Background 

Transcription factors (TFs) are key regulatory proteins that control gene expression by binding to specific DNA sequences and influencing transcription. One such factor, FOXE3, is a forkhead transcription factor expressed in the developing lens, where it plays a critical role in cell proliferation and differentiation during lens formation. To investigate FOXE3 function, it was overexpressed using an avian-specific RCAS vector. RCAS, derived from the Avian Sarcoma-Leukosis Virus (ASLV), enables sustained gene expression in chicken cells and is widely used in developmental biology research. This vector originates from Rous Sarcoma Virus (RSV), the first retrovirus ever discovered, known for causing cancer in chickens. As a retrovirus, RCAS carries its genetic material as RNA and relies on the enzyme reverse transcriptase to convert RNA into DNA within the host cell. The resulting DNA is integrated into the host genome, allowing the inserted gene—such as FOXE3—to be stably expressed in the target cells.

Research Questions and Rationale 

This project explores the role of FOXE3 in eye development, focusing on its potential influence on cell fate decisions. Key questions include: What is the role of FOXE3 in determining cell fate during eye development? Does FOXE3 suppress neural differentiation pathways? Lastly, can over expressing FOXE3 in the developing chick retina lead to retinal abnormalities?

Lens epithelial cells, which naturally express FOXE3, do not undergo neural differentiation, in contrast to retinal cells that depend on neural gene expression for proper development. This observation suggests that FOXE3 may play a role in repressing neural pathways. Therefore, we hypothesize that early over expression of FOXE3 in the retina will interfere with neural differentiation and lead to disruptions in normal retinal development.

Methods

Results 

Figure 2. A. Restriction map of enzymes used within both RCAS-GFP and RCAS FOXE3 GFP Vector B. Restriction Digest of RCAS vectors with FLAG-tagged human FOXE3T2AGFP; Gel Legend is shown below

Figure 3. 

Future Directions 

To investigate the effects of FOXE3 expression on neural development, RCAS viruses will be concentrated, titered, and used for chick embryo injections. Injections will be administered into the neural tube at stage 10 (approximately 33–38 hours post-fertilization), a critical window when the optic vesicles are forming and the overlying ectoderm is thickening into the lens placode. Embryos will be harvested at stage 23 (E4 or 96 hours post-fertilization), a point at which the optic cup has begun to differentiate into retinal tissue. Morphological differences in neural structure between control and FOXE3-overexpressing embryos will be assessed through hematoxylin and eosin (H&E) staining and section analysis. If no overt phenotypic changes are observed, immunohistochemistry will be employed to detect more subtle alterations in protein expression.

Career Readiness Skills 

Conducting research has significantly strengthened my critical thinking and problem-solving skills as I’ve learned to design experiments, analyze data, and draw meaningful conclusions. Collaborating with peers and faculty has enhanced my teamwork and collaboration, while presenting findings in written and oral formats has improved my communication skills. Additionally, navigating complex research tools and methodologies has advanced my technology proficiency, making me more adaptable in professional settings.

Acknowledgements 

MS41093 Robinson External Proposal Submission Incentive (EPSI) Facilities and Administration Recovery 

Katia Del-Rio Tsonis Lab