Axenfeld-Rieger syndrome-associated mutants of the transcription factor FOXC1 abnormally regulate NKX2-5 in model zebrafish embryos

Abstract

<jats:p>FOXC1 is a member of the forkhead family of transcription factors, and whose function is poorly understood. A variety of <jats:italic>FOXC1</jats:italic> mutants have been identified in patients diagnosed with the autosomal dominant disease Axenfeld-Rieger syndrome, which is mainly characterized by abnormal development of the eyes, particularly those who also have accompanying congenital heart defects (CHD). However, the role of FOXC1 in CHD, and how these mutations might impact FOXC1 function, remains elusive. Our previous work provided one clue to possible function, demonstrating that zebrafish <jats:italic>foxc1a</jats:italic>, an orthologue of human <jats:italic>FOXC1</jats:italic> essential for heart development, directly regulates the expression of <jats:italic>nkx2.5</jats:italic>, encoding a transcriptional regulator of cardiac progenitor cells. Abnormal expression of <jats:italic>Nkx2-5</jats:italic> leads to CHD in mice and is also associated with CHD patients. Whether this link extends to the human system, however, requires investigation. In this study, we demonstrate that FOXC1 does regulate human <jats:italic>NKX2-5</jats:italic> expression in a dose-dependent manner via direct binding to its proximal promoter. A comparison of FOXC1 mutant function in the rat cardiac cell line H9c2 and zebrafish embryos suggested that the zebrafish embryos might serve as a more representative model system than the H9c2 cells. Finally, we noted that three of the Axenfeld-Rieger syndrome FOXC1 mutations tested increased whereas a fourth repressed the expression of <jats:italic>NKX2-5</jats:italic>. These results imply that mutant FOXC1s might play etiological roles in CHD by abnormally regulating <jats:italic>NKX2-5</jats:italic> in the patients. And zebrafish embryos can serve as a useful in vivo platform for rapidly evaluating disease-causing roles of mutated genes.</jats:p>

Mechanistic Insights into Axenfeld–Rieger Syndrome from Zebrafish foxc1 and pitx2 Mutants

“…This expression pattern highlights the important role of neural crest cells in development and helps to explain the broad range of phenotypes observed in patients with ARS. The LPM also contributes to the development the heart and cardiovascular system and thus defects in this tissue arising from FOXC1 dysfunction may contribute to cardiovascular anomalies observed in ARS patients [ 21 , 26 , 27 ]. Although typically known as a developmental disease, foxc1a continues to be expressed in the adult zebrafish eye, including the anterior segment and retinal ganglion cell layer [ 28 ], possibly contributing to maintenance of adult ocular tissues.…”

“…In zebrafish, mutation of foxc1a alone or in combination with foxc1b leads to cardiac phenotypes that include hypoplastic myocardium and ventricular outflow tract, as well as defects in cardiac valve formation that are similar to that observed in patients with FOXC1 -attributible ARS [ 21 , 26 ]. While zebrafish studies generally support a role or foxc1 in heart development, reports differ in their analysis of heart morphology, assay different time points, and study different combinations of mutations.…”

“…Utilizing foxc1 homozygous loss of function embryos, a role for this gene in regulating cardiac progenitor specification and atrioventricular canal (AVC) formation has been proposed. Such studies show that Foxc1a directly binds to the promoter of nkx2.5 [ 26 ], a gene required for cardiac progenitor specification in the lateral plate mesoderm (LPM). The reduced expression of nkx2.5 in zebrafish foxc1 homozygous mutants provides mechanistic insight into the hypoplastic myocardium observed in ARS patients.…”

Cardiac anomalies in Axenfeld–Rieger syndrome

“…1,2 A number of extraocular manifestations have been described including craniofacial and dental anomalies, umbilical abnormalities, pituitary abnormalities, sensorineural hearing loss, and CHDs. 3,4 Approximately half of all cases are due to mutations in Forkhead Box C1 or paired-like homeodomain transcription factor 2. 2,3 Both Forkhead Box C1 and paired-like homeodomain transcription factor 2 encode transcription factors that play key roles during embryonic development.…”

“…3,5,6 Insight into the mechanisms by which mutations in Forkhead Box C1 and paired-like homeodomain transcription factor 2 lead to Axenfeld-Rieger syndrome has been largely derived from mouse and zebrafish models, highlighting abnormalities of neural crest cell migration as well as variation in the expression of genes critical to embryonic development. 2,4,[7][8][9][10][11][12] Both genes have been associated with determination of the left-right axis, with clear potential implications for cardiovascular development. 7,8,[13][14][15][16] Importantly, transcriptional activity of Forkhead Box C1 is negatively regulated by paired-like homeodomain transcription factor 2, helping to explain the overlapping phenotypes.…”