Loss of foxd3 Function Due to a Novel Insertional Mutation Results in Eye Abnormalities in Zebrafish

Abstract

Purpose: : The foxd3 gene encodes a forkhead-domain transcription factor that is expressed in neural crest precursors and in early migratory neural crest cells in different species. Neural crest-derived periocular mesenchyme makes a major contribution to the developing anterior segment of the eye, but the molecular mechanisms involved in normal development of these structures are not clear at this time. A role for foxd3 in eye development has not been evaluated. Using a new foxd3-deficient zebrafish line as well as morpholino-mediated translational inhibition, we examined a potential role of foxd3 in eye development.

Methods: : A zebrafish line containing a viral insertion into the foxd3 locus was obtained from Znomics, and the position of viral insertion was defined. Foxd3 insertional mutants were examined for phenotypic defects at 1-5dpf. Morpholino injection, histology, in situ hybridization were performed using standard protocols.

Results: : The 5.7kb viral insertion incorporated at position 267 of foxd3 coding region; the mutant protein is predicted to contain 33% of normal foxd3 sequence (including only 30% of its DNA-binding forkhead domain) and 11 additional amino acids encoded by the viral sequence. RT-PCR analysis demonstrated a major decrease of foxd3 transcript level in homozygous mutant embryos. Therefore the foxd3^267v mutation appears to result in a complete loss of foxd3 function. Homozygous mutants display a protruding jaw, loss of posterior pharyngeal arches, cardiac edema and embryonic lethality by 7dpf while heterozygous embryos appear to be normal. To analyze ocular development in homozygous mutants, histological analysis was performed and revealed a notably disorganized lens and retina after 3.5dpf in a subset of embryos. The foxd3^267v ocular phenotype was characterized by incomplete penetrance and variable expressivity. To explore ocular anomalies associated with foxd3, foxd3-deficient embryos (morphants) were generated through injection of morpholino and reveal a similar ocular phenotype. To further evaluate the ocular anomalies associated with foxd3 deficiency, expression of pitx2, lmx1b, foxe3, sox2, grem2, prox1 and other genes that are known to contribute to the development of different ocular structures is being analyzed.

Conclusions: : The new foxd3^267v mutation in zebrafish is associated with a complete loss of function of foxd3. Our data suggest that foxd3 plays a role in eye development in zebrafish. Further characterization of a phenotype in foxd3 mutants and morphants will help to reveal specific defects associated with foxd3 deficiency.

Keywords: genetics • anterior segment • transcription factors