Disruption of foxc1 genes in zebrafish results in dosage-dependent phenotypes overlapping Axenfeld-Rieger syndrome

Abstract

The Forkhead Box C1 (FOXC1) gene encodes a forkhead/winged helix transcription factor involved in embryonic development. Mutations in this gene cause dysgenesis of the anterior segment of the eye, most commonly Axenfeld-Rieger syndrome (ARS), often with other systemic features. The developmental mechanisms and pathways regulated by FOXC1 remain largely unknown. There are two conserved orthologs of FOXC1 in zebrafish, foxc1a and foxc1b. To further examine the role of FOXC1 in vertebrates, we generated foxc1a and foxc1b single knockout zebrafish lines and bred them to obtain various allelic combinations. Three genotypes demonstrated visible phenotypes: foxc1a−/− single homozygous and foxc1−/− double knockout homozygous embryos presented with similar characteristics comprised of severe global vascular defects and early lethality, as well as microphthalmia, periocular edema and absence of the anterior chamber of the eye; additionally, fish with heterozygous loss of foxc1a combined with homozygosity for foxc1b (foxc1a+/−;foxc1b−/−) demonstrated craniofacial defects, heart anomalies and scoliosis. All other single and combined genotypes appeared normal. Analysis of foxc1 expression detected a significant increase in foxc1a levels in homozygous and heterozygous mutant eyes, suggesting a mechanism for foxc1a upregulation when its function is compromised; interestingly, the expression of another ARS-associated gene, pitx2, was responsive to the estimated level of wild-type Foxc1a, indicating a possible role for this protein in the regulation of pitx2 expression. Altogether, our results support a conserved role for foxc1 in the formation of many organs, consistent with the features observed in human patients, and highlight the importance of correct FOXC1/foxc1 dosage for vertebrate development.

The Axenfeld–Rieger Syndrome Gene FOXC1 Contributes to Left–Right Patterning

“…native start sites (Figure S3), both mutations occur in the N-terminal transactivation domain where even small deletions profoundly reduce FOXC1 activity [75]. Furthermore, our novel mutations overlap previously published foxc1 null mutations (el542, el543, and el620 [60]) and phenocopy loss-of-function mutations in the Forkhead box DNA-binding domain [76]. For these reasons, we believe that the novel foxc1 mutations provide good models for foxc1 loss-of-function.…”

“…Firstly, previous studies whereby foxc1b was knocked down via morpholino oligonucleotides failed to resolve a phenotype despite foxc1a knockdown resulting in microphthalmia, somitic delay, and intracranial hemorrhage [ 46 , 66 , 86 ]. Furthermore, foxc1b mutants have also been described without resolving a phenotype [ 60 , 76 ]. Our findings demonstrate that foxc1b is required for cardiac situs in isolation.…”

“…Secondly, similar human mutations upstream of the Forkhead box DNA-binding domain are loss-of-function alleles that cause Axenfeld-Rieger syndrome [38,42,87]. Considering the significantly overlapping expression patterns of the foxc1 paralogs, it is unsurprising that genetic buffering is observed; this is evidenced by the presence of gut situs defects and an almost complete penetrance of hydrocephalus in foxc1a −/− ; foxc1b −/− double homozygotes, which is lacking in both our foxc1a single mutants and those previously published [76]. Hydrocephalus specifically is a key phenotype of the murine Foxc1 −/− mutant "congenital hydrocephalus" and an occasional finding in patients with heterozygous FOXC1 mutation or deletion [27,38].…”

The Ocular Neural Crest: Specification, Migration, and Then What?

“…Without question, both proteins show dosage sensitivity, as minute deletions of either Foxc1 or Pitx2 lead to ARS by way of haploinsufficiency, and a previous study reported an ARS phenotype that may also result from the genetic duplication of Foxc1 (Berry et al, 2006). In addition, zebrafish carrying a heterozygous Foxc1 gene deletion mutation showed ocular defects from haploinsufficiency consistent with the ocular manifestations in their human counterparts (Ferre-Fernández et al, 2020). Similarly, an assessment of the requirements for Pitx2 showed that heterozygotes for either hypomorphic or null alleles in the Pitx2 gene have eye abnormalities consistent with ARS, indicating a Pitx2 dosage requirement for eye development in mice (Gage et al, 1999a,b).…”

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

“…By 3 days post fertilization (dpf), rudimentary structures of the anterior segment are present and will continue to develop through the first month of life. Zebrafish with homozygous mutations in foxc1a and foxc1b display underdeveloped or absent anterior segments beginning around 3 dpf [ 39 ]. Additional ocular phenotypes, including microphthalmia ( Figure 1 I,J) and coloboma are often observed in double homozygous foxc1a / foxc1b zebrafish, which are typically not observed in ARS patients.…”