Electron microscopy reveals lack of rod-like outer segments or synapses in nrl−/−; tbx2b−/− double mutants. Using the metrics described below, no nrl−/−;tbx2b−/− double mutant larvae was found to have a cell that could confidently be described as a rod, despite assessing six individual larvae. In wild types, all six larvae examined had obvious rod photoreceptors. This reaffirms that although mutation of tbx2b promotes a rod-like phenotype, it is insufficient to rescue loss of nrl. Rod cells can be identified morphologically using EM from key distinctive features. Rod outer segments (A) are fully enclosed by the outer segment membrane (magenta arrow) and contain discrete stacked disks which can be identified by characteristic hairpin loops at their ends (green arrows). Cone outer segments (B, C) result from invaginations of the outer segment membrane, producing disk layers made of continuous membrane that are exposed to the extracellular matrix (red arrows). Rod synapses (D) are electron dense structures, appearing darker than the surrounding cone synapses, and contain a single, relatively large, synaptic ribbon (blue arrow). Cone synapses (E, F) are identifiable by an abundance of comparatively shorter synaptic ribbons (black arrows).
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