Download Visualization of an endogenous retinoic acid gradient across

LETTER
doi:10.1038/nature12037
Visualization of an endogenous retinoic acid gradient
across embryonic development
Satoshi Shimozono1, Tadahiro Iimura1, Tetsuya Kitaguchi2, Shin-ichi Higashijima3 & Atsushi Miyawaki1,2
protein (CFP) and yellow fluorescent protein (YFP), mutants of
Aequorea GFP (Fig. 1a). Alterations in the conformation of the LBD
in response to RA binding are converted into changes in fluorescence
resonance energy transfer (FRET) from CFP to YFP. Among the constructs containing the LBD from RAR-b (Supplementary Fig. 3a), the
GEPRA-B fusion protein showed the largest response in HeLa cells
(Supplementary Fig. 4). In situ calibration for the intracellular RA
concentration ([RA]i) demonstrated an apparent dissociation constant
(K9d) of 2 nM (Fig. 1b). Responses to the RA precursors retinal and
b
RA
O
RAR
LBD
OH
CFP
FRET efficiency
Ratio (GEPRA-B)
a
0.5
0.5
0.4
0.4
YFP
c
0.3
0.01
Transmission
YFP
1
10
0.1
Concentration (nM)
Ratio (CFP/FRET)
Ratio (GEPRA-G)
100
0.5
raldh2
cyp26s
e
6s
p2
h2
2
6
0.4
4
0.3
2
0
[RA]i (nM)
0
ld
≥3
0.4
1
0
GEPRA-G 0.45
Bud
raldh2
cyp26s
cy
0.5
ra
raldh2/cyp26s
Ratio
GEPRA-B
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Bud
Ratio
d
cy
p2
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0
[RA]i (nM)
In vertebrate development, the body plan is determined by primordial morphogen gradients that suffuse the embryo. Retinoic acid
(RA) is an important morphogen involved in patterning the
anterior–posterior axis of structures, including the hindbrain1–6
and paraxial mesoderm7,8. RA diffuses over long distances, and
its activity is spatially restricted by synthesizing and degrading
enzymes9. However, gradients of endogenous morphogens in live
embryos have not been directly observed; indeed, their existence,
distribution and requirement for correct patterning remain controversial10. Here we report a family of genetically encoded indicators for RA that we have termed GEPRAs (genetically encoded
probes for RA). Using the principle of fluorescence resonance
energy transfer we engineered the ligand-binding domains of RA
receptors to incorporate cyan-emitting and yellow-emitting fluorescent proteins as fluorescence resonance energy transfer donor
and acceptor, respectively, for the reliable detection of ambient
free RA. We created three GEPRAs with different affinities for
RA, enabling the quantitative measurement of physiological RA
concentrations. Live imaging of zebrafish embryos at the gastrula
and somitogenesis stages revealed a linear concentration gradient
of endogenous RA in a two-tailed source–sink arrangement across
the embryo. Modelling of the observed linear RA gradient suggests
that the rate of RA diffusion exceeds the spatiotemporal dynamics
of embryogenesis, resulting in stability to perturbation. Furthermore, we used GEPRAs in combination with genetic and pharmacological perturbations to resolve competing hypotheses on the
structure of the RA gradient during hindbrain formation and somitogenesis. Live imaging of endogenous concentration gradients
across embryonic development will allow the precise assignment of
molecular mechanisms to developmental dynamics and will accelerate the application of approaches based on morphogen gradients
to tissue engineering and regenerative medicine.
RA is a small lipophilic molecule that acts as a ligand for nuclear RA
receptors (RARs). RA is synthesized from retinal by retinaldehyde
dehydrogenase type 2 (Raldh2) and degraded into polar metabolites
by Cyp26 (Supplementary Fig. 1a). During early vertebrate embryogenesis, regions of RA synthesis and degradation were mapped by
detecting messenger RNA encoding these metabolizing enzymes. In
zebrafish, raldh2 is expressed in the mid-trunk, whereas cyp26 is
expressed at both the anterior and posterior ends11. We therefore
speculate that a two-tailed gradient of RA could form with the highest
concentration in the mid-trunk and tapering off at each end (Supplementary Fig. 2). Gradient formation of other morphogens, all of
which are genetically encoded peptides, could be observed by fusing
them to green fluorescent protein (GFP) and introducing them exogenously into embryos12–16. However, the non-peptidic structure of RA
makes it difficult to image directly17. It therefore remains unknown
whether postulated RA gradients exist, have a linear distribution and
are required for normal embryonic development.
To address this gap we developed GEPRAs. The ligand-binding
domains (LBDs) from mouse RARs were flanked by cyan fluorescent
500
1,000
1,500
Head-to-tail distance (μm)
Figure 1 | Development and characterization of GEPRAs. a, Schematic
representation of GEPRA. The light and dark yellow denote the unbound and
RA-bound states of GEPRA, respectively. b, RA titration curves for GEPRA-B
(red) and GEPRA-G (blue). Rmax (RA-depleted form) and Rmin (RA-saturated
form) values for GEPRA-B were slightly higher than those of GEPRA-G. Fitting
with the Michaelis–Menten equation yielded K9d values of 2 and 4 nM for
GEPRA-B and GEPRA-G, respectively. Each data point is the mean 6 s.d. for
nine experiments. c, Transmission and fluorescence (YFP and FRET ratio)
images of a five-somite embryo from the GEPRA-B transgenic zebrafish line.
d, e, Ratiometric [RA]i images (left), whole-mount in situ hybridization results
for raldh2 and cyp26s (centre), and graphs of the spatial distributions of raldh2
and cyp26s (right) in bud-stage embryos expressing GEPRA-B (d) and GEPRAG (e). Absolute [RA]i values are displayed on the right side of each graph.
Scale bars, 200 mm.
1
Laboratory for Cell Function Dynamics, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan. 2Life Function and Dynamics, ERATO, JST, 2-1 Hirosawa, Wako-city, Saitama,
351-0198, Japan. 3National Institutes of Natural Sciences, Okazaki Institute for Integrative Bioscience, National Institute for Physiological Sciences, Okazaki, Aichi, 444-8787, Japan.
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RESEARCH LETTER
sensitive in zebrafish embryos because the eYFP chromophore takes a
relatively long time to mature compared with the rapid timescale of
zebrafish embryogenesis.
We verified that the [RA]i gradient in the head region at the bud
stage (Fig. 1d, e) was contained within the hindbrain field (Supplementary Fig. 9). We also found that the signal representing high
[RA]i in the mid-trunk was detectable at 75% epiboly and developed
into a clear peak at the tailbud stage (Fig. 2a). Given these observations,
we were able to resolve a historical controversy about the putative RA
gradient in the hindbrain4–6. The major argument against the presence of this gradient is the observation that embryos depleted of
endogenous RA can be fully rescued with a uniform concentration
of exogenous RA5. To examine whether this approach resulted in a
rectangular RA distribution, we performed rescue experiments with
the GEPRA-B transgenic line (Fig. 2b). At 4 h after fertilization (hpf),
embryos were exposed to 10 mM DEAB and various concentrations of
RA. The embryos were assessed for [RA]i at the three-somite to foursomite stages and for morphology at 36 hpf. Treatment with 10 mM
DEAB (Fig. 2b, second column of images) nearly abolished the signal
representing high [RA]i and the imaged embryo developed a kinked
head, which is a characteristic of RA depletion in zebrafish2. When
10 nM RA was applied together with 10 mM DEAB (Fig. 2b, fourth
a
0
Time (h)
0.5
70%
1
75%
1.5
80%
2
85%
2.5
90%
Bud
Transmission
0.5
RA
(GEPRA-B)
Ratio
0
0.5
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0.3
0
b
1,000
Distance (μm)
10 μM DEAB
Control
4S
1 nM RA
10 μM DEAB
3S
4S
10 nM RA
10 μM DEAB
4S
Transmission
0.5
RA
(GEPRA-B)
0
Ratio
retinol were almost negligible (K9d ? 100 nM; Supplementary Fig. 1b).
We speculated that using multiple GEPRAs with different RA affinities
would allow us to measure [RA]i quantitatively and create two additional GEPRA variants. GEPRA-AA was generated by introducing two
amino-acid substitutions in the LBD of GEPRA-B. Of the constructs
containing the LBD from RAR-c, GEPRA-G produced the best results
(Supplementary Fig. 3b). GEPRA-AA and GEPRA-G produced K9d
values of 50 nM (Supplementary Fig. 5a) and 4 nM (Fig. 1b), respectively.
We generated transgenic zebrafish lines ubiquitously expressing
GEPRA-B or GEPRA-G. In a five-somite embryo expressing GEPRAB, the probe was uniformly distributed (Fig. 1c, YFP), but the CFP/FRET
ratio—that is, [RA]i—was highest in the mid-trunk and lowest in
the head and tail (Fig. 1c, ratio). Next we depleted embryos of RA
with 10 mM 4-(diethylamino)benzaldehyde (DEAB), an inhibitor of
Raldh2. DEAB abolished the GEPRA-B-derived high [RA]i signal in
the mid-trunk (Supplementary Fig. 6). This indicates that the probe
is RA-specific because DEAB should increase retinal and retinol
concentrations.
We compared signals representing [RA]i with the spatial expression
patterns of RA-metabolizing enzymes. Immediately after imaging with
GEPRA-B (Fig. 1d, left), bud-stage embryos were fixed and subjected
to in situ hybridization with probes specific for raldh2 and cyp26
species (cyp26s; a mixture of cyp26a1, cyp26b1 and cyp26c1)5 (Fig. 1d,
centre). Comparative spatial profiles indicated that the signals representing [RA]i were high in the raldh2-expressing region and low in
the cyp26s-expressing region (Fig. 1d, right). Two intermediate zones
flanked by the raldh2-expressing and cyp26s-expressing regions were
of particular interest. In the posterior zone, the signal representing
[RA]i was graded almost linearly. In the anterior zone, however, the
probe saturated near the raldh2-expressing region. To examine high
levels of [RA]i quantitatively, we imaged GEPRA-G transgenic zebrafish at the bud stage (Fig. 1e) and found linear [RA]i gradients in
both the anterior and posterior intermediate zones. We also expressed
GEPRA-AA transiently by injecting fertilized wild-type eggs with
mRNA. GEPRA-AA produced no [RA]i gradient in three-somite
embryos (Supplementary Fig. 5b). On the basis of the K9d values of
the three GEPRAs, the highest [RA]i situated anteriorly within the
raldh2-expressing region was estimated to be 6 nM (Fig. 1e). All of
the gradients identified by using both GEPRA-B and GEPRA-G
(Fig. 1d, e) reached the edges of the cyp26s-expressing regions, indicating that the steady-state distribution of RA is linear. This finding
supports the model in which a local source and a local sink together
generate a linear gradient in the flanked region based on a simple
diffusion model18 within the spatiotemporal parameters of embryogenesis19. We examined whether a rectangular distribution of [RA]i
could exist stably in the intermediate zones with a computer simulation. Assuming that the diffusion coefficient of RA and the width of
the zone were 10 mm2 s21 (ref. 20) and 200 mm, respectively, our simulation showed that a rectangular distribution created a linear gradient
in about 10 min (Supplementary Fig. 7), which is much faster than the
time scale of embryonic growth and supports a source–sink model in
the formation of a linear gradient by simple diffusion.
RA signalling in zebrafish embryos was previously observed using
an exogenous reporter gene system, in which an RA response element
(RARE) drives the expression of enhanced YFP (eYFP)21. However,
RARE–eYFP transgenic zebrafish did not produce specific fluorescence signals until very late, at roughly the 18-somite stage (Supplementary Fig. 8a), and even at the 20-somite stage eYFP fluorescence
was detected only in old somites. In contrast, the GEPRA-B signal
indicating a high [RA]i was distributed from the region near the otic
vesicle to the most posterior somite (Supplementary Fig. 8b). At this
stage, raldh2 mRNA accumulated in the eyes and all somites, whereas
mRNAs encoding cyp26s were abundant in the head region and tail tip
(Supplementary Fig. 8c); these patterns agree with the distribution
of GEPRA-B signals representing [RA]i. Thus, RARE–eYFP, which
unlike GEPRAs is an indicator of RA signalling and not [RA]i, is not
0.5
0.4
0
1,000
Head-to-tail distance (μm)
Morphology
at 36 hpf
Figure 2 | [RA]i gradient during hindbrain development. a, Time-lapse
[RA]i imaging of GEPRA-B in an embryo from 70% epiboly to bud stage. A
series of transmission images (top), [RA]i images (middle) and ratio profiles
(bottom) are shown. The most anterior and most posterior points represented
in the ratio profiles are indicated in the transmission images by cyan and green
dots, respectively. Times since the start of imaging are shown above the figures.
b, Visualization of [RA]i in GEPRA-B-bearing embryos (three-somite (3S) to
four-somite (4S) stage) with and without DEAB to deplete RA, and various
concentrations of exogenous RA as denoted above the images. In each column
reflecting the various treatment conditions, transmission and [RA]i images are
accompanied by a graph showing the spatial distribution of [RA]i along the
anterior–posterior axis of the body. A transmission image of a later
developmental stage (36 hpf) is also presented. A red arrowhead indicates a
kink in the head region. Scale bars, 200 mm.
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LETTER RESEARCH
a
10.5
11.5
Bud
12.5
2S
13.5
4S
14.5
7S
15.5
9S
16.5 hpf
11S
14S
Transmission
0.5
RA
(GEPRA-B)
Ratio
0
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0 1,000
b
Head-to-tail distance (mm)
10.5
11.5
12.5
13.5
14.5
15.5
16.5
Transmission
0.5
RA
(GEPRA-B)
Ratio
column), however, a signal reflecting high [RA]i was detected.
Although full recovery was not observed, a substantial [RA]i gradient
was created in the hindbrain field. The gradient was sufficient to allow
normal embryo development. Under the same conditions, 1 nM RA
(Fig. 2b, third column) resulted in a smaller [RA]i gradient and did not
rescue the zebrafish from the effects of RA depletion. The results were
reproduced for each treatment condition with multiple embryos (Supplementary Fig. 10). These perturbation experiments revealed that the
distribution of RA for hindbrain patterning is reliably governed by
local degradation of RA, as proposed previously4–6. Such [RA]i gradients depending on RA degradation were also observed during somitogenesis (Supplementary Fig. 11). In the zebrafish mutant giraffe (gir)
containing a mutation in the cyp26a1 gene, there are patterning defects
in various organs22. We injected GEPRA-B mRNA into gir embryos,
and did indeed find a global increase in [RA]i in six-somite embryos
(Supplementary Fig. 12).
Another essential morphogen that functions during the formation
of the embryonic axis includes the fibroblast growth factor (FGF)
family7,8,23,24. The interactions between RA and FGF signalling include
positive and negative feedback and feedforward mechanisms. To
examine how Fgf8 regulates endogenous RA gradients, we performed
[RA]i imaging experiments using embryos in which Fgf8 signalling
was suppressed. We injected fgf8 splice-blocking morpholino oligonucleotides (MOs)25 into one-cell-stage embryos to phenocopy the
well-characterized zebrafish mutant acerebellar (ace)26,27. As a result
of mutation of the fgf8 gene, homozygous ace embryos lack both a
cerebellum and organizer in the midbrain–hindbrain boundary while
retaining expression of some rhombomere-marker genes26. Because
RA signalling is required for the formation of posterior segments of
the hindbrain2–6, such as rhombomeres 5–7, we assessed whether the
RA gradient was retained in the hindbrain field of fgf8 morphants
expressing GEPRA-B. Time-lapse transmission images confirmed
effective knockdown of fgf8 expression based on a bulge in the area of
the developing midbrain (arrowheads)26 and shortened tail (Fig. 3a, b).
In the MO-treated embryos, the signal representing high [RA]i in the
mid-trunk was observed until 12.5 hpf (Fig. 3b). Normal posteriorization of their hindbrain26 was verified by using in situ hybridization
with two rhombomere markers (Supplementary Fig. 13). In addition,
the expression profiles of raldh2 and cyp26s were unaffected at 10 hpf
(Fig. 3c). After 13.5 hpf, however, high [RA]i was attenuated to baseline
levels in fgf8 morphants (Fig. 3b). We found that this decrease in [RA]i
was due to decreased levels of raldh2 expression (Fig. 3d), indicating
that fgf8 may have a role in the maintenance of raldh2 gene expression.
The downregulation of raldh2 expression was previously observed
in ace mutants28; however, the GEPRA system allowed us to directly
image the effect on the RA gradient after the loss of raldh2 expression.
The free diffusion of a lipophilic molecule such as RA may be
limited in aqueous environments. A large fraction of RA molecules
are probably transported intracellularly by interaction with cellular
RA-binding proteins10,29. On the assumption that most intracellular
RAs are bound to carrier proteins, GEPRAs detect the free form of RA.
The endogenous RA-buffering system is reminiscent of intracellular
Ca21 buffering in which loading cells with a large amount of highaffinity Ca21 probes does not markedly affect intracellular Ca21
dynamics30. Thus, the expression of GEPRAs can be increased without
disturbing endogenous RA dynamics or the normal embryonic development of fish. In support of this, GEPRA transgenic embryos developed
without apparent morphological aberrations.
Here we have generated fluorescent probes that allowed direct visualization of the endogenous RA gradient in live embryos. The existence
of an RA gradient is a matter of historical debate4–6. Our GEPRA
system clearly demonstrated that a two-tailed linear RA gradient exists
during early embryogenesis, and that Fgf8 is not required for RA
gradient formation in the hindbrain but is required for the maintenance of raldh2 expression and the RA gradient later in development.
The ability of GEPRA to directly reveal endogenous morphogen
0
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0.3
0
c
1,000
Control MO
Head-to-tail distance (mm)
fgf8 MO
10 hpf
d
10 hpf
Control MO
fgf8 MO
14 hpf
14 hpf
14 hpf
14 hpf
raldh2
raldh2
10 hpf
10 hpf
cyp26s
cyp26s
Figure 3 | [RA]i gradient is affected by fgf8 expression. a, b, Time-lapse
[RA]i imaging of GEPRA-B-bearing embryos from 10.5 to 16.5 hpf after
injections of control MO (a) or fgf8-specific MO (b). Transmission images (top
row), [RA]i images (middle row) and ratio profiles (bottom row) are presented.
The control embryo showed normal somitogenesis; somite stages are labelled in
the transmission images (a). A bulge in the area of the developing midbrain in
the embryo injected with the fgf8-specific MO is indicated by black arrowheads;
a signal representing a high [RA]i in the trunk is indicated by white arrows
(b). c, d, Comparative in situ hybridizations showing expression of raldh2 and
cyp26s in embryos at 10 hpf (c) and 14 hpf (d) after injections with control or
fgf8-specific MO. All images are lateral views. Scale bars, 200 mm.
gradients such as RA in live embryos will allow a greater understanding of their roles and mechanisms in patterning the vertebrate
embryo and will support gradient-based approaches in medicine and
bioengineering.
METHODS SUMMARY
The genes for GEPRAs were constructed from complementary DNAs encoding
the LBD of mouse RAR-b or RAR-c, and subcloned into pCS2 for expression in
HeLa cells and for in vitro synthesis of mRNA or into pT2KXIGDin for generation
of transgenic zebrafish lines. [RA]i imaging was performed with an inverted confocal microscope equipped with a 310 objective lens and a 440-nm laser. With the
aperture fully open, non-confocal fluorescence images were acquired. The dualemission ratio imaging was performed under exactly the same conditions for in
situ [RA]i calibration (HeLa cells) and in vivo time-lapse two-dimensional [RA]i
imaging (zebrafish embryos). Thus, absolute [RA]i values in the two-dimensional
image can be obtained from the ratios by using the curves shown in Fig. 1b. RA,
retinol, retinal and DEAB were administrated with 0.1% dimethylsulphoxide
(DMSO). The morphology of an embryo was observed with the confocal microscope simultaneously with [RA]i or with a stereomicroscope afterwards. The
transgenic zebrafish line (Tg(RARE–gata2:NTD–eYFP)ld1)21 was obtained from
the Zebrafish International Resource Center (University of Oregon). The eYFP
fluorescence was observed with an inverted confocal microscope equipped with a
473-nm or 488-nm laser. cDNA encoding raldh2, cyp26a1, cyp26b1, cyp26c1,
krox20, hoxd4, otx2 or myoD in pCS2 was used to construct the probe for in situ
hybridization. All embryos were allowed to grow after live [RA]i imaging, unless
they were subjected to in situ hybridization. Embryos developed without any
morphological aberrations, indicating that GEPRA expression and light exposure
are not morphogenic.
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RESEARCH LETTER
Full Methods and any associated references are available in the online version of
the paper.
Received 22 November 2012; accepted 25 February 2013.
Published online 7 April 2013.
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Supplementary Information is available in the online version of the paper.
Acknowledgements The authors thank Y. Wada, R. Aoki, M. Sugiyama, F. Picazo and
members of the Brain Science Institute Research Resource Center for technical
assistance; C. Yokoyama and A. Terashima for critical reading of the manuscript; the
FANTOM Consortium for the cDNA clones; and the Zebrafish International Resource
Center for the transgenic zebrafish. This work was partly supported by grants from
Japan Ministry of Education, Culture, Sports, Science and Technology Grant-in-Aid for
Scientific Research on Priority Areas ‘Fluorescence Live Imaging’ and ‘Cell Innovation’
and the Human Frontier Science Program.
Author Contributions S.S. and A.M. conceived and designed the study. S.S. performed
all the experiments, analysed the data and designed the manuscript. T.I. supervised the
experiments on somitogenesis. T.K. and S.H. generated transgenic zebrafish lines. A.M.
designed and wrote the manuscript, and supervised the project.
Author Information DNA sequences of GEPRAs are deposited in the DNA Data Bank of
Japan (DDBJ) under accession numbers AB787561–AB787563. Reprints and
permissions information is available at www.nature.com/reprints. The authors declare
no competing financial interests. Readers are welcome to comment on the online
version of the paper. Correspondence and requests for materials should be addressed
to A.M. ([email protected]).
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METHODS
Materials. RA, retinol, retinal and DEAB were purchased from Sigma-Aldrich.
These compounds were dissolved in dimethylsulphoxide (DMSO) to prepare
stock solutions. The compounds were administered in a final concentration of
0.1% DMSO.
Gene construction. cDNA encoding the LBD of RAR-b (FANTOM3 (ref. 31),
6820403N24) was amplified using primers containing 59 SphI and 39 SacI sites.
The digested product was ligated to a BamHI/SphI fragment encoding CFP
and a SacI/XhoI fragment encoding YFP. The ligated product was subcloned into
the BamHI/XhoI sites of the pCS2 vector for mammalian expression. In this
plasmid, genes encoding the donor and/or acceptor were replaced to improve
the RA indicator. Similarly, cDNA encoding RAR-c (FANTOM3 (ref. 31),
F730319B19) was used to make a SphI/SacI fragment encoding the LBD.
Together with the BamHI/SphI fragment encoding CFP and a SacI/EcoRI fragment encoding YFP, cDNA coding for the indicator was subcloned into the
BamHI/EcoRI sites of the pCS2 vector. Introduction of two amino-acid substitutions (R269A and S280A)32 in the LBD of RAR-b was performed as described
previously33.
Characterization of the indicators. HeLa cells were grown in DMEM medium
supplemented with 10% FBS. cDNAs were transfected into cells by using
Lipofectamine and Plus reagent (Invitrogen). Two days after transfection, the
medium was exchanged with DMEM/F12 without phenol red. Dose–response
curves were determined with nine transfected cells.
Generation of GEPRA transgenic zebrafish lines. GEPRA cDNAs were subcloned into the pT2KXIGDin vector, and transgenic lines were created as
described previously34. Fish were maintained at 28.0 uC.
In vivo RA imaging. A glass bead (Iuchi BZ-1) was placed on a coverslip, and
1% agarose (Takara L03) in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.4 mM CaCl2,
0.16 mM MgSO4) was poured on the coverslip and allowed to harden. The glass
bead was then removed to generate a round chamber. An embryo that had been
anaesthetized with Tricaine at more than 16 hpf was placed in the chamber and
covered with 0.3% agarose in E3 medium. The chamber was submerged in E3
medium containing Tricaine. Time-lapse two-dimensional imaging was performed
in the xy–t mode with an FV1000 (Olympus) confocal inverted microscope system
equipped with a 310 objective lens (numerical aperture 0.4; UPlanApo) and a 440nm laser. With the aperture fully open, non-confocal fluorescence images were
acquired. The ratio imaging of embryos was performed under exactly the same
conditions as for calibration experiments with HeLa cells. Thus, absolute [RA]i
values in the image can be obtained from the ratios using the curves shown in
Fig. 1b. Image processing was performed with ImageJ software (http://rsb.info.nih.
gov/ij/). After the background had been subtracted, ratio images were generated and
median-filtered.
Estimation of GEPRA concentration in embryos. Confocal fluorescence images
were acquired with a GEPRA transgenic embryo and a series of YFP solutions of
various concentrations with excitation at 488 nm. The size of the confocal aperture
was about 2 Airy disks. By comparing the intensities of the confocal fluorescence
images, the GEPRA concentration in the embryo was estimated to be approximately 0.1 mM. Considering the concentration35 and affinity for RA (refs 36, 37) of
cellular RA-binding proteins, which were reported to be 10 mM and 0.13–2 nM,
respectively, only a small fraction of RA molecules should be bound to GEPRA.
Transgenic RARE–eYFP zebrafish. Transgenic zebrafish (RARE–gata2:NTD–
eYFP)ld1)21 were obtained from the Zebrafish International Resource Center
(University of Oregon). Fluorescence was observed with an FV1000 confocal
microscope (Olympus) with a 488-nm laser or an FV10i confocal microscope
(Olympus) with a 473-nm laser.
Depletion of endogenous RA and application of exogenous RA. Embryos were
incubated in E3 medium containing 0.1% DMSO (vehicle), 10 mM DEAB, 1 nM
RA with 10 mM DEAB, or 10 nM RA with 10 mM DEAB from 4 to 24 hpf. After
24 hpf, embryos were bathed in E3 medium. RA imaging was performed at 11 hpf
(roughly the three-somite stage). The imaged embryos were kept at 28.0 uC until
their morphologies were examined at 36 hpf with a stereomicroscope (MZ16 F;
Leica) equipped with a charge-coupled device (CCD) camera (DP50; Olympus).
In situ hybridization. cDNA encoding raldh2, cyp26a1, cyp26b1, cyp26c1, krox20,
hoxd4, otx2 or myoD was amplified from a cDNA library prepared from 10-hpf or
24-hpf embryos and cloned into the pCS2 vector. Probes were labelled with a
digoxigenin or fluorescein labelling mix (Roche Diagnostics) and detected with
alkaline phosphatase-conjugated antibodies (1:5,000; Roche Diagnostics). The
colorimetric reaction was performed with BM purple reagent (Roche Diagnostics). Images were captured with a stereomicroscope (MZ16 F; Leica)
equipped with a CCD camera (DP50; Olympus). Fluorescence and in situ hybridization images were linearly registered with the Image Processing Toolbox in
MATLAB software (MathWorks).
Knock down of fgf8. Splice-site-targeted morpholino oligonucleotides for the fgf8
gene (E2I2 (59-TAGGATGCTCTTACCATGAACGTCG-39) and E3I3 (59-CAC
ATACCTTGCCAATCAGTTTCCC-39)) were used to block its pre-mRNA splicing in zebrafish embryos25. One-cell-stage embryos were injected with both E2I2
and E3I3 (2.5 ng of each) or a control oligonucleotide (5 ng; 59-CCTCTTA
CCTCAGTTACAATTTATA-39).
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