The forkhead box transcription factors,
foxi1 and
foxi2, are present at 28 hpf in WT embryos in multiple sites, including the dorsal and ventral CMZ, respectively, and the otic vesicle and pharyngeal arches. Zebrafish
foxi1 has a key role in otic placode formation and jaw development through maintenance of survival of neural crest cell populations.
56–60 Full elucidation of the developmental roles of
foxi2 has yet to occur; expression in the mouse neural retinal layer has been described, and
Xenopus Foxi2 was shown recently to be required for activation of Foxi1e, critical for consequent ectodermal gene expression.
61,62 gdf6a−/− embryos have significant alterations in ocular expression of
foxi1 and
foxi2, as
foxi1 is lost from the dorsal CMZ, and
foxi2 expands to encompass the ventral and dorsal CMZ. The highly specific expression pattern of these transcription factors, coupled with loss of
foxi1 when dorsal retinal identity is not initiated, suggests that the CMZ has similar dorsal-ventral patterning to the retina. We are aware of no previous reports of CMZ patterning and cellular identity, which implies that stem cells in these areas may have differential proliferative or inductive potential. With
foxi1's known role in maintenance of neural crest cell survival, and roles of other
fox's in cell cycle control,
57,60,61 we hypothesized that
foxi1 and
foxi2 have a role in regulation of cell cycle progression in the CMZ and consequent ocular size. We examined possible anomalies in ocular proliferation and apoptosis in the previously described
foo mutant (
foxi1hi3747Tg )
63 that contains a retrovirus-induced mutation, and observed no changes in ocular size and proliferation (data not shown). It is possible that
foxi1 has redundant roles with
gdf6a and other dorsal genes. Alternatively, expansion of
foxi2 expression may compensate for loss of
foxi1. If the latter is correct, then loss of
foxi2 should result in reduced eye size in
gdf6a mutants. This, indeed, is the case as transient knockdown of
foxi2 coupled with loss of one functional copy of
gdf6a resulted in the occurrence of microphthalmia, revealing a novel requirement for this gene in controlling ocular size. The further reduction of eye size in
gdf6a homozygous mutants injected with Foxi2 morpholino, compared to that of homozygous mutants alone further supported our hypothesis of a compensatory role of
foxi2 in the determination of eye size in
gdf6a mutants. Of great interest for the future is elucidation of developmental roles of
foxi2 and its transcriptional targets in the eye, as currently there is a lack in literature on this topic.