The ponli enhancer activates transcription in the zebrafish green, red and blue cone photoreceptors

Abstract

Purpose: The ponli gene encodes a membrane-associated apical polarity protein. The Ponli protein is restrictively expressed the inner segment membrane regions of green, red, and blue cone photoreceptors in zebrafish. Ponli is expected to play a critical role in the cone mosaic patterning in the zebrafish. It is unknown how the distinct expression pattern of the ponli gene is established and maintained in the zebrafish retina. Thus, we set out to identify the cis-regulatory elements of the ponli gene, which we hypothesize should be conserved among different teleost species that express the ponli gene.

Methods: The conserved cis-elements were predicted by aligning the sequences of ponli genes of five teleost species using the ClustalW2 sequence alignment program. The functions of these predicted cis-elements were investigated via mutation analyses in vivo. The expression levels and patterns of reporter genes and the endogenous ponli gene were analyzed by confocal microscopy, real-time PCR, and Western blotting.

Results: We showed that the promoter of the ponli gene resides in a 130-bp sequence upstream of its transcription start site and its enhancer is located in the 5.7-kb first intron. These cis-regulatory elements drive specific expression of mCherry in green, red, and blue cones in the Tg(ponli^{130-bp promter+5.7-kb enhancer}:HA-mcherry)^pt118b transgenic fish. The first intron of the ponli gene contains a 200-bp region that is sequentially and functionally conserved among five teleosts. This critical region is likely a central part of the ponli enhancer that determines the cell-type specificity. In addition, the expression level of the ponli cis-regulatory elements is moderate, thousands times lower than that of the green opsin promoter.

Conclusions: The ponli enhancer provides us with an entry point to study how vertebrate cone and rod photoreceptors differ in their biochemical, structural, and functional properties by expressing distinct gene profiles. The ponli enhancer may potentially serve as a handle to reveal specific interactions between cis- and trans-acting transcriptional regulatory elements in these cones. In addition, the ponli enhancer provides us with a unique tool to specifically and moderately express transgenes in the zebrafish green, red, and blue cones.

Keywords: 648 photoreceptors • 500 differentiation • 698 retinal development