Abstract
Purpose :
MicroRNAs (miRs) function as important regulators of photoreceptor development and physiology. The purpose of this study was to determine if photoreceptor sensory cilium development requires miR-129-3p, a key regulator of ciliogenesis.
Methods :
Both morpholinos and TALENs were used to inactivate miR-129-3p in zebrafish. Zebrafish contain two mir-129 paralogs, mir-129-1 and mir-129-2. We designed TALENs against each locus and selected mutations that are predicted to inactivate the seed sequence of each miR-129-3p paralog without significantly altering the pre-miRNA hairpin structure. We used PNA to examine cone outer segment length in 5dpf larvae of both single and double mir-129 mutants.
Results :
At 5 days post fertilization (dpf), zebrafish larvae injected with morpholinos targeting miR-129-3p exhibited phenotypes consistent with malfunctioning cilia, including shortened photoreceptor outer segments, kidney cysts, ectopic otoliths, and severe ventral body curvature. Mir-129-1-3p-/-; mir-129-2-3p-/- fish, which are predicted to lack all functional miR-129-3p, were viable and were produced in the expected Mendelian ratio. At 5 dpf, these mutant animals lacked the severe body curvature and kidney cysts that were readily apparent in morphant fish, and PNA staining showed normal cone outer segment lengths.
Conclusions :
Contrary to our hypothesis and in stark contrast to miR-129-3p morphant zebrafish, miR-129-3p mutants have no overt cilia phenotypes at 5 dpf. This suggests that the morpholino may non-selectively target additional RNAs that can compensate for loss of miR-129-3p in mutant fish.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.