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Dev Dyn
2010 Dec 01;23912:3172-81. doi: 10.1002/dvdy.22451.
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Characterization of a novel type I keratin gene and generation of transgenic lines with fluorescent reporter genes driven by its promoter/enhancer in Xenopus laevis.
Suzuki KT
,
Kashiwagi K
,
Ujihara M
,
Marukane T
,
Tazaki A
,
Watanabe K
,
Mizuno N
,
Ueda Y
,
Kondoh H
,
Kashiwagi A
,
Mochii M
.
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We investigated the characteristics of a novel type I keratin gene in Xenopus laevis during ontogenesis. The transcript was first detected in the posterior region at the late neurula stage, and then restricted to the fin and external gill during embryogenesis. To examine the transcriptional regulation of the keratin gene in vivo, we generated transgenic lines with fluorescent reporter genes driven by its 4.2-kb upstream sequence. The promoter/enhancer activity recapitulated the endogenous gene expression during embryogenesis. Sequential deletion analyses revealed that the regions proximal to the promoter were essential for fin-specific expression. Reporter expression was detected in various organs, including the fin and gill. In particular, robust expression was observed in the developing limbs and gill. The reporter fluorescence rapidly decreased with internal gill resorption during metamorphosis. The transgenic lines carrying the promoter/enhancer should represent valuable tools for elucidating the formation, development and resorption of various organs, especially the gill.
Figure 2. Spatiotemporal expression patterns of Fin and Gill Keratin (FGK) gene during embryogenesis. A: Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of FGK gene expression. The numbers indicate the Nieuwkoop and Faber (NF) developmental stages. Total RNA from each sample was subjected to semiquantitative RT-PCR. The ornithine decarboxylase (ODC) gene was amplified as an internal control. B: Whole-mount in situ hybridization of FGK gene expression. All photographs are lateral views. The signals of FGK expression are shown by the arrows. The arrowhead indicates the external gill at NF40.
Figure 3. Deletion and enhancer analyses of the 4.2-kb upstream sequence of the Fin and Gill Keratin (FGK) gene in F0 transgenic embryos. A–D: Representative enhanced green fluorescent protein (EGFP) fluorescence images of F0 embryos. Transgenic embryos express the EGFP reporter gene under the control of the entire 4.2-kb upstream sequence of the FGK gene at Nieuwkoop and Faber (NF) stages 22 (A), 28 (B), 35/36 (C), and 41 (D). The arrowhead in (D) indicates the external gill. E: Summary of the successive deletion analyses of the upstream sequence of the FGK gene. Left top: VISTA analyses of the 4.2-kb upstream sequences of the FGK gene and its X. tropicalis homologue xtKRT16prov. Left: Diagrams of the successive deletion constructs of the 4.2-kb upstream sequence. The region from −563 bp to −476 bp is indicated by the dotted red lines. Right: Graph showing the percentages of fin-specific EGFP expression at NF stage 40 for the different deletion constructs. The actual numbers of embryos with fin-specific EGFP expression and total numbers of EGFP-positive embryos are shown on the right side of the graph. F: An enhancer analysis of the proximal sequence from the FGK promoter including regions IV and V. The three deleted elements in regions IV and V (−567/−342, −567/−177 and −567/−78 bp) were fused to the CMV minimal promoter in pEGFP1-1 and subjected to the transgenic analysis. G: Genomic sequence of the region proximal to the FGK promoter. The upstream sequence and TATA box are shown in black and red, respectively. Regions IV and V are represented by orange and green boxes, respectively. The arrow indicates the transcription start site. Bold underlines indicate putative transcription factor binding sites predicted by the JASPAR program.Download figure to PowerPoint
Figure 4. Enhanced green fluorescent protein (EGFP) fluorescence images of F0 tadpoles at premetamorphic stages. A,B: Dorsal fluorescence views of F0 transgenic tadpoles with the EGFP reporter gene driven by the entire 4.2-kb upstream sequence of the Fin and Gill Keratin (FGK)gene at stages 52 (A) and 54 (B). The insets are high-magnification images of the boxed olfactory epithelium and hindlimbs. O, olfactory epithelium; L, lens; IG, internal gill; HL, hindlimb.Download figure to PowerPoint
Figure 5. Changes in enhanced green fluorescent protein (EGFP) fluorescence in F1 offspring embryos during embryogenesis. A–J: The images are lateral (A–F,H), ventral (G,J) and dorsal (I) views at NF stages 24 (A), 30 (B), 33 (C), 40 (D–G), and 44 (H–J). O, olfactory epithelium; L, lens, EG, external gill; BA, branchial arch.Download figure to PowerPoint
Figure 6. Attenuation of enhanced green fluorescent protein (EGFP) fluorescence in F1 offspring tadpoles during spontaneous metamorphosis. A–F: The images are dorsal (left column) and ventral (right column) views at Nieuwkoop and Faber (NF) stages 59 (A,B), 61 (C,D) and 62 (E,F). G,H: Bright (G) and fluorescence (H) dorsal views at NF stage 64. It should be noted that the EGFP fluorescence of the internal tissues cannot be observed because of the thickly developed adult skin.
Figure 7. Down-regulation of enhanced green fluorescent protein (EGFP) reporter gene expression in F1 offspring tadpoles during thyroid hormone (TH)-induced metamorphosis. A–H: NF stage 52 transgenic F1 tadpoles were reared in the absence (A,C,E,G) or presence (B,D,F,H) of 5 nM T3 for 5 days, and then fluorescence images were taken. A–D: The images are dorsal (A,B) and ventral (C,D) views. E–H: High magnification images of the internal gill (E,F) and hindlimb (G,H) are also shown. O, olfactory epithelium; L, lens; IG, internal gill; FL, forelimb; GF, gill filament; FP, filter plate.
krt16 (keratin 16, type I) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, lateral view, anteriorleft, dorsal up.
krt16 (keratin 16, type I) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anteriorleft, dorsal up.
krt16 (keratin 16, type I) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 40, lateral view, anteriorleft, dorsal up.
