Abstract
Purpose :
FOXC1 is a transcription factor involved in heart, craniofacial and ocular development in vertebrates. Mutations in FOXC1, along with mutations in PITX2, cause Axenfeld-Rieger syndrome (ARS) and explain approximately half of the ARS cases. However, there is still a significant number of patients with an unknown genetic cause. Expression and activity of transcription factors involved in development are finely controlled by their regulatory elements that are often evolutionarily conserved. It has been shown that mutations in those regulatory elements could be also pathogenic. The goal of this project is to discover regulatory elements for FOXC1 using zebrafish as a vertebrate model and relate this information to human eye development and disease.
Methods :
BLAST alignments involving regions surrounding human FOXC1 and two orthologous zebrafish genes foxc1a and foxc1b were carried out to identify conserved sequences. CRISPR-Cas9 was used to generate zebrafish lines carrying deletions for the identified conserved elements. The obtained lines were characterized by gross morphology examination, OCT and histology analysis. Expression of foxc1a and foxc1b genes and encoded proteins was assessed by RT-qPCR, in situ hybridization with RNAscope probes and immunohistochemistry.
Results :
We identified 3 elements downstream of human FOXC1 and 1 element upstream of FOXC1 that were conserved in zebrafish foxc1a or in both foxc1a and foxc1b. The deletion of a 152kb intergenic region comprising all 3 downstream elements (ΔCED1-3) resulted in a downregulation of both the foxc1a transcript and protein in developing zebrafish embryos. Homozygous ΔCED1-3 larvae die at 1 month post fertilization and present with enlarged anterior chambers of the eye and a significant pericardial and other organs’ edema. Deletion of an 82.7Kb region containing only 2 out of the 3 downstream conserved elements (ΔCED2-3) produced a similar but milder phenotype. Deletion of elements upstream either foxc1a (ΔCEU1a) and foxc1b (ΔCEU1b) did not produce a visible phenotype.
Conclusions :
The identified downstream conserved elements are essential for normal foxc1a expression and their deletion results in a phenotype consistent with foxc1/FOXC1 deficiency in zebrafish and humans. Further studies of these regions in human patients is likely to explain additional ARS cases.
This is a 2021 ARVO Annual Meeting abstract.