SPECIFIC AIMS T cell immunodeficiency, congenital alopecia, and nail dystrophy is a rare genetic disorder that results from a mutation in the FOXN1 gene. The titular phenotype associated with the disease is caused by lack of differentiation of epithelial cells that make up the thymus, surround hair follicles, and are involved in nail growth [1]. The FOXN1 gene codes for the Forkhead Box 1 Helicase Transcription factor which regulates transcription by binding to specific DNA sequences with its binding domain [2]. While this transcription factor is well known for its role in thymopoiesis and hair follicle differentiation, it also seems to play an important role in wound healing [3]. One way in which this role manifests is in FOXN1 deficient mice as they have been shown to heal at an accelerated rate and do not scar [4]. While FOXN1 is known to be involved in wound healing, how it regulates this process has yet to be fully understood.
Our primary goal is to elucidate specific mechanisms that FOXN1 is involved in during epithelial cell differentiation and how they pertain to wound healing. The long-term goal of this project is to form a broader understanding of the epithelial environment during wound healing to improve outcomes for wounded individuals. To understand FOXN1’s role in wound healing, I will use zebrafish as a model, as their nearly transparent skin allows for fluorescent imaging of molecular processes and their external development allows for characterization of development processes. I hypothesize that mutations in FOXN1 will lead to upregulation of genes related to proliferation and cell movement during wound healing and downregulation of proteins associated with differentiation during development.
AIM 1: Find conserved amino acids among FOXN1 orthologs Approach: I will use NCBI BLAST to identify orthologs of FOXN1 within other organisms and use Clustal Omega to align their sequences to determine conserved amino acids across species within the Forkhead Domain and the overall protein. CRISPR-Cas9 will then be used to create mutations in the known mutation site R320W, another in the FHD, K316W, and one outside the FHD, S535W. To validate the model, a wound healing assay will be completed on each mutant and WT, determining the change in wound area throughout time. Hypothesis: FOXN1 WT and S535W zebrafish will show lower rates of re-epithelialization than those with R320W and K316W mutations. Rationale: Mutations in the FH domain likely leads to defects in DNA-binding, which is important to the FOXN1’s transcription factor activity.
AIM 2: Identify differentially expressed genes in FOXN1 mutants during wound healing Approach: This approach will use validated mutant and control zebrafish. We will take epidermal samples from unwounded zebrafish and wounded (at the wound edge) zebrafish and will utilize RNA-sequencing to determine differential gene expression. Relative gene expression from the validated FOXN1-deficient zebrafish will be compared to that of the WT. Differentially expressed genes will then be sorted by GO. Differentially expressed genes found to be involved in cell movement and proliferation will then be mutated in FOXN1-deficient zebrafish using CRISPR-Cas9 and a wound healing assay will be performed to validate these genes’ roles in re-epithelialization. Hypothesis: FOXN1 mutants will have increased expression of genes related to cell movement and proliferation. Rationale: Re-epithelialization requires skin cells to move and proliferate to cover wounds.
AIM 3: Identify proteins important for epithelial development that cause faster re-epithelialization Approach: This approach will also use the validated mutant and control zebrafish lines. Epithelial samples will be taken from each zebrafish line at 2dpf, 6dpf, and 12dpf. These cells will then be independently digested and labeled using iTRAQ before being analysis using mass-spectrometry to determine the proteins present, then sorted by GO. Differentially expressed proteins found to be involved in epithelial cell differentiation will then be mutated using CRISPR-Cas9 and validated using the wound healing assay described in aim 1. Hypothesis: FOXN1 mutant epithelial cells will show decreased levels of proteins associated with differentiation throughout zebrafish development. Rationale: Lack of differentiation of epithelial cells in the mutant zebrafish may play a role in the accelerated wound healing phenotype, so proteins involved in the early differentiation process may affect the wound healing process later.
Through these aims, I hope to determine FOXN1’s regulatory role in epithelial cell differentiation and it’s contribution to wound healing. This will help us better understand how the epithelial environment plays a role in wound healing. In the future, CRISPRa could be utilized in a way to help rescue the phenotype of FOXN1 mutants.
Romano, R., Palamaro, L., Fusco, A., Iannace, L., Maio, S., Vigliano, I., Giardino, G., & Pignata, C. (2012). From murine to human nude/SCID: the thymus, T-cell development and the missing link. Clinical & developmental immunology, 2012, 467101. https://doi.org/10.1155/2012/467101
Bukowska, Joanna, et al. “FOXN1 in skin development, homeostasis and wound healing.” International Journal of Molecular Sciences, vol. 19, no. 7, 4 July 2018, p. 1956, https://doi.org/10.3390/ijms19071956.
Gawronska-Kozak, Barbara. “Scarless skin wound healing in Foxn1 deficient (nude) mice is associated with distinctive matrix metalloproteinase expression.” Matrix Biology, vol. 30, no. 4, 1 May 2011, pp. 290–300, https://doi.org/10.1016/j.matbio.2011.04.004.