LETTER doi:10.1038/ nature12037 Visualization of an endogenous retinoic acid gradient across embryonic development Satoshi Shimozono, Tadahiro limura', Tetsuya Kitaguchi, Shin-ichi Higashijima& Atsushi Miyawaki In vertebrate development, the body plan is determined by prim- protein(CFP) and yellow fluorescent protein (YFP), mutants of ordial morphogen gradients that suffuse the embryo Retinoic acid Aequorea GFP(Fig 1a). Alterations in the conformation of the LBD (RA)is an important morphogen involved in patterning the in response to RA binding are converted into changes in fluorescence anterior-posterior axis of structures, including the hindbrain- resonance energy transfer(FRET) from CFP to YFP. Among the con and paraxial mesoderm". RA diffuses over long distances, and structs containing the LBD from RAR-P( Supplementary Fig 3a),the its activity is spatially restricted by synthesizing and degrading GEPRA-B fusion protein showed the largest response in HeLa cells zymes. However, gradients of endogenous morphogens in live( Supplementary Fig. 4). In situ calibration for the intracellular RA embryos have not been directly observed; indeed, their existence, concentration([RA)demonstrated an apparent dissociation constant distribution and requirement for correct patterning remain con- (K'a) of 2 nM( Fig. 1b). Responses to the RA precursors retinal and troversial. Here we report a family of genetically encoded indica- tors 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 人 receptors to incorporate cyan-emitting and yellow-emitting fluor escent proteins as fluorescence resonance energy transfer donor CFP and acceptor, respectively, for the reliable detection of ambient 图 free RA. We created three geRas with different affinities for FRET efficiency RA, enabling the quantitative measurement of physiological RA Concentration(nM) concentrations. Live imaging of zebrafish embryos at the gastral Ratio(CFP/F 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. Further more,we used GEPRAs in combination with genetic and pharma cological perturbations to resolve competing hypotheses on the d GEPRA-B 0.5 raldh2/cyp26s structure of the ra gradient during hindbrain formation and somi- genesis. Live imaging of endogenous concentration gradients across embryonic development will allow the precise assignment of molecular mechanisms to developmental dynamics and will acce- s plication 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. la). During early vertebrate embryo- ⑤ genesis, regions of RA synthesis and degradation were mapped by 05001,0001,500 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 ends ". We therefore Figure 1 Development and characterization of GEPRAs a, Schematic speculate that a two-tailed gradient of RA could form with the highest RA-bound states of GEPRA, respectively. b, RA titration curves for GEPRA-B concentration in the mid-trunk and tapering off at each end(Sup- (red) and GEPRA-G(blue). Rmax(RA-depleted form)and Rmin(RA-saturated plementary Fig. 2) Gradient formation of other morphogens, all of form)values for GEPRA-B were slightly higher than those of GEPRA-G Fitting which are genetically encoded peptides, could be observed by fusing with the Michaelis-Menten equation yielded K'a values of 2 and 4 nM for them to green fluorescent protein(GFP) and introducing them exo EPRA-B and GEPRA-G, respectively. Each data point is the mean +s d for hakes it difficult to image directly ? It therefore remains unknown images of a five-somite embryo from the GEPRA-: transgenic zebrafish line. whether postulated RA gradients exist, have a linear distribution and for raldh2 and cyp 26s (centre), and graphs of the spatial distributions of raldh2 are required for normal embryonic development. and cyp26s(right)in bud-stage embryos expressing GEPRA-B(d)and GEPRA- domains(LBDs)from mouse RARs were flanked by cyan fluorescent Scale bars, 200ule a values are displayed on the right side of each graph To address this gap we developed GEPRAs. The ligand-binding G(e). Absolute RA na351-0198, Japan?Life Function and Dynamics, ERATO, JST, 2-1 Hirosawa, Wako-city Saitam 351-0198, Japan. National Institutes of Natural Sciences, Okazaki te for Integrative Bioscience, National Institute for Physiological Sciences, Okazaki, Aichi, 444-8787, Japan. 0 MONTH 2013 VOL 000I NATURE I @2013 Macmillan Publishers Limited. All rights reserved
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 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 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 1 Laboratory for Cell Function Dynamics, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan. 2 Life Function and Dynamics, ERATO, JST, 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan. 3 National Institutes of Natural Sciences, Okazaki Institute for Integrative Bioscience, National Institute for Physiological Sciences, Okazaki, Aichi, 444-8787, Japan. YFP Ratio (CFP/FRET) a d 0 0.5 e Bud GEPRA-B raldh2/cyp26s raldh2 cyp26s 0 0.5 0 0.45 Transmission Bud b c GEPRA-G cyp26s raldh2 O OH CFP YFP RA RAR LBD FRET efficiency Ratio (GEPRA-B) 0.01 1 10 100 Ratio (GEPRA-G) 0.4 0.5 0.4 0.5 0.3 0.1 Concentration (nM) Ratio 0.4 0.5 raldh2 cyp26s cyp26s [RA]i (nM) ≥3 2 1 0 500 1,000 1,500 Head-to-tail distance (μm) 0.4 0.3 Ratio 6 2 4 [RA]i (nM) 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. 00 MONTH 2013 | VOL 000 | NATURE | 1 ©2013 Macmillan Publishers Limited. All rights reserved
RESEARCHLETTER retinol were almost negligible(K'd>> 100 nM; Supplementary Fig. 1b). sensitive in zebrafish embryos because the eYFP chromophore takes a We speculated that using multiple GEPRAs with different RA affinities relatively long time to mature compared with the rapid timescale of ould allow us to measure[RAJi quantitatively and create two addi- zebrafish embryogene tional GEPRA variants GEPRA-AA was generated by introducing two We verified that the [RAl gradient in the head region at the bud amino-acid substitutions in the LBD of GEPRA-B Of the constructs stage(Fig. ld, e) was contained within the hindbrain field(Sup- containing the LBD from RAR-Y, GEPRA-G produced the best results plementary Fig. 9). We also found that the signal representing high (Supplementary Fig. 3b). GEPRA-AA and GEPRA-G produced K'd [RAJi in the mid-trunk was detectable at 75% epiboly and developee values of 50 nM(Supplementary Fig 5a)and 4 nM(Fig 1b), respectively. into a clear peak at the tailbud stage(Fig 2a). Given these observations, We generated transgenic zebrafish lines ubiquitously expressing we were able to resolve a historical controversy about the putative RA GEPRA-B or GEPRA-G In a five-somite embryo expressing GEPRA- gradient in the hindbrain-. The major argument against the pre B, the probe was uniformly distributed(Fig. Ic, YFP), but the CFP/FRET sence of this gradient is the observation that embryos depleted of ratio-that is, [RAJ-was highest in the mid-trunk and lowest in endogenous ra can be fully rescued with a uniform concentration he head and tail (Fig. Ic, ratio). Next we depleted embryos of ra of exogenous RA. To examine whether this approach resulted in a th 10 uM 4-(diethylamino)benzaldehyde(DEAB), an inhibitor of rectangular RA distribution, we performed rescue experiments with Raldh2. DEAB abolished the GEPRA-B-derived high [RAli signal in the GEPRA-B transgenic line(Fig 2b). At 4h after fertilization(hpf), the mid-trunk(Supplementary Fig. 6). This indicates that the probe embryos were exposed to 10 HM DEAB and various concentrations of is RA-specific because DEAB should increase retinal and retinol RA. The embryos were assessed for [RAli at the three-somite to fou concentrations somite stages and for morphology at 36hpf Treatment with 10 HM We compared signals representing [RAJi with the spatial expression DEAB(Fig 2b, second column of images)nearly abolished the signal patterns of RA-metabolizing enzymes. Immediately after imaging with representing high [RAl and the imaged embryo developed a kinked GEPRA-B(Fig Id, left), bud-stage embryos were fixed and subjected head, which is a characteristic of RA depletion in zebrafish. When entre). Comparative spatial profile "p26b1 and cyp26 )'E p26 10nM RA was applied together with 10 HM DEAB(Fig2b, fourth to in situ hybridization with probes specific for raldh2 and species(cyp26s; a mixture of cyp26a1 cated that the signals repre ting (RAJi were high in the raldh2-expressing region and low in the cyp26s-expressing region(Fig. ld, right). Two intermediate zones flanked by the raldh2-expressing and cyp26s-expressing regions wereTransmission of particular interest. In the posterior zone, the signal representing 00000O [RAl was graded almost linearly In the anterior zone, however, the probe saturated near the raldh2-expressing region. To examine high levels of [RA] quantitatively, we imaged GEPRA-G transgenic zebra fish at the bud stage(Fig. le)and found linear [RAJi gradients in both the anterior and posterior intermediate zones. We also expressed GEPRA-AA transiently by injecting fertilized wild-type eggs with :Nwm∧kk~% mRNA. GEPRA-AA produced no [RAJi gradient in three-somite embryos( Supplementary Fig. 5b). On the basis of the K'a values of he three GEPRAs, the highest [RAl situated anteriorly within the b Control O HM DEAB 10 HM DEAB raldh2-expressing region was estimated to be 6nM(Fig. le). All of the gradients identified by using both GEPRA-B and GEPRA (Fig. Id, e)reached the edges of the cyp26s-expressing regions, indi- Transmis cating 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 ffusion model within the spatiotemporal parameters of embry genesis. We examined whether a rectangular distribution of [RAJ (GEPRA-B could exist stably in the intermediate zones with a computer simu the zone were 10 um lation showed that a rectangular distribution created a linear gradient in about 10 min( Supplementary Fig. 7), which is much faster than the d-to-tail distance (um) time scale of embryonic growth and supports a source-sink model he formation of a linear gradient by simple diffusion. RA signalling in zebrafish embryos was previously observed using (RArE) drives the expression of enhanced YFP (eYFP). However, (RAl imaging of GEPRA-B in an embryo from 70% epiboly to bud stage.A RARE-eYFP transgenic zebrafish did not produce specific fluores- series of transmission images(top), [RAli images(middle)and ratio profiles cence signals until very late, at roughly the 18-somite stage(Sup-(bottom) are shown. The most anterior and most posterior points represented plementary Fig 8a), and even at the 20-somite stage e YFP fluorescence in the ratio profiles are indicated in the transmission images by cyan and green as detected only in old somites. In contrast, the GEPRA-B signal dots, respectively. Times since the start of imaging are shown above the figure indicating a high (RA] was distributed from the region near the otic b, Visualization of IRAl in GEPRA-B-bearing embryos( three-somite (3S) concentrations of exogenous RA as denoted above the images In each column stage, raldh2 mRNA accumulated in the eyes and all somites, whereas reflecting the various treatment conditions, transmission and [RAl images are mRNAs encoding cyp26s were abundant in the head region and tail tip accompanied by a graph showing the spatial distribution of (RA), along the (Supplementary Fig. 8c); these patterns agree with the distribution anterior-posterior axis of the body. A transmission image of a later of GEPRA-B signals representing [RAJ. Thus, RARE-eYFP, which developmental stage(36hpf) is also presented. A red arrowhead indicates a nlike GEPRAs is an indicator of RA signalling and not [RAli is not kink in the head region Scale bars, 200 um. 2I NATURE I VOL 00000 MONTH 2013 @2013 Macmillan Publishers Limited. All rights reserved
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 s 21 (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 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 0.5 0.4 0 1,000 Head-to-tail distance (μm) Control 10 μM DEAB 1 nM RA 10 μM DEAB 10 nM RA 10 μM DEAB Ratio Transmission RA (GEPRA-B) Morphology at 36 hpf a b 0 0.5 4S 3S 4S 4S Transmission RA (GEPRA-B) 75% 90% Bud Time (h) 0 0.5 1 1.5 2 2.5 0 0.5 70% 80% 85% 0 1,000 Distance (μm) Ratio 0.3 0.5 0.4 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. RESEARCH LETTER 2 | NATURE | VOL 000 | 00 MONTH 2013 ©2013 Macmillan Publishers Limited. All rights reserved
LETTERRESEARCH Although full recovery was not observed, a substantial (RA) gradient as created in the hindbrain field. The gradient was sufficient to allow Transmission normal embryo development. Under the same conditions, InM RA (Fig 2b, third column)resulted in a smaller [RAl gradient and did not rescue the zebrafish from the effects of RA depletion. The results were (GEPRA-B produced for each treatment condition with multiple embryos( Sup- plementary Fig. 10). These perturbation experiments revealed that the distribution of RA for hindbrain patterning is reliably governed by local degradation of RA, as proposed previously-6 Such [RAJi gradi- ents depending on Ra degradation were also observed during somi- b ogenesis(Supplementary Fig. 11). In the zebrafish mutant giraffe (gir) containing a mutation in the cyp 26al gene, there are patterning defect in various organs. We injected GEPRA-B mRNA into gir embryos, 000 and did indeed find a global increase in [RAJi in six-somite embryo (Supplementary Fig 12). (GEPRA-B) Another essential morphogen that functions during the formation of the embryonic axis includes the fibroblast growth factor(FGF) family32324. The interactions between RA and FGF signalling include positive and negative feedback and feedforward mechanisms. To Head-to-tail distance (mm) examine how Fgf8 regulates endogenous RA gradients, we performed [RAl imaging experiments using embryos in which Fgf8 signalling was suppressed. We injected gf8 splice-blocking morpholino ol nucleotides(MOs)into one-cell-stage embryos to phenocopy the ●- raldh2 well-characterized zebrafish mutant cerebellar (ace).. 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 genes2.Because RA signalling is required for the formation of posterior segments of the hindbrain2-6 such as rhombomeres 5-7, we assessed whether the Figure 3 [RAli gradient is affected by fgf expression. a,b, Time-lapse RA gradient was retained in the hindbrain field of fgf8 morphants RAl imaging of GEPRA-B-bearing embryos from 10.5 to 16.5hpf after xpressing GEPRA-B. Time-lapse transmission images confirmed injections of control Mo (a)or fgf8-specific MO (b) effective knockdown of fgf8 expression based on a bulge in the area of row), (RAJ, images(middle row) and ratio profiles(bottom row)are presented. the developing midbrain(arrowheads)and shortened tail(Fig 3a, b). The control embryo showed normal somitogenesis; somite stages are labelled In the MO-treated embryos, the signal representing high [RAJi in the the transmission images(a). A bulge in the area of the developing midbrain in mid-trunk was observed until 12.5 hpf(Fig. 3b). Normal posteriori- the embryo injected with the fgf8-specific MO is indicated by black arrowheads; zation of their hindbrain 6 was verified by using in situ hybridization a signal representing a high [RAli in the trunk is indicated by white arrows with two rhombomere markers(Supplementary Fig. 13). In addition, c, d, Comparative in situ hybridizations showing expression of raldh2 and the expression profiles of raldh2 and cyp26s were unaffected at 10hpf p26s in embryos at 10 hpf (c)and 14hpf(d)after injections with control or (Fig. 3c). After 13.5 hpf, however, high(RA) was attenuated to baseline fgf8-specific MO. All images are lateral views. Scale bars, 200 um. rels in fgf8 morphants( Fig. 3b). We found that this decrease in (RAl gradients such as RA in live embryos will allow a greater under was due to decreased levels of raldh2 expression(Fig. 3d), indicating standing of their roles and mechanisms in patterning the vertebrate that fgf& may have a role in the maintenance of raldh2 gene expression. embryo and will support gradient-based approaches in medicine and The downregulation of raldh2 ression was previous in ace mutants; however, the GEPRA system allowed us to directly image the effect on the Ra gradient after the loss of raldh2 expression. METHODS SUMMARY The free diffusion of a lipophilic molecule such as RA may be The genes for GEPRAs were constructed from complementary DNAs encoding limited in aqueous environments. A large fraction of RA molecules the LBD of mouse RAR-B or RAR-y, and subcloned into pCS2 for expression in are probably transported intracellularly by interaction with cellular Hela cells and for in vitro synthesis of mRNA or into pT2KXIGAin for generation RA-binding proteins 0. 29.On the assumption that most intracellular of transgenic zebrafish lines. [RAh imaging was performed with an inverted RAs are bound to carrier proteins, GEPRAs detect the free form of RA. focal microscope equipped with a x10 objective lens and a 440-nm laser. with Ca2t buffering in which loading cells with a large amount ofhe r aperture fully open, non-confocal fluorescence images were acquired. The dual- The endogenous RA-buffering system is reminiscent of intracellula emission ratio imaging was performed under exactly the same conditions for in affinity Ca probes does not markedly affect intracellular Ca imaging (zebrafish embryos). Thus, absolute (RAI values in the two-dimensional disturbing endogenous RA dynamics or the normal embryonic deve- retinol, retinal and DEAB were administrated with 0.1% dimethylsulphoxide lopment of fish. In support of this, GEPRA transgenic embryos developed (DMSO). The morphology of an embryo was observed with the confocal micro- without apparent morphological aberrations scope simultaneously with [RAli or with a stereomicroscope afterwards. The oafish line(Tg(RARE NTD-eYFP)ldl) was obtained from alization of the endogenous RA gradient in live embryos. The existence the Zebrafish Intemational Resource Center (University of Oregon). The eYFP 473-nm or 488-nm laser. cDNA encoding raldh2, cyp26al, cyp26b1, cyp26cl system clearly demonstrated that a two-tailed linear RA gradient exists krox20, hoxd4, otx2 or myoD in pCS2 was used to construct the probe for in situ during early embryogenesis, and that Fgf8 is not required for RA hybridization. All embryos were allowed to grow after live [RA), imaging, unless radient formation in the hindbrain but is required for the mainten- they were subjected to in situ hybridization. Embryos developed without any nce of raldh2 expression and the Ra gradient later in development. morphological aberrations, indicating that GEPRA expression and light exposure The ability of GEPRA to directly reveal endogenous morphogen are not morphogenic. 00 MONTH 2013 VOL 000 NATURE 3 @2013 Macmillan Publishers Limited. All rights reserved
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]iwas 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 offish. 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 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 forin 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. 0 0.5 Transmission RA (GEPRA-B) 10.5 11.5 12.5 13.5 14.5 15.5 16.5 d a b c raldh2 cyp26s Control MO fgf8 MO 14 hpf Control MO fgf8 MO raldh2 cyp26s 10 hpf 10 hpf 10 hpf 10 hpf 14 hpf 14 hpf 14 hpf Bud 2S 4S 7S 9S 11S 14S Transmission RA (GEPRA-B) 10.5 11.5 12.5 13.5 14.5 15.5 16.5 hpf 0 0.5 0 1,000 0.4 0.5 Ratio Head-to-tail distance (mm) 0 1,000 Head-to-tail distance (mm) Ratio 0.3 0.4 0.5 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. LETTER RESEARCH 00 MONTH 2013 | VOL 000 | NATURE | 3 ©2013 Macmillan Publishers Limited. All rights reserved
RESEARCHLETTER Full Methods and any associated references are available in the online version of 20. Eichele, G& Thaller, C Characterization of concentration gradients morphologically active retinoid in the chick limb bud. J Cell Biol. 105, 1917-1923 Received 22 November 2012; accepted 25 February 2013 21. Perz.Edwards, A, Hardison, N L& Linney, E Retinoic acid-mediated gene 22. Emoto, Y, Wada, H, Okamoto, H, Kudo, A. Imai, Y. Retinoic acid-metabolizing 2. Begemann, G, Marx, M, Mebus, K, Meyer, A& Bastmeyer, M. Beyond the neckless 23 Cord in zebrafish. Dev. Biol 278, 415-427(200 tories of hindbrain and spina 1. Stern, C D. Foley, A C Molecular dissection of Hox gene induction and Sawada, A etal. Fgf/MAPK signaling is a crucial positional cue in somite boundary 24. Dubrulle, J, McGrew, M J. Pourquie, O FGF signaling controls somite boundary 3. Maves, L& Kimmel, C. B Dynamic and sequential patterning of the zebrafish on clock control of spatiotemporal Hox gene 4.eLm2=25 32(2001 oundaries of retinoic acid 25. Draper, B w, Morcos, P.A.& Kimmel, C. B Inhibition of zebrafish fgf8 pre-mRNA gos: a quantifiable method for gene knockdown. 5. Hernandez, R E, Putzke A P, Myers, J. P, Margaretha, L& Moens, C B Cyp26 26. Brand, M et al Mu 123,179-190(1996) Izymes generate the re em necessary for hindbrain 27. Reifers, F et al Fgf8 is mutated in zeb 6. White, R J, Nie, Q, Lander, A D. Schilling, T. F Complex regulation of cyp26a1 somitogenesis. Development 125, 2381-2395 (1998 reates a robust retinoic acid gradient in the zebrafish embryo. PLoS Biol. 5, e304 28. Hamade. A etal retine Dev. bio.289,127-140(200 7. Diez del Corral, R etal Opposing FGF and retinoid pathways control ventral neural 29. Napoli, J. L Interactions of retinoid binding proteins and enzymes in retinoid of segmental patterning by retinoic acid 30. Haugland, R. P &Johnson, L D in Fluorescent and Luminescent Probes for Biological signaling during Xenopus somitogenesis. Dev Cell 6, 205-218(2004). Activity(ed Mason, W. T)40-50(Academic, 1999) 9. Aulehla, A& Pourquie, O Signaling gradients during paraxial mesoderm Supplementary Information is available in the online version of the paper. development. Cold Spring Harb. Perspect Biol. 2, a000869 (2010) nents The authors thank y Wada, R. Aoki, M. s Picazo and 11. Kudoh, T, Wilson, S W&Dawid, L B Distinct roles for Fgf, Wnt and retinoic acid in assistance: C Yokoyama and A. Terashima for critical readi posteriorizing the neural ectoderm. Development 129, 4335-4346(2002) for the cdna clones: and the Zebrafish Center for the transgenic zebrafish. This work was partly supported by grants sen,A& Gonalez-Gaitan M. Gradient formation of the and the Human Frontier Science Program D W. Stabil e130,141-152(2007 15. Yu, S.R. et al. Fgf8 morp rms by a source-sink mechanism with experiments on somitogenesis. T.K. and S H. generated transgenic zebrafish lines. A M 26 freely diffusing molecules. Nature 461, 533-536(20U underlies a reaction- designed and wrote the manuscript, and supervised the project. iffusion patterning system Science 336, 721-724(2012 Author Information DNA sequences of GEPRAs sited in the DNA Data Bank of le, P. Retinoic acid signalling during development. Developme 139,843-858(2012) re.com/reprints.Theauthorsdeclare l8. Crick, F. Diffusion in embryogenesis. Nature 225, 420-423(1970). o competing financial interests. Readers are welcome to comment on the online 19. Wolpert, L Positional information and the spatial pattern of cellular differentiation. version of the paper Correspondence and requests for materials should be addressed J. Theor.Bio25,1-47(1969) to A.M. (matsushi@brain riken jp). 4I NATURE I VOL 00000 MONTH 2013 @2013 Macmillan Publishers Limited. All rights reserved
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. 1. Stern, C. D. & Foley, A. C. Molecular dissection of Hox gene induction and maintenance in the hindbrain. Cell 94, 143–145 (1998). 2. Begemann, G., Marx, M., Mebus, K., Meyer, A. & Bastmeyer, M. Beyond the neckless phenotype: influence of reduced retinoic acid signaling on motor neuron development in the zebrafish hindbrain. Dev. Biol. 271, 119–129 (2004). 3. Maves, L. & Kimmel, C. B. Dynamic and sequential patterning of the zebrafish posterior hindbrain by retinoic acid. Dev. Biol. 285, 593–605 (2005). 4. Sirbu, I. O., Gresh, L., Barra, J. & Duester, G. Shifting boundaries of retinoic acid activity control hindbrain segmental gene expression. Development 132, 2611–2622 (2005). 5. Hernandez, R. E., Putzke, A. P., Myers, J. P., Margaretha, L. & Moens, C. B. Cyp26 enzymes generate the retinoic acid response pattern necessary for hindbrain development. Development 134, 177–187 (2007). 6. White, R. J., Nie, Q., Lander, A. D. & Schilling, T. F. Complex regulation of cyp26a1 creates a robust retinoic acid gradient in the zebrafish embryo. PLoS Biol. 5, e304 (2007). 7. Diez del Corral, R. et al. Opposing FGF and retinoid pathways control ventral neural pattern, neuronal differentiation, and segmentation during body axis extension. Neuron 40, 65–79 (2003). 8. Moreno, T. A. & Kintner, C. Regulation of segmental patterning by retinoic acid signaling during Xenopus somitogenesis. Dev. Cell 6, 205–218 (2004). 9. Aulehla, A. & Pourquie´, O. Signaling gradients during paraxial mesoderm development. Cold Spring Harb. Perspect. Biol. 2, a000869 (2010). 10. White, R. J. & Schilling, T. F. How degrading: Cyp26s in hindbrain development. Dev. Dyn. 237, 2775–2790 (2008). 11. Kudoh, T., Wilson, S. W. & Dawid, I. B. Distinct roles for Fgf, Wnt and retinoic acid in posteriorizing the neural ectoderm. Development 129, 4335–4346 (2002). 12. Teleman, A. A. & Cohen, S. M. Dpp gradient formation in the Drosophila wing imaginal disc. Cell 103, 971–980 (2000). 13. Entchev, E. V., Schwabedissen, A. & Gona´lez-Gaita´n, M. Gradient formation of the TGF-b homolog Dpp. Cell 103, 981–991 (2000). 14. Gregor, T., Wieschaus, E. F., McGregor, A. P., Bialek, W. & Tank, D. W. Stability and nuclear dynamics of the bicoid morphogen gradient. Cell 130, 141–152 (2007). 15. Yu, S. R. et al. Fgf8 morphogen gradient forms by a source–sink mechanism with freely diffusing molecules. Nature 461, 533–536 (2009). 16. Mu¨ller, P. et al. Differential diffusivity of Nodal and Lefty underlies a reactiondiffusion patterning system. Science 336, 721–724 (2012). 17. Rhinn, M. & Dolle´, P. Retinoic acid signalling during development. Development 139, 843–858 (2012). 18. Crick, F. Diffusion in embryogenesis. Nature 225, 420–423 (1970). 19. Wolpert, L. Positional information and the spatial pattern of cellular differentiation. J. Theor. Biol. 25, 1–47 (1969). 20. Eichele, G. & Thaller, C. Characterization of concentration gradients of a morphologically active retinoid in the chick limb bud. J. Cell Biol. 105, 1917–1923 (1987). 21. Perz-Edwards, A., Hardison, N. L. & Linney, E. Retinoic acid-mediated gene expression in transgenic reporter zebrafish. Dev. Biol. 229, 89–101 (2001). 22. Emoto, Y., Wada, H., Okamoto, H., Kudo, A. & Imai, Y. Retinoic acid-metabolizing enzyme Cyp26a1 is essential for determining territories of hindbrain and spinal cord in zebrafish. Dev. Biol. 278, 415–427 (2005). 23. Sawada, A. et al. Fgf/MAPK signaling is a crucial positional cue in somite boundary formation. Development 128, 4873–4880 (2001). 24. Dubrulle, J., McGrew, M. J. & Pourquie´, O. FGF signaling controls somite boundary position and regulates segmentation clock control of spatiotemporal Hox gene activation. Cell 106, 219–232 (2001). 25. Draper, B. W., Morcos, P. A. & Kimmel, C. B. Inhibition of zebrafish fgf8 pre-mRNA splicing with morpholino oligos: a quantifiable method for gene knockdown. Genesis 30, 154–156 (2001). 26. Brand, M. et al. Mutations in zebrafish genes affecting the formation of the boundary between midbrain and hindbrain. Development 123, 179–190 (1996). 27. Reifers, F. et al. Fgf8 is mutated in zebrafish acerebellar (ace) mutants and is required for maintenance of midbrain–hindbrain boundary development and somitogenesis. Development 125, 2381–2395 (1998). 28. Hamade, A. et al.Retinoic acid activatesmyogenesis in vivo through Fgf8 signalling. Dev. Biol. 289, 127–140 (2006). 29. Napoli, J. L. Interactions of retinoid binding proteins and enzymes in retinoid metabolism. Biochim. Biophys. Acta 1440, 139–162 (1999). 30. Haugland, R. P. & Johnson, I. D. in Fluorescent and Luminescent Probes for Biological Activity (ed. Mason, W. T.) 40–50 (Academic, 1999). 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. (matsushi@brain.riken.jp). RESEARCH LETTER 4 | NATURE | VOL 000 | 00 MONTH 2013 ©2013 Macmillan Publishers Limited. All rights reserved
LETTERRESEARCH METHODS cellular RA-binding proteins, which were reported to be 10 HM and 0. 13-2 nM, Materials. RA, retinol, retinal and DEAB were purchased from Sigma-Aldrich. espectively, only a small fraction of RA molecules should be bound to GEPRA These compounds were dissolved in dimethylsulphoxide(DMsO)to prepa Transgenic RARE-eYFP zebrafish Transgenic zebrafish(RARE-gata2: NTD- stock solutions. The compounds were administered in a final concentration of YFP)ldi) were obtained from the Zebrafish International Resource Center 0. 1% DMSO University of Oregon). Fluorescence was observed with an FV1000 confocal Gene construction. cDNA encoding the LBD of RAR-B(FANTOM3 (ref. 31), microscope (Olympus)with a 488-nm laser or an FVIOi confocal microscope ig primers con Depletion of endogenous RA and application of exogenous RA. Embryos we product was ligated to a BamHI/SphI fragment encoding CFP incubated in E3 medium containing 0. 1% DMSO(vehicle), 10 HM DEAB, I nM and a Sacl/Xhol fragment encoding YFP. The ligated product was subcloned into RA with 10 HM DEAB, or 10nM RA with 10 HM DEAB from 4 to 24hpf After the BamHI/Xhol sites of the pCS2 vector for mammalian expression. In this 24hpf, embryos were bathed in E3 medium. RA imaging was performed at ll hpf the Ra indicator. Similarly, cDNA encoding RAR-Y(FANTOM3 (ref. 31). (roughly the three-somite stage). The imaged embryos were kept at 280C until their morphologies were examined at 36 hpf with a stereomicroscope(Mz16F F730319B19)was used to make a Sphl/Sac fragment encoding the LBD. Leica) equipped with a charge-coupled device(CCD)camera(DP50: Olympus) Together with the BamHI/Sphl fragment encoding CFP and a Sacl/EcoRI frag. In situhybridization. cDNA encoding raldh2, cyp26a1, cyp26b1, cyp26cl, krox20, ment encoding YFP, cDNA coding for the indicator was subcloned into the hoxd4, otx2 or myod was al ed from a cDNA library prepared from 10-hpf or BamHl/EcoRI sites of the pCS2 vector. Introduction of two amino-acid substitu- 24-hpf embryos and cloned into the pCS2 vector. Probes were labelled with a tions(R269A and S280A)in the LBD of RAR-B was performed as described digoxigenin or fluorescein labelling mix(Roche Diagnostics)and detected with alkaline phosphatase-conjugated antibodies(1: 5,000: Roche Diagnostics). The Characterization of the indicators. HeLa cells were grown in DMEM medium colorimetric reaction was performed with BM purple reagent(Roche Dia- upplemented with 10% FBS. CDNAs were transfected into cells by using gnostics). Images were captured with a stereomicroscope (MZ16 F: Leica) ipofectamine and Plus reagent rogen). Two days after transfection, the equipped with a CCD camera(DP50: Olympus) Fluorescence and in situ hybridi- medium was exchanged with DMEM/F12 without phenol red. Dose-response zation images were linearly registered with the Image Processing Toolbox in urves were determined with nine transfected cells MATLAB software(MathWorks). neration of GEPRA transgenic zebrafish lines. GEPRA CDNAs were sub- Knock down of fgt Splice- site-targeted morpholino oligonucleotides for the ff cloned into the PT2KXIGAin vector, and transgenic lines were created as gene(E212(5'-TAGGATGCTCTTACCATGAACGTCG-3")and E313(5'-CAC described previously. Fish were maintained at 280C. ATACCTTGCCAATCAGTTTCCC-3))were used to block its pre-mRNA splic- In vivo RA imaging. A glass bead (luchi BZ-1)was placed on a coverslip, and ing in zebrafish embryos". One-cell-stage embryos were injected with both E212 0.16mM MgSO)was poured on the coverslip and allowed to harden. The glass CCTCAGTTACAATTTATN-/ Control oligonucleotide(5ng:5'-CCTCTTA 1% agarose(Takara L03)in E3 medium(5mM Nacl, 0. 17 mM KCl,0.4 mM CaCl2, and E313(2.5 ng of each) or a bead was then removed to generate a round chamber. An embryo that had been naesthetized with Tricaine at more than 16hpf was placed in the chamber and 31. Camino al. The transcriptional landscape of the mammalian genome. covered with 0.3% agarose in E3 medium. The chamber was submerged in E3 32. Zhang, Z P et al Role of Ser289 in RARy and its homologous amino acid residue medium containing Tricaine. Time-lapse two-dimensional imaging was perform RARa and RARp in the binding of retinoic acid. Arch. Biochem. Biophys. 380, in the xy-t mode with an FV1000 (Olympus)confocal inverted microscope syster nm laser. with the aperture fully open, non-confocal fluorescence images were acquired. The ratio imaging of embryos was performed under exactly the same 34. Urasaki, A, Morvan, G& Kawakami, K Functional dissection of the Tol2 conditions as for calibration experiments with HeLa cells. Thus, absolute [RAl gow//).After the background had been subtracted, ratio images were generated and 35 174. 630 sequence in the subterminal region essential for transposition. Genesis values in the image can be obtained from the ratios using the curves shown in g.1b.ImageprocessingwasperformedwithImagesoftware(http://rsb.infonih Mech.Dev.58,27-38(199) Estimation of GEPRA concentration in embryos Confocal fluorescence images 36. Dong, D, Ruuska, S E, Levinthal, D.J. Noy, N Distinct roles for cellular retinoic uired with a GEPRA transgenic embryo and arious concentrations with excitation at 488 nm. The size of the confocal aperture 274,23695-23698(1999) was about 2 Airy disks. By comparing the intensities of the confocal fluorescence 37. Norris, A W, Cheng, L, Giguere, V- Rosenberger, M&Li,E. Measurement of nages, the GEPRA concentration in the embryo was estimated to be approxi inding proteins by fluorometric titration. Biochim. Biophys. Acta 1209, 10-18 mately 0.1 uM. Considering the concentrationand affinity for RA(refs 36, 37)of @2013 Macmillan Publishers Limited. All rights reserved
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 440- nm 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, ratioimageswere 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). 31. Carninci, P. et al. The transcriptional landscape of the mammalian genome. Science 309, 1559–1563 (2005). 32. Zhang, Z. P. et al. Role of Ser289 in RARc and its homologous amino acid residue of RARa and RARb in the binding of retinoic acid. Arch. Biochem. Biophys. 380, 339–346 (2000). 33. Sawano, A. & Miyawaki, A. Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis. Nucleic Acids Res. 15, e78 (2000). 34. Urasaki, A., Morvan, G. & Kawakami, K. Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition. Genesis 174, 639–649 (2006). 35. Gustafson, A. L., Donovan, M., Annerwall, E., Dencker, L. & Eriksson, U. Nuclear import of cellular retinoic acid-binding protein type I in mouse embryonic cells. Mech. Dev. 58, 27–38 (1996). 36. Dong, D., Ruuska, S. E., Levinthal, D. J. & Noy, N. Distinct roles for cellular retinoic acid-binding proteins I and II in regulating signaling by retinoic acid. J. Biol. Chem. 274, 23695–23698 (1999). 37. Norris, A. W., Cheng, L., Gigue`re, V., Rosenberger, M. & Li, E. Measurement of subnanomolar retinoic acid binding affinities for cellular retinoic acid binding proteins by fluorometric titration. Biochim. Biophys. Acta 1209, 10–18 (1994). LETTER RESEARCH ©2013 Macmillan Publishers Limited. All rights reserved
