First published online 26 November 2003
doi: 10.1242/dev.00877
Development 131, 57-71 (2004)
Published by The Company of Biologists 2004
Characterization of the pufferfish Otx2 cis-regulators reveals evolutionarily conserved genetic mechanisms for vertebrate head specification
Chiharu Kimura-Yoshida1,
Kuniko Kitajima1,
Izumi Oda-Ishii1,
E Tian2,
Misao Suzuki3,
Masayuki Yamamoto4,
Tohru Suzuki5,
Makoto Kobayashi4,
Shinichi Aizawa6 and
Isao Matsuo1,*
1 Head Organizer Project, Vertebrate Body Plan Group, RIKEN Center for
Developmental Biology, 2-2-3 Minatojima Minami Cho, Chuou-Ku, Kobe, Hyougo
650-0047, Japan
2 Department of Morphogenesis, Institute of Molecular Embryology and Genetics,
Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan
3 Division of Transgenic Technology, Center for Animal Resources and
Development, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan
4 The Center for Tsukuba Advanced Research Alliance and Institute of Basic
Medical Sciences, University of Tsukuba, Tsukuba 305-8577, Japan
5 Nutrition Division, National Research Institute of Aquaculture, Fisheries
Research Agency, Nansei-cho, Watarai-gun, Mie 516-0193, Japan
6 Animal Resources and Genetic Engineering Team and Vertebrate Body Plan Group,
RIKEN Center for Developmental Biology, 2-2-3 Minatojima Minami Cho, Chuou-Ku,
Kobe, Hyougo 650-0047, Japan
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Fig. 3. Schematic diagram of the lacZ transgene constructs used to
identify cis-acting regions throughout the Fotx2 genomic locus. The
translational start site is indicated (0). Each fragment examined for
cis-activity is denoted by the bars marked F1-F13. Restriction enzyme sites
used are indicated above. The colored genomic fragments consistently display
specific lacZ activity at 10.5 dpc. By contrast, the fragments in
black possess no cis-acting ability at 10.5 dpc. Tissues in which expression
is driven by these fragments are indicated in colored letters below. The
number of lacZ-positive embryos in the specific regions among
transgenic embryos is indicated on the right with transgene names. In all
these embryos, the patterns of expression were reliably identical although
their levels were variable. B, BamHI; Bs, BstEII; EV,
EcoRV; N, NotI; Ns, NspV; X, XbaI.
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Fig. 1. Identity of the Fugu rubripes Otx2 gene. (A) Comparison of
Fugu and mouse Otx2 genes. Filled boxes represent coding
exons. Homeobox regions are shown in gray. (B) Comparison of the deduced amino
acid sequences of the Fugu Otx2 (FrOtx2), zebrafish
Otx2 (DrOtx2), Xenopus Otx2 (XlOtx2) and
mouse Otx2 (MmOtx2). Homeodomains highly conserved between
all Otx2 genes are shaded. Otx protein sequences were retrieved from
the GenBank database. Degenerate PCR primers are indicated by arrows and Otx
tail motifs are underlined. (C) Phylogenetic tree of Otx/Crx family proteins.
The tree was constructed using the NJ method. Protein sequences were retrieved
from the GenBank database. Bf, amphioxus Branchiostoma floridae; Dr,
zebrafish Danio rerio; Fr, Fugu rubripes; Hs, Homo
sapiens; Lj, Lampetra japonica; Mm, Mus musculus; Pm,
Petromyzon marius; Sc, dogfish Scyliorhinus canicula; Xl,
Xenopus laevis.
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Fig. 2. Expression analysis of the Fotx2 gene in wild-type pufferfish
embryos. (A) Expression patterns of the Fotx2 gene analyzed by RTPCR.
The ß-actin fragment was amplified as a control for the quality
of cDNA. (B-E) Expression patterns of the Fugu genes analyzed by
whole-mount in situ hybridization. Pufferfish no tail expression at
44 hpf (B). Fotx2 expression at 44 (C), 54 (D) and 78 hpf (E). MBT,
midblastula transition; a, anterior; an, anterior neural tube; e, eye; fb,
forebrain; m, mesencephalon; mhb, mid-hindbrain boundary; s, somites; p,
posterior; y, yolk.
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Fig. 4. Analysis of lacZ expression with seven transgene constructs. (A-G)
Lateral views of 10.5 dpc embryos harboring lacZ transgene
constructs, following ß-galactosidase staining. (A) F3placZ, (B)
F4placZ, (C) F5placZ, (D) F8placZ, (E)
F9placZ, (F) F11placZ and (G) F12placZ transgenic
embryos. d, diencephalon; E, eye; m, mesencephalon; hy, hyoid arch; ma,
mandibular arch; ms, cephalic mesenchyme; n, nasal pits; t, telencephalon, v,
trigeminal ganglion.
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Fig. 5. Developmental changes in lacZ expression with the F3placZ
construct. Lateral and frontal views of the F3placZ transgenic
embryos at 8.5 (A,B), 9.5 (C), 10.5 (D), 12.5 (I,J) and 13.5 (M) dpc, and a
dorsal view at 13.5 dpc (N), following ß-galactosidase staining.
Transverse sections of transgenic embryos at 10.5 (E-H) and 12.5 (K,L) dpc.
Sagittal sections of embryos at 10.5 (H) and 12.5 (L) dpc. Whole-mount in situ
hybridization analysis of noggin mRNA indicates that mouse noggin expression
is evident in the roof of the neural tube, including at the level of the
diencephalon (O, arrowheads). Lateral view of the F3hsplacZ
transgenic embryo at 10.5 dpc, following ß-galactosidase staining (P).
cp, choroid plexus; d, diencephalon; he, cortical hem; lt, lamina terminalis;
lv, lateral ventricle; m, mesencephalon; t, telencephalon.
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Fig. 6. Developmental changes in lacZ expression with the F8placZ
construct. Lateral (A,C), frontal (B,D,H) and dorsal (K) views of
F8placZ transgenic embryos following ß-galactosidase staining.
Transverse and sagittal sections of the transgenic embryos at 10.5 (E-G) and
12.5 (I,J) dpc. (A,B) 9.5, (C-G) 10.5, (H-J) 12.5 and (K) 13.5 dpc transgenic
embryos. Whole-mount in situ hybridization analysis of Wnt3a mRNA
indicates that mouse Wnt3a expression is detected in the prospective
dorsal diencephalon and cortical hem (arrowhead; L). cp, choroid plexus; d,
diencephalon; he, cortical hem; m, mesencephalon; t, telencephalon.
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Fig. 7. Developmental changes in lacZ expression with the F5placZ
construct. Lateral (A,B,D,H) and dorsal (I) views of the transgenic embryos
following ß-galactosidase staining. Sagittal (C,G) and transverse (E,F)
sections of the transgenic embryos. (A) 9.5, (B,C) 10.5, (D-G) 11.5 and (H,I)
13.5 dpc transgenic embryos. lacZ expression is detected in the
ventral diencephalon at 9.5 dpc (arrowhead; A). At 10.5 dpc, lacZ
expression is present in the ZLI (white arrowheads; B). lacZ activity
occurs in the mammillary recess (arrowhead; C). At 11.5 dpc, transgene
activity remains in the lateral mesencephalon, ZLI (arrowheads) and the
ventral diencephalon, including in the retromammillary region (D-G). d,
diencephalon; m, mesencephalon; t, telencephalon.
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Fig. 8. Developmental changes in lacZ expression with the
F11placZ construct. Lateral (A-C,I) and dorsal (J) views of the
transgenic embryos following ß-galactosidase staining. Transverse and
sagittal sections of the transgenic embryos at 10.5 (D-G) and 12.5 dpc (H).
(A) 9.5, (B, D-G) 10.5, (C) 11.5, (H) 12.5 and (I,J) 13.5 dpc transgenic
embryos. (E,F,G) Transverse section through the cephalic region of a 10.5 dpc
transgenic embryo, showing lacZ expression in the trigeminal
ganglions (V) and the ventral portion of the spinal cord (arrowheads). (K)
Whole-mount in situ hybridization analysis of mouse Otx2 expression
at 9.5 dpc. Otx2 expression is also detected in the trigeminal nerves
and the first branchial groove (arrowheads). d, diencephalon; m,
mesencephalon; p, p1 diencephalon; t, telencephalon; V1, opthalmic branch of
the trigeminal nerve; V2, maxillar branch of the trigeminal nerve.
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Fig. 9. Developmental changes in lacZ expression with the
F12placZ construct. Lateral (A,B,E,F) and frontal (C,G) views of
transgenic embryos following ß-galactosidase staining. Transverse and
sagittal sections of the transgenic embryos at 11.5 (D) and 12.5 (H-K) dpc.
(A) lacZ expression is detected initially in the cranial portion of
the first branchial groove and nasal regions (arrowhead) at 9.5 dpc. bg,
branchial groove; d, diencephalon; e, eye; m, mesencephalon; ma, mandibular
arch; ha, hyoid arch; ie, inner ear; na, nasal pits; oe, olfactory epithelium;
pi, pinna; pl, pigment layer of retina; t, telencephalon.
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Fig. 10. Schematic representation of Otx2-lacZ reporter expression in the
mouse brain, based on the neuromeric model of Puelles and Rubenstein
(Puelles and Rubenstein, 1993 )
and Bulfone et al. (Bulfone et al.,
1993). The expression patterns of the transgenes depicted in this
figure are reconstructed from the analysis of whole-mount and serial sections
of ß-galactosidase-stained transgenic embryos, and are represented in the
following colors: F8placZ, red; F3placZ, blue;
F5placZ, green; F11placZ, yellow. ACH, archicortex; CB,
cerebellum; CP, choroid plexus; DT, dorsal thalamus; ET, epithalamus I,
isthmus; M, mesencephalon; NCX, neocortex; PT, pretectum; r1-7, rhombomere
1-7; ZLI, zona limitans intrathalamica.
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Fig. 11. Transient expression analysis of F3 and F8 cis-regions fused with the GFP
reporter gene in transgenic zebrafish. Lateral and dorsal views of transgenic
fish embryos transiently harboring the F3pGFP (A-E) and
F8pGFP (F,G) constructs. GFP activity with F3pGFP at 8.5
(A), 12 (B) and 32 (E) hpf. Bright (C) and dark (D) field observations of
F3pGFP transgenic fish at 18 hpf. F8pGFP expression at 18
(F) and (G) 30 hpf. a, anterior; di, diencephalon; e, eye; mhb, mid-hindbrain
boundary; ov, optic vesicle; tec, tectum; tel, telencephalon; p,
posterior.
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Fig. 12. Identification of conserved sequence elements. (A,B) The 69 kb
Fugu genomic sequences and the 1 Mb mouse genomic sequences, which
correspond to –500 to +500 kb of the Otx2 locus were examined
by Pipmaker analysis. Regions of significant similarity are indicated by
horizontal lines. Conserved sequences and the hypothetical coding region are
defined by E1-E8 and by a black bar (Q9NX78), respectively. Colored bars
denote the genomic fragments that display specific lacZ activity in
Fig. 3. (C,D) Lateral views of
F11placZ (C) and E6/7dplacZ (D) transgenic embryos at 10.5
dpc following ß-galactosidase staining.
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© The Company of Biologists Ltd 2004
