Proximal and Distal Sequences Control UV Cone Pigment Gene Expression in Transgenic Zebrafish

Wenqin Luo,John Williams,Philip M Smallwood,Jeffrey W Touchman,Laura M Roman,Jeremy Nathans

doi:10.1074/jbc.m400161200

Abstract

The molecular basis of cone photoreceptor-specific gene expression is largely unknown. In this study, we define cis-acting DNA sequences that control the cell type-specific expression of the zebrafish UV cone pigment gene by transient expression of green fluorescent protein transgenes following their injection into zebrafish embryos. These experiments show that 4.8 kb of 5'-flanking sequences from the zebrafish UV pigment gene direct expression specifically to UV cones and that this activity requires both distal and proximal sequences. In addition, we demonstrate that a proximal region located between -215 and -110 bp (with respect to the initiator methionine codon) can function in the context of a zebrafish rhodopsin promotor to convert its specificity from rod-only expression to rod and UV cone expression. These experiments demonstrate the power of transient transgenesis in zebrafish to efficiently define cis-acting regulatory sequences in an intact vertebrate.

Highlights

The molecular basis of cone photoreceptor-specific gene expression is largely unknown
These experiments show that 4.8 kb of 5؅-flanking sequences from the zebrafish UV pigment gene direct expression to UV cones and that this activity requires both distal and proximal sequences
We have shown that 5Ј-flanking sequences from the zebrafish UV pigment gene direct expression to UV cones, that this activity requires both distal and proximal sequences, and that at least one proximal element located within the interval Ϫ215 to Ϫ110 bp can function in the context of a rhodopsin promotor to convert its specificity from rod-only expression to rod and UV cone expression

Summary

Introduction

The molecular basis of cone photoreceptor-specific gene expression is largely unknown. We define cis-acting DNA sequences that control the cell type-specific expression of the zebrafish UV cone pigment gene by transient expression of green fluorescent protein transgenes following their injection into zebrafish embryos. We report an in vivo analysis of cis-acting DNA sequences that control expression of the zebrafish UV pigment in the appropriate photoreceptor type. These experiments take advantage of (a) the efficiency with which transiently transgenic fish can be generated by microinjection of plasmid DNA and (b) the rapid development of the zebrafish retina, in which all terminal cell fate decisions are complete within ϳ3 days postfertilization (dpf1; [13]). The data reveal a requirement for both proximal and distal sequences 5Ј of the UV pigment gene, and they further show that a small proximal sequence can mediate UV cone-specific expression in the context of the rhodopsin promotor

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Conclusion