Expression of a 12-kb promoter element derived from the zebrafish enolase-2 gene in the zebrafish visual system

Abstract

We recently cloned the zebrafish neuronal enolase-2 gene and showed that a 12-kb eno2 promoter element was sufficient to drive transgene expression widely in CNS neurons in vivo from 48h post-fertilization through adulthood. The aim of the present study was to establish the expression pattern of the 12-kb eno2 promoter element in the zebrafish visual system. Endogenous eno2 mRNA was detected in the developing retina from 2 days post-fertilization (dpf), and by 12dpf was localized to the retinal ganglion cell, inner and outer nuclear layers. Similar to endogenous eno2, GFP expression in the retina of Tg(eno2:GFP) larvae was first evident at 2dpf, and by 12dpf intense GFP expression was seen in the retinal ganglion cell and photoreceptor layers, with weaker expression in the inner nuclear layer. We identified cell types expressing the eno2 promoter element by using two complementary strategies: (i) double label immunofluorescence analysis of Tg(eno2:GFP) zebrafish, and (ii) generation of double transgenic zebrafish expressing red fluorescent protein under transcriptional control of the 12-kb eno2 promoter and GFP under a rod- or cone-specific promoter. The 12-kb eno2 promoter was expressed in retinal ganglion cells, amacrine cells, including a subset that co-expressed tyrosine hydroxylase, and rod photoreceptors. These data suggest that abnormalities of vision should be sought in transgenic models of diseases generated using this promoter. Owing to the specific expression of fluorescent reporters in neuronal subpopulations, Tg(eno2:GFP) and Tg(eno2:mRFP) zebrafish may be useful for studies of retinal lamination, neuronal differentiation and synapse formation in the visual system.

Figure 1. Expression of endogenous eno2 in the retina

RNA in situ hybridization was employed in order to analyze endogenous eno2 mRNA expression in the retina at 72 hpf (A–C) and 12 dpf (D–F) A–C: Panel A shows a low power superior view of two whole mount 72hpf larvae (rostral left) that were hybridized with a cRNA probe specific to eno2 (upper larva) or a sense control probe (lower larva). Hybridized probe was detected using a histochemical reaction with a purple/blue reaction product. The region of each image encompassing the left eye is shown at higher magnification in panels B and C, as indicated by the dotted outline. D–F: Panel D shows a low power view of two 15μm cryosections of eyes from 12dpf larvae that were hybridized with cRNA probe specific to eno2 (upper sample) or a control sense probe (lower sample). Hybridized probe was detected with a histochemical reaction producing a purple/blue product. Representative high magnification fields from similar sections are shown in panels E and F. Key: GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer

Figure 2. GFP expression in the retina of Tg(eno2:egfp)^Pt404 zebrafish larvae

A: The image shows a Z-plane projection of multiple confocal images of the head region of a live 5dpf Tg(eno2:egfp)^Pt404 larva (rostral left, dorsal up) demonstrating GFP expression. B: Single plane confocal image of GFP expression in the retina of a live 72hpf Tg(eno2:egfp)^Pt404 larva. C – F: Indirect immunofluorescence and confocal microscopy were employed to detect GFP expression in fixed 20μm thick retinal cryosections derived from Tg(eno2:egfp)^Pt404 zebrafish at (C) 2dpf; (D) 3dpf; (E) 5dpf; and (F) 12dpf. Key: R, retina; TeO, optic tectum; HB, hindbrain ON, optic nerve; L, lens; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer.

Figure 3. Expression of GFP in retinal ganglion cells and their central projections in a Tg(eno2:egfp)^Pt404 zebrafish larva

A single plane confocal micrograph is shown of a cranial oblique coronal cryosection derived from a Tg(eno2:egfp)^Pt404 zebrafish at 5dpf. Double indirect immunofluorescence was employed to detect GFP expression (green) and Zn8 immunoreactivity (red) simultaneously. Key: OT, optic tract; otherwise same as figures 1 and 2.

Figure 4. Expression of GFP in the inner nuclear layer of Tg(eno2:egfp)^Pt404 retina

Double Indirect immunofluorescence and confocal microscopy were employed to detect GFP expression and cell markers of interest in cryosections derived from 12dpf Tg(eno2:egfp)^Pt404 zebrafish. A: Lin7 (red; bipolar cells; left panel); eno2:egfp (green, center panel); overlay, right panel. Key same as figure 2. B: Tyrosine hydroxylase (red; subset of amacrine cells; upper left panel); eno2:egfp (green, middle left panel); overlay (lower panel). Arrows mark TH-immunoreactive cells. A lower magnification image is shown on the right, indicating the region from which the images on the left were taken. Key same as figure 2.

Figure 5. Generation of Tg(mRFP) zebrafish

A: The schematic illustration shows the genomic organization of the eno2 gene and the DNA construct used to generate the transgenic Tg(eno2:mrfp)^Pt407 zebrafish shown in panels B and C B: The 12kb eno2:mrfp construct was microinjected into single-cell zebrafish embryos and stable transgenic lines were derived as described in the text. The genotype of transgenic founders was confirmed by genomic PCR using eno2:mrfp-specific primers. The picture shows an ethidium bromide stained agarose gel. Lane 1, genomic DNA from wild-type strain AB* zebrafish spiked with mRFP plasmid; lane 2 genomic DNA from wild-type zebrafish; lane 3 genomic DNA from Tg(eno2:mrfp)^Pt407 zebrafish. C: mRFP expression in living stable Tg(eno2:mrfp)^Pt407 zebrafish larva by epifluorescence microscopy. An oblique lateral view of a Tg(eno2:mrfp)^Pt407 larva is shown at 5 days post-fertilization (rostral left, dorsal up). The yolk sac, Y, is autofluorescent. The major GFP-expressing divisions of the nervous system are labelled; Key: Tel, telencephalon; TeO, optic tectum; HB, hindbrain; SC, spinal cord; R, retina.

Figure 6. Expression of the 12kb eno2 promoter in photoreceptors of transgenic zebrafish

A: Double indirect immunofluorescence and confocal microscopy were employed to detect zpr1 immunoreactivity (a marker of red and green-sensitive cone photoreceptors; red; left panel) and eno2:egfp expression (green; middle panel) in cryosections derived from 12dpf Tg(eno2:egfp)^Pt404 zebrafish. B, C: Tg(eno2:mrfp)^Pt407 zebrafish were crossed with Tg(sws1:egfp) or Tg(rhodopsin:egfp) zebrafish that express GFP in either UV cone or rod photoreceptors respectively. GFP and mRFP were detected by double indirect immunofluorescence and confocal microscopy in larvae at 12dpf. Each set of images shows GFP (left, green); mRFP (middle, red); overlay (right).