Background 

Transcription factors (TFs) are regulatory proteins that control gene expression by binding to specific DNA sequences and influencing transcription. One area of development TF become increasingly important is cranial placodes. During early development, cranial placodes arise from a shared region of multipotent surface ectoderm. For example, both the lens placode and olfactory placode originate from this common surface ectoderm tissue before differentiating into distinct cell types. In the lens, FOXE3 is a forkhead transcription factor that is specifically expressed in the lens, a non nerual tissue, where it regulates cell proliferation and differentiation. In contrast, the olfactory placode gives rise to both neural and non-neural tissues but does not naturally express FOXE3. To study gene function in this system, the RCAS method, an avian specific retrovirus-based approach, can be used to deliver genes into cells to be incorporated into the host genome and subsequently expressed.

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?

The rationale for this project is based on evidence from FOXE3 null mice, which showed increased expression of neural genes and decreased expression of epithelial markers compared to wild type. This may suggest that FOXE3 may normally function to maintain epithelial identity and suppress neural gene programs during development. However, FOXE3 is not endogenously expressed in all ectoderm-derived tissues, such as the olfactory placode, raising the question of whether it can actively suppress neural development in a tissue where it is normally absent.

To test this, we used the RCAS retroviral system to introduce FOXE3 into early embryonic tissue. Embryos were injected at stage 10, when the surface ectoderm has not yet fully differentiated into distinct structures such as the lens and olfactory placodes. This timing allows FOXE3 to be expressed prior to lineage commitment. Tissues were then harvested at embryonic day 4 (E4), a developmental stage when early neurogenesis is occurring.

Methods

Results 

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

Figure 3. 

Future Directions 

Future studies will further refine the temporal and cellular characterization of FOXE3 function during development. Analysis of embryos at embryonic day 6 (E6) will be important to determine whether the effects observed at earlier stages persist into later phases of neurogenesis or instead reflect transient developmental changes. In parallel, antibody staining approaches will be repeated and optimized to improve the identification of post-mitotic neuronal populations, allowing for a more precise assessment of neuronal differentiation. Quantitative analysis of neuronal and non-neuronal cell populations within the olfactory epithelium will also be performed to assess potential shifts in tissue composition that may not be evident through morphological evaluation alone. Additionally, refinement of viral delivery methods will be necessary to ensure more consistent and efficient RCAS-mediated expression, thereby improving experimental reproducibility across samples.

Nace Competencies & Acknowledgements 

Career and self-development: by pushing me to set independent goals and reflect on setbacks

Communication skills: through presenting findings, discussing troubleshooting strategies with peers, and translating my work and literature findings into my Honors Thesis

Professionalism: by requiring consistent time management and accountability for experimental outcomes. 

Thank you to KDRT Lab & Nour Sayed 

Funding NIH R21 EY031092 Grant