tracheal system, and others, like mice, are too expensive and time consuming to maintain. Also, in flies, the phenotype seems to be fairly consistant. In mammals, the phenotypes vary so much that is difficult to draw solid conclusions or repeat results. As mentioned, flies have a well conserved Fgfr2 homolog, Breathless (Btl), and I began by looking at the STRING network for Btl. The proteins in this network had a number of different term associations when I used AmiGO to search the biological processes in which they are involved, but only one protein, Stumps, was associated with cell migration in the tracheal system. Doing a simple literature search of Btl and Stumps, I found the two genes, when mutated seperately, produce very similar phenotypes. Also, I found the two interact with one another in a yeast-two-hybrid assay. Therefore, it is likely the two genes are working in the same pathway. To further make sure Stumps was a good candidate, I did an alignment in T-Coffee with it and its homologs humans, mice, and mosquitoes. Using Prosite, I found that certain areas of the alignment corresponded to a protein domain known as the DBB domain. This domain is only found in Stumps and its homologs. Prosite also provided information about the domain's binding abilities, and according to that, the DBB domain is capable of binding to a tyrosine phosphatase site. These sites are involved in phosphate transferring, and there is a domain within the Fgfr2 protein, a tyrosine kinase domain, that is responsible for transferring phosphate groups. This provides a link between Btl and Stumps and how they might be interacting. Knowing this information, I revisited my hypothesis to narrow it and focus on future experiments that might lead to more insight.
Hypothesis 2: Disruption of the Btl/Stumps interaction causes a disruption of cell migration which causes the trachea phenotype of Crouzon Syndrome
With this more directed hypothesis, I would perform a number of experiments to test its validity. First, I propose an experiment to test whether or not migration is the culprit of the poor tracheal development. Using flies with a mutated Btl gene which display the phenotype seen here, it would be simple to take transverse sections (with a one cell thickness) of the tracheal system and stain the tissue for epithelial cells. This could be compared to stained tissue sections of wild-type flies, and a cell count could be performed. If the same number of cells exists just in different formations between the control and mutant tissue, it would indicate migration is a likely cause for the truncated phenotype. Further migration experiments would be done using a Boyden Chamber Assay (1). Using the Boyden Chamber method, I could test the ability of the cells which express Btl and/or Stumps to migrate in response to different stimuli. This could be compared with cells where Btl and/or Stumps is knocked down to see if their migration patterns differ. If the migration patters differ.
Next, I would propose would be an immunofluorescence test to see if Btl and Stumps are colocalizing in tracheal tissue. By using an antibody of one color to detect the presence of Btl and an antibody of another color to detect Stumps I could compare the expression of the two proteins in wild-type and mutant tissue to see if the interaction between them differs. A decrease in colocalization in the mutant would suggest the disruption of this interaction is involved in the mutant phenotype.
In summary, my goal is to determine the role of cell migration in the Breathless mutant tracheal phenotype and to understand how Stumps is working in the Breathless pathway.
Future directions
Continuing experiments in this area would include a protein domain interaction experiment. By testing the interaction of these two proteins by their individual domains, we could learn about exactly where they are interacting and what exactly they might be doing via that interaction. Another experiment might look at the other players in the Ras pathway because these have been linked to Btl and Stumps (2). Lastly, the future of this research will need to focus on learning about wild-type development. Cell differentiation, proliferation, and migration in the trachea and upper airways is very poorly understood by research today. Understanding how normal tracheal development occurs will be essential to making breakthroughs in any diseases that affect this area of development.
When it comes to Crouzon Syndrome and the involvement of the Fgfr2 gene, there are a number of possibilities for improper development. It is clear that a lot of research is yet to be done in this area. Perhaps it is cell migration, as I hypothesized, and studying the migration pathway in flies will just be the start to a breakthrough in Crouzon research. However, there are many, many possible hypotheses and several other model organisms that will probably have to be explored before clinical research for this disease really takes off.
When it comes to Crouzon Syndrome and the involvement of the Fgfr2 gene, there are a number of possibilities for improper development. It is clear that a lot of research is yet to be done in this area. Perhaps it is cell migration, as I hypothesized, and studying the migration pathway in flies will just be the start to a breakthrough in Crouzon research. However, there are many, many possible hypotheses and several other model organisms that will probably have to be explored before clinical research for this disease really takes off.
References
[1] Chen, H. C. Boyden chamber assay. 2005. Methods of Molecular Biology. 294:15-22
PMID: 15576901
[2] Imam, F., Sutherland, D., Huang, W., Krasnow, MA. Stumps, a Drosophila gene required for fibroblast growth factor (FGF)-directed migration of tracheal and mesodermal cells. 1999. Genetics. 152(1):307-18
PMID: 10224263
PMID: 15576901
[2] Imam, F., Sutherland, D., Huang, W., Krasnow, MA. Stumps, a Drosophila gene required for fibroblast growth factor (FGF)-directed migration of tracheal and mesodermal cells. 1999. Genetics. 152(1):307-18
PMID: 10224263