First published online May 5, 2005
Journal of Experimental Biology 208, 1915-1925 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01570
Genomic sequences encoding two types of medaka hemopexin-like protein Wap65, and their gene expression profiles in embryos
Makiko Nakaniwa1,
Makoto Hirayama1,
Atsushi Shimizu2,
Takashi Sasaki2,
Shuichi Asakawa2,
Nobuyoshi Shimizu2 and
Shugo Watabe1,*
1 Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School
of Agricultural and Life Sciences, The University of Tokyo, Tokyo113-8657,
Japan
2 Department of Molecular Biology, Keio University School of Medicine, 35
Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
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Fig. 1. Schematic representation of mWap65-1-hrGFP5k and
mWap65-2-hrGFP5k constructs. (A) Structures of mWap65-1 and
mWap65-2. The initiation methionine codon (ATG) of mWap65-1
and mWap65-2 is located at 29 bp and 89 bp downstream the putative
transcription start point, respectively. Promoters used in this study were DNA
fragments of 5 kb in the 5'-flanking region for both mWap65s.
Black boxes and lines indicate exons and introns, respectively. Numbers under
the boxes indicate exon numbers from the 5' end. (B) Structures of
mWap65-1-hrGFP5k and mWap65-2-hrGFP5k constructs. DNA
fragments of 5 kb in the 5'-flanking regions for both mWap65s
were inserted into the phrGFP vector. pA, polyA signal.
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Fig. 2. Comparison of genomic organization of mWap65-1 and
mWap65-2 with that of the human hemopexin gene. Exons, solid
rectangles; introns, lines. Black and gray boxes indicate coding and
non-coding regions, respectively. Numbers under the rectangles indicate the
number of exons from the 5' end. The genomic organization of the human
hemopexin gene was constructed based on work by Takahashi et al.
(1985 ), and the NCBI's
LocusLink
(http://www.ncbi.nlm.nih.gov/genome/guide/human/).
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Fig. 3. PipMaker output comparison for mWap65-1 with mWap65-2 and
the human hemopexin gene. Similarities of mWap65-1 with
mWap65-2 and human hemopexin gene are shown on the vertical axis with
percentages of sequence identity. The horizontal arrow indicates the direction
of transcription. Exons are shown as tall black boxes; short white boxes
represent CpG islands where the ratio CpG/GpC lies between 0.6 and 0.75; gray
boxes represent ratios exceeding 0.75. Simple, simple repeat; SINE, short
interspersed repetitive element.
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Fig. 4. The nucleotide sequences of 5'-flanking regions of mWap65-1
(A) and mWap65-2 (B). The nucleotide assigned +1 is the putative
transcription start point. Putative transcription factor binding sites
determined by TFSEARCH are underlined. SRY, sex determining region Y; USF,
upstream stimulatory factor; Nkx-2.5, homeobox protein NK-2 homolog; Cdx1,
caudal-type homeobox gene 1; MZF1, myeloid zinc finger 1; Oct-1,
octamer-binding transcription factor 1; XFD-1, Xenopus fork head
domain factor 1; C/EBP , CCAAT/enhancer binding protein ; HFH-2,
fork head homolog 2; HNF-3ß, hepatocyte nuclear factor-3ß; Evi-1,
ectopic viral integration site 1 encoded factor 1; GATA-1, globin
transcription factor-1; AML-1a, acute myeloid leukemia-1a; Pbx-1, pre-B-cell
leukemia transcription factor-1; Prx-2, paired related homeobox 2;  EF1,
-crystallin/E2-box factor 1; Brn-2, brain-2; Sox-5, SRY-related high
mobility group protein (HMG)-box gene 5; HNF-1, hepatocyte nuclear factor 1;
c-Ets-1, v-ets erythroblastosis virus E26 oncogene homolog 1.
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Fig. 5. Changes in the relative accumulated mRNA levels of mWap65-1 (A)
and mWap65-2 (B) in medaka embryos during development. Total RNAs
were isolated from embryos at various stages of 9 to 39. The relative levels
were determined using those of ß-actin as the control. The
values were expressed as the ratios to the lowest value obtained in this
study.
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Fig. 6. Whole-mount in situ hybridization of mWap65-1 and
mWap65-2. Embryos at stage 32 (somite completion) were hybridized
with DIG-labeled antisense probes specific for mWap65-1 (A-C) and
mWap65-2 (D-E) mRNA. B and C show a higher magnification of A, and E
is a higher magnification of D. Arrowheads indicate positive signals. lv,
liver; pf, pectoral fin; tb, tail bud. Scale bars, 240 µm.
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Fig. 7. Transient expression of GFP in medaka embryos injected with
mWap65-1-hrGFP5k construct and examined under dark-field (A,C,E,G)
and light-field (B,D,F,H) optics. (A,B) Stage 16 (late gastrula); (C,D) stage
23 (12 somite); (E,F) stage 26 (22 somite); (G,H) stage 29 (34 somite). White
arrowheads indicate cells showing GFP fluorescence. Embryonic shield and body
(outlined) are shown with black arrowheads. Red lines surround liver anlage.
Scale bars, 240 µm.
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Fig. 8. Transient expression of GFP in medaka embryos injected with
mWap65-2-hrGFP5k construct. (A,B) Stage 16 (late gastrula); (C,D)
stage 26 (22 somite); (E,F) stage 30 (35 somite); (G,H) stage 31 (gill blood
vessel formation). Transgenic embryos were exposed under dark- (A,C,E,G) and
light-field illumination (B,D,F,H). White arrowheads indicate cells showing
GFP fluorescence. Embryonic shield is indicated by a black arrowhead. Red
lines surround liver anlage. Scale bars, 240 µm.
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Fig. 9. Transient expression of GFP in the medaka larva after hatching (11 days
post-fertilization), which had been injected with the
mWap65-1-hrGFP5k plasmid. (A) Dark-field and (B) light-field
illumination. Arrow indicates liver. Scale bars, 450 µm.
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© The Company of Biologists Ltd 2005
