Given the timing of the changes in the expression of
shh,
pax6, and
vsx2 and the increased apoptosis in the inferior ocular fissure, we hypothesized that
cyp1b1 overexpression had an early effect on the spatial patterning of the optic cup. In humans, heterozygous mutations in
SHH are associated with holoprosencephaly and a spectrum of eye malformations, including anophthalmia, microphthalmia, and colobomas.
44–46 Shh is expressed in the floor plate of the diencephalon and is required for patterning the optic cup through the specific spatial expression of transcription factors.
47 Shh regulates
Vax2 and
Pax2 in the optic stalk and ventral neural retina to maintain the expression of these transcription factors within the more proximal structures of the eye.
48,49 Mice with
Vax2 or
Pax2 mutations show the inhibition of basement membrane breakdown in the fissure, resulting in colobomas. In contrast,
Shh inhibits
Pax6, restricting its expression to the distal optic cup, which is critical for lens induction from the surface ectoderm.
50,51 Human mutations in
PAX6 are most commonly associated with aniridia (OMIM 706108), an autosomal dominant congenital eye disease characterized by cataract, limbal stem cell deficiency, glaucoma, and hypoplasia of the iris, optic nerve, and fovea. While large disruptions in gene function resulting from frameshift or nonsense mutations lead to these widespread ocular defects,
52,53 rare missense
PAX6 mutations have been associated with colobomas.
54 In mice,
Pax6 regulates other genes associated with colobomas, such as
Vsx2 (
Chx10),
Maf1, and
Six3.
55–57 The current studies showed that in zebrafish,
cyp1b1 overexpression increased
shha gene expression in the midline floor plate, while decreasing
pax6a,
pax6b, and
vsx2 gene expression in the developing retina. Notably, RA is also a key regulator of ocular fissure closure, as both increased and decreased RA result in colobomas in mice and zebrafish.
58–61 Exposure to exogenous RA inhibits
Shh expression in the midline floor plate.
62 We observed that the overexpression of
cyp1b1 not only increased RA levels throughout the developing head and eye, but also increased
shha expression. Furthermore, genetically or pharmacologically decreasing endogenous RA synthesis to compensate for
cyp1b1 overexpression did not rescue the observed colobomatous defects. It is unclear whether these signaling pathways are affected only in the overexpression state or represent normal endogenous targets of
cyp1b1. We did not observe alterations in the levels of
shh,
pax6, or
vsx2 with decreased Cyp1b1, suggesting that there are additional targets of Cyp1b1 that regulate fissure patency. Taken together, the results of the present study provide evidence that the overexpression of
cyp1b1 regulates ocular fissure closure through an RA-independent mechanism involving
shh and
pax6.