The developmental progression of eight opsin spectral signals recorded from the zebrafish retinal cone layer is altered by the timing and cell type expression of thyroxin receptor β2 (trβ2) gain-of-function transgenes

Abstract

AbstractZebrafish retinal cone signals shift in spectral shape through larval, juvenile, and adult development as expression patterns of eight cone-opsin genes change. An algorithm extracting signal amplitudes for the component cone spectral types is developed and tested on two thyroxin receptor β2 (trβ2) gain-of-function lines crx:mYFP-2A-trβ2 and gnat2:mYFP-2A-trβ2, allowing correlation between opsin signaling and opsin immunoreactivity in lines with different developmental timing and cell-type expression of this red-opsin-promoting transgene. Both adult transgenics became complete, or nearly complete, ‘red-cone dichromats’, with disproportionately large LWS1 opsin amplitudes as compared to controls, where LWS1 and LWS2 amplitudes were about equal, and significant signals from SWS1, SWS2, and Rh2 opsins were detected. But in transgenic larvae and juveniles of both lines it was LWS2 amplitudes that increased, with LWS1 cone signals rarely encountered. In gnat2:mYFP-2A-trβ2 embryos at 5 days post fertilization (dpf), red-opsin immunoreactive cone density doubled, but red-opsin amplitudes (LWS2) increased < 10%, and green, blue and UV opsin signals were unchanged, despite co-expressed red opsins, and the finding that an sws1 UV-opsin reporter gene was shut down by the gnat2:mYFP-2A-trβ2 transgene. By contrast both LWS2 red-cone amplitudes and the density of red-cone immunoreactivity more than doubled in 5 dpf crx:mYFP-2A-trß2 embryos, while UV-cone amplitudes were reduced 90%. Embryonic cones with trβ2 gain-of-function transgenes were morphologically distinct from control red, blue or UV cones, with wider inner segments and shorter axons than red cones, suggesting cone spectral specification, opsin immunoreactivity and shape are influenced by the abundance and developmental timing of trβ2 expression.Significance StatementAs different combinations of eight cone-opsin mRNAs are successively expressed during zebrafish development and maturation, the composite cone-ERG spectral signal shifts. Amplitudes of each of the eight resulting cone signals are inferred computationally from the composite signal, both in controls and in two thyroxin-receptor β2 (trβ2) gain-of-function transgenics, crx:mYFP-2A-trβ2 and gnat2:mYFP-2A-trβ2, trβ2 being a transcription factor required for expression of the red-cone opsins LWS1 and LWS2. Adult transgenics become red cone dichromats with excess LWS1 amplitudes, but larvae and juveniles evoke excess LWS2 amplitudes. Controls retain 5 to 6 cone signals of changing composition throughout development. The progression of transgene-induced amplitude alterations is slower in gnat2:mYFP-2A-trβ2, with supernormal red-opsin antigenicity not immediately correlating with red-cone signaling.