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Nucleic Acids Res
2010 Aug 01;3814:4635-50. doi: 10.1093/nar/gkq230.
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Identification and characterization of alternative promoters of zebrafish Rtn-4/Nogo genes in cultured cells and zebrafish embryos.
Chen YC
,
Wu BK
,
Chu CY
,
Cheng CH
,
Han HW
,
Chen GD
,
Lee MT
,
Hwang PP
,
Kawakami K
,
Chang CC
,
Huang CJ
.
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In mammals, the Nogo family consists of Nogo-A, 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-n/Nogo-C1, Rtn4-m/Nogo-C2 and Rtn4-l/Nogo-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-l/Nogo-B mRNA in the brain, brachial arches, eyes, muscle, liver 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 and GATA6 were involved in the control of P1 promoter activity in the liver and intestine, while Myf5 and MyoD for the control of P1 and P3 promoter activities in muscles. These data demonstrate that zebrafish Rtn4/Nogo transcripts might be generated by coupling mechanisms of alternative first exons and alternative promoter usage.
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’).
Acevedo,
A new role for Nogo as a regulator of vascular remodeling.
2004, Pubmed
Acevedo,
A new role for Nogo as a regulator of vascular remodeling.
2004,
Pubmed
Amacher,
Multiple regulatory elements contribute differentially to muscle creatine kinase enhancer activity in skeletal and cardiac muscle.
1993,
Pubmed
André,
Intestinal fatty acid binding protein gene expression reveals the cephalocaudal patterning during zebrafish gut morphogenesis.
2000,
Pubmed
Apone,
Muscle gene E-box control elements. Evidence for quantitatively different transcriptional activities and the binding of distinct regulatory factors.
1995,
Pubmed
Becker,
Axonal regrowth after spinal cord transection in adult zebrafish.
1997,
Pubmed
Black,
Transcriptional control of muscle development by myocyte enhancer factor-2 (MEF2) proteins.
1998,
Pubmed
Brösamle,
Nogo-Nogo receptor signalling in PNS axon outgrowth and pathfinding.
2009,
Pubmed
Chen,
Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1.
2000,
Pubmed
Chen,
Recapitulation of zebrafish sncga expression pattern and labeling the habenular complex in transgenic zebrafish using green fluorescent protein reporter gene.
2009,
Pubmed
Chou,
Expression and characterization of a brain-specific protein kinase BSK146 from zebrafish.
2006,
Pubmed
Denovan-Wright,
cDNA sequence and tissue-specific expression of a basic liver-type fatty acid binding protein in adult zebrafish (Danio rerio).
2000,
Pubmed
Diekmann,
Analysis of the reticulon gene family demonstrates the absence of the neurite growth inhibitor Nogo-A in fish.
2005,
Pubmed
Filbin,
Myelin-associated inhibitors of axonal regeneration in the adult mammalian CNS.
2003,
Pubmed
Fournier,
Identification of a receptor mediating Nogo-66 inhibition of axonal regeneration.
2001,
Pubmed
Groves,
Fgf8 drives myogenic progression of a novel lateral fast muscle fibre population in zebrafish.
2005,
Pubmed
Hammond,
Signals and myogenic regulatory factors restrict pax3 and pax7 expression to dermomyotome-like tissue in zebrafish.
2007,
Pubmed
Her,
In vivo studies of liver-type fatty acid binding protein (L-FABP) gene expression in liver of transgenic zebrafish (Danio rerio).
2003,
Pubmed
Her,
Zebrafish intestinal fatty acid binding protein (I-FABP) gene promoter drives gut-specific expression in stable transgenic fish.
2004,
Pubmed
Higashijima,
High-frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin.
1997,
Pubmed
Hinits,
Differential requirements for myogenic regulatory factors distinguish medial and lateral somitic, cranial and fin muscle fibre populations.
2009,
Pubmed
Holtzinger,
Gata4 regulates the formation of multiple organs.
2005,
Pubmed
Hu,
Nogo-A interacts with the Nogo-66 receptor through multiple sites to create an isoform-selective subnanomolar agonist.
2005,
Pubmed
Josephson,
Nogo-receptor gene activity: cellular localization and developmental regulation of mRNA in mice and humans.
2002,
Pubmed
Kato,
Characterization of the promoter for the mouse alpha 3 integrin gene.
2002,
Pubmed
Kawakami,
A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish.
2004,
Pubmed
Kawakami,
Transgenesis and gene trap methods in zebrafish by using the Tol2 transposable element.
2004,
Pubmed
Kawakami,
Transposon tools and methods in zebrafish.
2005,
Pubmed
Kim,
Axon regeneration in young adult mice lacking Nogo-A/B.
2003,
Pubmed
Klinger,
Identification of Nogo-66 receptor (NgR) and homologous genes in fish.
2004,
Pubmed
Kritz,
In vivo modulation of Nogo-B attenuates neointima formation.
2008,
Pubmed
Kuang,
ER stress triggers apoptosis induced by Nogo-B/ASY overexpression.
2006,
Pubmed
Lin,
The transcription factor Six1a plays an essential role in the craniofacial myogenesis of zebrafish.
2009,
Pubmed
Liu,
Structure, linkage mapping and expression of the heart-type fatty acid-binding protein gene (fabp3 ) from zebrafish (Danio rerio).
2003,
Pubmed
Liu,
Differential expression of duplicated genes for brain-type fatty acid-binding proteins (fabp7a and fabp7b) during early development of the CNS in zebrafish (Danio rerio).
2004,
Pubmed
McKinsey,
MEF2: a calcium-dependent regulator of cell division, differentiation and death.
2002,
Pubmed
Miao,
Identification of a receptor necessary for Nogo-B stimulated chemotaxis and morphogenesis of endothelial cells.
2006,
Pubmed
Morrisey,
GATA6 regulates HNF4 and is required for differentiation of visceral endoderm in the mouse embryo.
1998,
Pubmed
Oertle,
Genomic structure and functional characterisation of the promoters of human and mouse nogo/rtn4.
2003,
Pubmed
Pare,
The mouse fetoprotein transcription factor (FTF) gene promoter is regulated by three GATA elements with tandem E box and Nkx motifs, and FTF in turn activates the Hnf3beta, Hnf4alpha, and Hnf1alpha gene promoters.
2001,
Pubmed
Pierce,
Nucleotide sequence of a cDNA clone coding for an intestinal-type fatty acid binding protein and its tissue-specific expression in zebrafish (Danio rerio).
2000,
Pubmed
Qi,
Pro-apoptotic ASY/Nogo-B protein associates with ASYIP.
2003,
Pubmed
Rao,
Distinct regulatory elements control muscle-specific, fiber-type-selective, and axially graded expression of a myosin light-chain gene in transgenic mice.
1996,
Pubmed
Reiter,
Multiple roles for Gata5 in zebrafish endoderm formation.
2001,
Pubmed
Reiter,
Gata5 is required for the development of the heart and endoderm in zebrafish.
1999,
Pubmed
,
Xenbase
Schnapp,
Induced early expression of mrf4 but not myog rescues myogenesis in the myod/myf5 double-morphant zebrafish embryo.
2009,
Pubmed
Schwab,
Nogo and axon regeneration.
2004,
Pubmed
Ticho,
Three zebrafish MEF2 genes delineate somitic and cardiac muscle development in wild-type and mutant embryos.
1996,
Pubmed
Wanner,
Reevaluation of the growth-permissive substrate properties of goldfish optic nerve myelin and myelin proteins.
1995,
Pubmed
Yiu,
Signaling mechanisms of the myelin inhibitors of axon regeneration.
2003,
Pubmed
Zhao,
GATA6 is essential for embryonic development of the liver but dispensable for early heart formation.
2005,
Pubmed