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James Lister, Samantha A Spencer; Contribution of multiple MITF gene family members to RPE development in zebrafish. Invest. Ophthalmol. Vis. Sci. 2017;58(8):106.
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© ARVO (1962-2015); The Authors (2016-present)
The microphthalmia-associated transcription factor (MITF) is required for development of the retinal pigment epithelium (RPE) of the eye in mammals and birds. However, null mutations in the zebrafish MITF ortholog mitfa display normal development of the RPE. Zebrafish possess a second MITF ortholog, mitfb, but mitfa;mitfb double mutants also undergo normal RPE development. The purpose of this study was to determine if a third member of the zebrafish MITF/MiT family, tfec, expressed strongly in the presumptive RPE, may function in its development, alone or in conjunction with mitfa and mitfb.
Wild-type zebrafish embryos, and embryos bearing mutations in mitfa and/or mitfb, were injected with antisense morpholino oligonucleotides targeting the tfec gene to induce a transient knockdown of Tfec protein, and observed through early development (up to 5 days post-fertilization.) Wild-type embryos were also injected with mRNA encoding Cas9 endonuclease along with a CRISPR guide RNA targeting tfec. CRISPR/Cas9-injected embryos were raised to adulthood and screened for germline transmission of mutation of the locus, followed by outcrossing of prospective founders and sequencing of lesions. Stable tfec CRISPR lines were established and bred to mitfa and mitfb mutants to generate double and triple mutants. Morphogenesis of the eye of mutant embryos was analyzed by microscopy and expression of marker genes was examined by whole-mount in situ hybridization and immunocytochemistry.
Single tfec morphants/mutants display delayed melanization of the RPE, with tfec mutants showing a more dramatic effect. In comparison to tfec single mutants, tfec;mitfa double mutants have more severe defects in pigmentation and in ocular morphogenesis, with variable colobomata not observed in the tfec single mutants. Finally, eye development appears to be most severely compromised in mitfa;mitfb triple mutants. Expression of a subset of RPE markers show alterations as a result of manipulations of MiT gene activity.
Knockdown of tfec with antisense morpholino oligonucleotides, or by a CRISPR/Cas9 approach, indicates that tfec is vital for the development of the RPE. Together our data indicate that these three MiT factors make overlapping but differential contributions to RPE and eye development, with tfec surprisingly being the most important of the three, followed by mitfa, and mitfb having the least significance.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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