Bret Gardner and Dr. Michael Stark, Department of Physiology and Developmental Biology
Neural tube defects (NTDs) are among the most common congenital malformations, afflicting .5- 8/1000 live births (Saitsu, 2003). Examples of such defects include spina bifida and anencephaly, resulting from failed closure of the posterior and anterior neural tube, respectively. The neural tube is the fetal precursor to the central nervous system. Currently the most common and effective method for preventing such defects consists of periconceptional folate intake. In developed countries, sufficient folate (folic acid or vitamin B12) is readily obtained from multivitamins, enriched cereals, or other items in a balanced diet. This preventative treatment, however, remains ineffective in 30-50% of cases and in some of these instances maternal folate levels fall within the normal range (Greene, 2009). The reasons behind continued incidence of NTDs remain nebulous. The aim of my study that was partially funded by this ORCA Grant was to further elucidate the role that folate plays during neurulation using the chicken as a model organism. By completing and publishing this research, I hope to provide beneficial information to increase the effectiveness of treatments and potentially develop additional treatments to further reduce the prevalence of NTDs.
As with much scientific research, the procedures outlined in the grant proposal did not go exactly as planned. We initially proceeded as previously described, bathing chicken embryos in a solution of folate-FITC and PBS and later imaging them with fluorescent microscopy. However, these embryos failed to demonstrate any cell autonomous or consistent fluorescence which would have indicated active uptake of folate. Protocols were manipulated, varying incubation times, folate concentration, and even performing tissue culture to completely submerge a section of live tissue in the bath of folate. Even with these modifications, results remain elusive.
Rather than abandoning the project or readily admitting defeat, we are still designing experimental approaches and will continue working on this project throughout the Winter semester of 2011. Current efforts involve constructing a probe to identify mRNA expression of the folate binding protein via in situ hybridization. While this has been performed with great success previously in mouse (Saitsu, 2003) this method has not been published with chicken as a model organism. In this process, we are currently designing primers to amplify the gene from the complete chicken genome, isolate the gene, clone it into a vector, and then create our own probe for the folate binding protein. We hope that we may effectively use this probe to create an accurate temporal and spatial map of folate binding protein expression in chicken during neurulation.
Other efforts to further identify the role of folate will use the folate-FITC conjugate originally described, while using mouse embryos. Injection of this conjugate into pregnant mice will allow active uptake of folate to be monitored during various stages of neurulation. This monitoring will serve as a beneficial supplement to the mRNA expression map of folate binding protein currently available.
Finally, ongoing efforts also include further exploration of signaling pathways that have been shown to play a role in neural tube closure. We are using quantitative real-time PCR to monitor expression levels of Pax3 at different time points, preceding and following neural tube closure. Preliminary results indicate Pax3 is expressed as hypothesized and that isoforms of Pax3 are expressed at varying levels when these time points are compared. Manipulation of Pax3 expression in order to observe any effects on folate binding protein activity are still planned.
Pursuing this project has been a tremendous learning experience as part of my undergraduate career. As I plan to pursue a career in biomedical research, having the opportunity to be involved in primary research, proposing a personal project, identifying its efficacy for benefiting humanity, and struggling to obtain results are marvelous opportunities. I have learned to work diligently and think creatively to overcome unsuccessful procedures. I have learned to work closely with my mentor and my peers, relying on their greater experience and unique insights as I seek for further understanding and direction. Finally, and of great importance, I have learned that my love for research and discovery is not simply a passing whim, but rather a passion I am excited to pursue.