Fig. 2.

Successful generation of a cone-specific MCU overexpression zebrafish model. a Schematic of the MCU OE construct. The cone transducin promoter (TαCP, gnat2) drives expression of zebrafish MCU cDNA in all cone subtypes. The MCU cDNA is tagged with a T2A sequence followed by RFP. The T2A sequence causes ribosomes to stall and release the nascent MCU polypeptide with some added peptides from the T2A sequence before translating the RFP separately. Thus, RFP is present in the cytosol of cones with MCU overexpression. b Mitochondria-enriched retinal lysate of WT and MCU OE retinas probed with antibodies for MCU, MTCO1, and SDH. Each lane contains 8 µg of protein from lysate of two pooled retinas from a single fish. c Quantification of relative MCU signal as a function of protein concentration and relative to other mitochondrial markers from the type of analysis shown in B (n = 4 fish). Both exogenous and endogenous MCU were used for total MCU quantification in the MCU OE retina. The mean is reported and bars = standard error. ***p < 0.001 using Welch’s t-test. d Immunohistochemistry of a larval zebrafish retina expressing the MCU construct in A using MCU and mitochondrial cytochrome oxidase (MTCO1) antibodies. Scale bar = 5 µm