Nucleic Acids Res
August 1, 2010;
Identification and characterization of alternative promoters of zebrafish Rtn-4/Nogo genes in cultured cells and zebrafish embryos.
In mammals, the Nogo
family consists of Nogo
-B and Nogo
-C. However, there are three Rtn-4/Nogo
-related transcripts were identified in zebrafish. In addition to the common C-terminal region, the N-terminal regions of Rtn4
-C2 and Rtn4
-B, respectively, contain 9, 25 and 132 amino acid residues. In this study, we isolated the 5''-upstream region of each gene from a BAC clone and demonstrated that the putative promoter regions, P1-P3, are functional in cultured cells and zebrafish embryos. A transgenic zebrafish Tg(Nogo
-B:GFP) line was generated using P1 promoter region to drive green fluorescent protein (GFP) expression through Tol2-mediated transgenesis. This line recapitulates the endogenous expression pattern of Rtn4
-B mRNA in the brain
, brachial arches, eyes
and intestines. In contrast, GFP expressions by P2 and P3 promoters were localized to skeletal muscles of zebrafish embryos. Several GATA and E-box motifs are found in these promoter regions. Using morpholino knockdown experiments, GATA4
were involved in the control of P1 promoter activity in the liver
, while Myf5
for the control of P1 and P3 promoter activities in muscles. These data demonstrate that zebrafish Rtn4
transcripts might be generated by coupling mechanisms of alternative first exons and alternative promoter usage.
Nucleic Acids Res
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Figure 1. Three Rtn4/Nogo-related transcripts were generated by alternative promoter usage and alternative RNA splicing. Genomic organization of the zebrafish Rtn4/Nogo gene was shown. Exons are indicated by boxes numbered 1–7. Solid boxes indicate the Rtn4/Nogo coding region, whereas open boxes represent the 5′- and 3′-untranslated regions. Introns and the 5′-flanking regions are indicated by solid lines. All three isoforms have identical sequences derived from exons 2 to 7 but not exon 1. Exon 1a was used for Rtn4-l/Nogo-B, exon 1b for Rtn4-m/Nogo-C2 and exon 1c for Rtn4-n/Nogo-C1. The 5′-upstream promoter regions of each exon 1 are, respectively, designated P1, P2 and P3.
Figure 2. Activity of the 5′-upstream region of the P1 promoter in cultured cells. (A) Restriction map and possible transcription factor-binding motifs in the P1 promoter (−4885/−13). (B) COS-1 and C2C12 cells were cotransfected with 1 μg of each reporter construct and pSV-β-galactosidase, respectively. Cell lysates were prepared at 48 h after transfection and subjected to a luciferase activity assay. pGL3-Basic was used as the negative control.
Figure 3. Activity of the 5′-upstream region of the P1 promoter in zebrafish embryos. P1(-4885/-13)-GFP was microinjected into zebrafish embryos at the one-cell stage. Zebrafish embryos at 48-h post-fertilization (hpf) with GFP signals were selected for image analysis. Embryos are shown in lateral view with the anterior to the left and dorsal to the top. H, heart; S, skin; M, muscle; N, neuron; NC, notochord. Scale bars represent 100 (panels a and b) and 20 μm (panels c–j).
Figure 4. Expression patterns of the GFP in the transgenic zebrafish Tg(Nogo-B:GFP) line. Microinjection of the expression construct, P1(−4885/−13)-GFP, into zebrafish embryos at the one-cell stage and generation of a transgenic GFP line via Tol2-mediated transgenesis are described in the text. (A) contains images from the Tg(Nogo-B:GFP) transgenic line at different developmental stages. Merged bright field and fluorescence images are shown in panels a’–d’, while fluorescence images are shown in panels a–d. (B) Localization of Rtn4-l/Nogo-B mRNA in Tg(Nogo-B:GFP) fish at different developmental stages. ba, brachial arches; e, eyes; hb, hindbrain; i, intestine; l, liver; m, muscle; mhb, midbrain–hindbrain boundary.
Figure 5. Activity of the 5′-upstream regions of the P2 and P3 promoters in cultured cells. Restriction map and putative transcription factor-binding motifs of the P2 (−3230/−1) (A) and P3 promoters (−3014/−1) (B) are shown. (C) Transfection of expression constructs into COS-1 and C2C12 cells and luciferase activity were assayed in the same way as described in Figure 3.
Figure 6. Activity of the 5′-upstream regions of the P2 and P3 promoters in zebrafish embryos. (A) P2(−3230/−1)-GFP, P2(−1213/−1)-GFP, P3(−3014/−1)-GFP and P3(−1292/−1)-GFP were separately microinjected into zebrafish embryos at the one-cell stage. Zebrafish embryos at 48-hpf with GFP signals were selected for image analysis. For comparison, embryos injected with the α-actin promoter (panel e) displayed strong GFP expression specifically in muscles. Merged bright-field and fluorescence images are shown in panels a’–e’, while fluorescence images are shown in panels a–e. Scale bars indicate 100 μm. (B) Zebrafish embryos at 4 dpf mentioned above were subjected to cryosection and labelled with different antibodies as follow. The primary antibodies were mAb F59 (anti-MyHC, slow muscle) at 1: 20, mAb EB165 (anti-MyLC, fast muscle) at 1 : 200 and rabbit anti-GFP at 1: 200 dilution. After washing, slides were incubated with peroxidase-tagged secondary antirabbit antibodies at 1 : 200 dilution and stained with Fast DAB. (C) Expression patterns of GFP and Rtn4-n/Nogo-C1 in the transgenic zebrafish Tg(Nogo-C1:GFP) line. Images were taken from the Tg(Nogo-C1:GFP) transgenic line at 3 dpf. Merged bright-field and fluorescence images are shown in panel (a), while fluorescence images are shown in panel (a’). Expression of Rtn4-n/Nogo-C1 (panel b) and GFP (panel c) mRNA in the transgenic zebrafish Tg(Nogo-C1:GFP) line was analysed by whole-mount in situ hybridization. ba, brachial arches; e, eyes; i, intestine; m, muscle; mhb, midbrain-hindbrain boundary.
Figure 7. Loss of MyoD and Myf5 ablates somatic fast muscle and knockdown of GATA4 and GATA6 results in loss of GFP signal in the liver and intestine. (A) P1(−4885/−13)-GFP, P3(−3014/−1)-GFP and P3(−1292/−1)-GFP were separately injected or each coinjected with myod/myf5 double MOs into zebrafish embryos at the one-cell stage. Alternatively, myod/myf5 double MOs were injected to the transgenic Tg(Nogo-B:GFP) line at the one-cell stage. Zebrafish embryos at 48 hpf with GFP signals were selected for image analysis. Merged bright-field and fluorescence images are shown in panels (a’–h’), while fluorescence images are shown in panels (a–h). Scale bars indicate 100 μm. (B) Those embryos mentioned above at 3 dpf were subjected to whole-mount in situ hybridization using GFP as probe. All myod/myf5 double morphants did not show GFP signal. (C) GATA4/GATA6 double MOs were injected into zebrafish embryos of transgenic Tg(Nogo-B:GFP) line at one- to two-cell stage. The GATA4/GATA6 double-morphants and the parental transgenic line at 4 dpf were subjected to whole-mount in situ hybridization using GFP (panels a and b) and LFABP/iFABP (panels c and d) as probe. The liver and intestine were enlarged in panels (a’–d’).
A new role for Nogo as a regulator of vascular remodeling.