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Enhancer of split-related-2 mRNA shows cyclic expression during somitogenesis in Xenopus laevis

Enhancer of split-related-2 mRNA shows cyclic expression during somitogenesis in Xenopus laevis Volume 2 † Number 1 † March 2009 10.1093/biohorizons/hzp006 ......................................................................................................................................................................................................................................... Research article Enhancer of split-related-2 mRNA shows cyclic expression during somitogenesis in Xenopus laevis Rachel Blewitt* Department of Biology, University of York, Heslington, York, YO10 5YW, UK. * Corresponding author: Molecular Haematology Unit, The Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK. Email: rachel.blewitt@imm.ox.ac.uk Supervisor: Dr Betsy Pownall, Department of Biology, University of York, Heslington, York YO105YW, UK. ........................................................................................................................................................................................................................................ Somitogenesis is responsible for production of the segmented body plan typical of vertebrate embryos. The somites are blocks of meso- derm, produced by this process, that give rise to the vertebrae and ribs, the dermis of the skin and all the skeletal muscle of the body. Many genes that regulate somitogenesis have been identified in chick and mouse, whereas considerably fewer are known in Xenopus laevis. The expression of Hairy/Enhancer of split-related genes is known to cycle during somitogenesis and provides a mechanism for the regular formation of somites. In this project, in situ hybridizations were carried out on bowline, Thylacine1, Enhancer of split-related-1 (ESR1), ESR2 and ESR-5 in order to study their expression in relation to somitogenesis. All genes were found to be expressed during somi- togenesis, even as early on as late gastrula stages in some cases. In addition, the expression of ESR2 is shown to be oscillating in the presegmented mesoderm of neurula and early-tailbud embryos. This study has identified ESR2 as the second known gene (after esr9) to show periodic oscillations of gene expression which can be considered as cycling during somitogenesis in X. laevis. Key words: somitogenesis, enhancer of split related, Xenopus laevis, cycling, somitomere. ........................................................................................................................................................................................................................................ to the dermis of the skin, the vertebrae and ribs and all the Introduction skeletal muscle of the body, respectively. Pairs of somites The segmented, or metameric, body plan characteristic of all condense from the anterior of the PSM at regular intervals vertebrates is apparent during embryonic development as in a strict anterior – posterior manner—a process known as blocks of mesodermal cells called somites. The generation somitogenesis. of these transient somites is a crucial step in development Three major phases of somitogenesis can be described: as they not only give rise to the segmented vertebrae of the first, the PSM is created as the paraxial mesoderm cells are adult, the dermis of the skin and all of the skeletal muscle produced and organized into two bars of mesenchymal of the body, but, moreover, somites also provide positional tissue during gastrulation, with one lying either side of the cues to the migrating neural crest cells. This means that the midline. This is followed by the establishment of a segmental somites are key players in establishing the overall metameric pattern and then finally the formation of somitic bound- body plan. aries. It is now widely accepted that segmental units called Lying bilaterally to the midline structures (the neural somitomeres exist in the PSM prior to the formation of tube and notochord), the somites originate from a region somites, and so the PSM can be thought of as two distinct in the posterior of the embryo known as the presegmented regions: the posterior tailbud domain and the more anterior mesoderm (PSM). Somites initially form as undetermined somitomeric domain, with the boundary between these two balls of epithelial cells initially exhibiting only antero- regions termed the determination front. posterior identity, and later, in response to signals from Cooke and Zeeman first proposed a model for the somi- surrounding tissues including the neural tube, notochord togenesis mechanism, named the clock and wavefront model. and dorsal ectoderm, the cells will differentiate into the The theory behind this model is that there is a molecular dermatome, scleratome and myotome which will give rise oscillator or ‘clock’ with gradients of signalling molecules ......................................................................................................................................................................................................................................... 2009 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 22 Bioscience Horizons † Volume 2 † Number 1 † March 2009 Research article ......................................................................................................................................................................................................................................... interacting to generate dynamic gene expression patterns new pair of somites. Many other cycling genes have now 4 15 16 within the PSM. More recently, Pourquie ´ explains that in been identified, including lunatic Fringe, Hes1 and 17 18 19 this model the PSM cells are thought to oscillate between a c-Hey2 in chick, Lunatic fringe and Hes7 in mice, 20, 21 permissive and a non-permissive state for somite formation, her1 in fish and enhancer of split-related-9 (esr9)in as controlled by a cell-autonomous temporal clock. The for- frogs. Hairy/enhancer of split (HES) bHLH transcription mation of a somite is thought to be triggered when permiss- factors are targets of Notch signalling, while Lunatic fringe ive anterior PSM cells are hit by a ‘wavefront’ of maturation, is a glycosyl transferase required for Notch signalling. which moves posteriorly along the axis of the embryo. These genes all exhibit similar behaviour to c-hairy1, indicat- The formation of a somite begins in PSM cells at the deter- ing the segmentation clock may be conserved among ver- mination front, where there is an intersection of the opposing tebrates. However, there is no obvious rule as to which fibroblast growth factor (FGF) posterior – anterior signal particular gene(s) in this family will cycle in each species, and the retinoic acid (RA) anterior – posterior signal gradi- for example, neither c-hairy1 or Lunatic fringe frog homo- ents. FGF8 is thought to produce a moving wavefront, logs cycle in the Xenopus PSM, indicating the conservation similar to that hypothesized by Cooke and Zeeman; the of cycling genes may not be as strong as previously overexpression of FGF8 can inhibit somitogenesis by thought. causing an anterior extension of posterior genes, indicating Although extensive research has been carried out on somi- that FGF8 may control the activation of the segmentation togenesis in chick and mouse, little is known about oscillat- programme by maintaining the posterior identity of PSM ing genes in Xenopus laevis. The only gene in X. laevis that cells. Similarly, RA is believed to regulate somite formation shows the dynamic cycling expression pattern associated as the altered expression of RA causes significant distortions with somitogenesis is esr9. To better understand the in the size and morphology of somites. These somite process of somitogenesis in frogs, I identified expressed changes are preceded by alterations in a basic helix – loop – sequence tags representing clones coding for ESR1, ESR2 helix (bHLH) transcription factor expression during segmen- and ESR-5 which were used to generate probes for in situ tation, which is thought to arise from the altered ability of hybridization. ESR-5 is a well characterized gene known to RA to inhibit the FGF signalling pathway at the determi- be expressed in the PSM and in the anterior half of the nation front. This is achieved by increasing the expression two most posterior somitomeres (S-II and S-III). ESR1 is a of MKP3, a protein known to dephosphorylate, and thus well-documented neural marker with expression also seen inactivate, ERK (a member of the MAPK pathway) in the tailbud, highlighting the possibility of its role in somi- which is essential for FGF signalling. Consequently, RA is togenesis. In contrast, there are no documented expression thought to play a key role in regulating gene expression pat- analyses of the ESR2 gene in X. laevis. In addition, the terns during segmentation. The Notch signalling pathway expression pattern of Thylacine1 and bowline mRNA were also has a crucial role in somitogenesis, through investigated, as they provide good molecular markers of Notch-dependent intercellular communication. This signal- somitomeres S-II and S-III. ling pathway is mediated through the Hairy/Enhancer of There are many different ways to order and number somi- split oscillator (the ‘clock’), and couples surrounding cells tomeres. In this report, somitomeres will be referred to in the 11 9 in order to produce an ordered, synchronized oscillation. same way Moreno and Kintner do, as S0 – S-III with S-III Mesp family genes, including Mesp2-like Thylacine1,are being the most posterior somitomere and S0 the most also bHLH transcription factors that appear to play a anterior. crucial role in the segmentation process in chick, mouse, fish and frog, as they are essential for the development of Methods the paraxial mesoderm. Acting upstream of the Notch cascade, they regulate somite boundary positioning at the Obtaining DNA determination front and establish the somite anterior – DNA sequences and clone numbers for required genes were posterior polarity. It is thought that RA signalling is able retrieved from the National Center for Biotechnology to regulate somitogenesis by controlling the Mesp family Information and clones ordered from the National Institute genes, evident as Mesp promoters are capable of responding for Basic Biology (NIBB), using both the clone number and to RA signalling. These cells at the determination front are DNA sequence. The plasmids were supplied, dried on then competent to respond to Notch pathway genes acting as paper and were extracted by soaking in water, vortexing segmentation cues and form somitomeres. and heating repeatedly, before storing at 2208C. Oscillatory waves of gene expression in the PSM was first observed in chick, where a gene coding for a bHLH tran- Escherichia coli Transformation scription factor c-hairy1, a chick homologue of the Drosophila pair-rule gene hairy, exhibited dynamic rhythmic Plasmids were introduced to Promega competent Escherichia waves of expression, repeating with the formation of each coli cells and shaken in Luria – Bertani (LB) broth before ......................................................................................................................................................................................................................................... 23 Research article Bioscience Horizons † Volume 2 † Number 1 † March 2009 ......................................................................................................................................................................................................................................... incubating on antibiotic plates and picking individual colo- 50 units of human chorionic gonadotropin (hCG), 48 – nies to select for transformed E. coli. These were then 72 h before the eggs are needed, then injecting a further shaken overnight in LB broth with ampicillin to allow 250 units of hCG in the night before the eggs are required. growth of the transformed E. coli ( protocol obtained from By gently squeezing the female with pressure on her http://www.promega.com/tbs/tb095/tb095.pdf ). A puri- abdomen and lower back, the eggs were expelled, and then fied plasmid solution was obtained using ‘QIAprep Spin the sperm solution was immediately pipetted over the eggs Miniprep’ according to the manufacturer’s protocol (which for fertilization. After 5 min, the embryos were flooded can be found online: http://www1.qiagen.com/HB/ with NAM/10 (10% NAM, 5% 0.1 HEPES) and left for QIAprepMiniprepKit_EN). The concentration of the final at least 40 min to allow for cortical rotation before a 2.5% plasmid solution was then determined using a NanoDrop cysteine solution ( pH 7.8) was used to de-jelly the eggs. spectrophotometer. These were then washed extensively with buffered water to remove all traces of cysteine, followed by one wash in Diagnostic DNA Digests NAM/10 before putting the embryos in trays lined with Diagnostic DNA digests using plasmid and gene sequences to 1% agarose and covering with NAM/10 for culturing. identify restriction enzyme (RE) sites were used to check the Fertilized embryos were recognized by their rigidity as the correct gene was in each plasmid, to determine the orien- vitelline membrane thickens, and any unfertilized eggs were tation of each gene in the plasmid and also the size of the discarded. insert. A linearizing RE was then identified for each. Fixing Making Probe Template Embryos were collected at the required stage after removing DNA template for the probe was generated by mixing 5 mg their vitelline membranes and were fixed in MEMFA (0.1 M DNA, 10% 10 buffer and 3% linearizing RE, and incubat- MOPS, 2 mM EDTA, 1 mM MgSO and 3.7% formal- ing at 378C for 90 min to digest the DNA before checking dehyde) for 60 min before being washed in 100% EtOH that all DNA has cut to completion by running on an and stored in fresh EtOH at 2208C. agarose gel. The digestion was then cleaned up using In situ Hybridization ‘QIAquick Gel Extraction kit’ according to manufacturer’s protocol, leaving a purified DNA template. Embryos were rehydrated in a graded series of EtOH washes with PBSAT (PBS, 0.1% Tween) and washed extensively in Making Antisense Probe PBSAT. They were permeabilized with Proteinase K treat- Antisense RNA probe was generated by mixing 20% tran- ment at a concentration of 10 mg/ml 20 – 35 min. After scription buffer, 5% 10 digoxigenin NTP mix (containing rinsing in 0.1% triethanolamine ( pH 7.8), acetic anhydride 10 mM of each ATP, CTP and GTP, 6.5 mM UTP and was added to make a 0.25% solution, and then again to 3.5 mM DIG-11-UTP), 10 mM dithiothreitol (DDT), 4% make a 0.5% solution. After refixing in 3.7% formal- RNasin, 6% appropriate polymerase and 50 mg/ml DNA dehyde/PBSAT, embryos were washed extensively in template, and incubating at 378C for 2 h to allow adequate PBSAT and then equilibrated in hybridization buffer (50% time for transcription, before checking the transcript on formamide, 5 SSC, 1 mg/ml total yeast RNA, 100 mg/ml agarose gel. DNA template was removed from the mixture heparin, 1 Denharts, 0.1% Tween, 0.1% Chaps and by adding RNase-free DNase before precipitating the probe 10 mM EDTA). After prehybridizing in fresh hybridization with 11% 5 M ammonium acetate and 72% ethanol buffer for 2 h at 608C, embryos were incubated overnight (EtOH) and leaving overnight at 2208C. The pellet was in probe solution ( probe plus hybridization buffer). spun down at 48C before washing in 70% EtOH, drying Embryos were rinsed twice in hybridization buffer at 608C and redissolving in dH O, to leave a purified antisense to remove any residual probe before a series of rinses were RNA probe solution. Probes were hydrolysed, if necessary performed, still at 608C, with 2 SSC (0.1% Tween) and by adding an RNase-free solution of 40 mM NaHCO / then 0.2% SSC (0.1% Tween) and then at room temperature 60 mM Na CO and leaving at 608C for 20 – 30 min. with maleic acid buffer (MAB: 100 mM MAB, 150 mM 2 3 NaCl, 0.1% Tween, pH 7.8). Following blocking in MAB, Fertilizations 2% Block (Blocking Reagent; Roche) and 20% heat-treated Mature male X. laevis were anaesthetized with 0.05% ben- lamb serum at room temperature for 2 h to prevent non- zocaine before removing the testes and storing them in specific antibody binding, embryos were rocked overnight normal amphibian media (NAM) (110 mM NaCl, 2 mM at 48C in MAB þ Block þ HT lamb serum þ 1/2000 . . KCl, 1 mM Ca(NO ) 4H O, 1 mM MgSO 7H O, dilution of sheep anti-digoxigenin antibody coupled to alka- 3 2 2 4 2 0.1 mM Na EDTA). One-fourth to one-eighth of a testicle line phosphatase. They were washed several times with MAB was macerated in a few drops of NAM to produce a sperm to remove any excess antibody and rinsed twice in alkaline solution. Ovulation was induced in females by injecting phosphatase buffer (100 mM Trizma, 50 mM MgCl , ......................................................................................................................................................................................................................................... 24 Bioscience Horizons † Volume 2 † Number 1 † March 2009 Research article ......................................................................................................................................................................................................................................... 100 mM NaCl, 0.1% Tween, pH9.5) in order to equilibrate Thylacine1 Expression Is Seen Exclusively in S-II and S-III embryos before application of the substrate. Embryos were In situ hybridization using the Thylacine1 antisense probe incubated in BM purple for as long as necessary to detect reveals the specific expression of this gene in the developing the antibody (between a few hours and a few days), as BM somitomeres. At stage 12, two very faint stripes can be seen purple substrate turns blue and precipitates in the presence above the blastopore, marking the early segmentation in the of the antibody. After the colour developed, embryos were gastrula embryo (arrowheads, Fig. 1A). By stage 16, washed twice in PBSAT to remove any residual BM purple Thylacine1 expression is much stronger and more apparent, and fixed overnight in 3.7% formaldehyde/PBSAT. marking three somitomeres in this case (Fig. 1B). The Embryos were later bleached in 5% H O /PBSAT rolling 2 2 marking of more than two somitomeres is not an unexpected under bright light until pigment was gone and then washed result as Sparrow et al. note that between one and three several times in PBSAT to remove all traces of H O before 2 2 stripes are usually observed, with two being the most fre- fixing again in 3.7% formaldehyde/PBSAT. quent. At stages 26 and 31 (Fig. 1C and D, respectively), two distinct lines can be seen in each, showing expression Embryo Photography of Thylacine1 in S-II and S-III. The more posterior of the two somitomeres seen at stage 31 shows an extension of Photographs of embryos were taken in PBSA on 1% agarose- Thylacine1 expression ventrally on the tailbud (marked by lined trays using Spot camera with a Leica MZFLIII stereo- a black asterisk on Fig. 1D) which has not previously been microscope, and Spot Advanced computer software. reported. Pictures were cropped and light levels adjusted using Adobe Photoshop. Expression Analysis of bowline in X. laevis Reveals a Novel Expression Pattern in the Somites and Somitomeres Results In a stage 12 embryo, in situ hybridization using an antisense Isolation of cDNAs probe for bowline reveals a faint circumblastoporal cDNAs coding for ESR1, ESR2 and ESR-5 were provided by expression pattern (Fig. 2A). This expression in a gastrula M.E. Pownall and are being used in other current studies stage embryo has not been described in any literature; in (unpublished data), while Thylacine1 and bowline cDNAs fact, the earliest noted expression pattern is the presence of were obtained from NIBB using their unique clone bilateral stripes in a stage 13 embryo. The fact that this numbers (Table 1). Information about the RE sites of each expression is circumblastoporal and does not show any gene and plasmid details were collected using their sequences specific stripe pattern as observed by Kondow et al. in ( plasmid sequences obtained from the I.M.A.G.E consor- stage 13 embryos raises the question of whether this is a tium; http://image.hudsonalpha.org/html/vectors.shtml) true representation of bowline mRNA expression in a gas- and used to determine both the orientation of the gene and trula embryo, or is background colour that has developed the best linearizing RE to generate a probe template due to the absence of true mRNA expression; further work (Table 1). From this information, diagnostic digests were is needed to decipher this. By stage 16, however, bilateral carried out and subsequently, linearized probe templates stripes can be clearly seen marking S-II and S-III (shown by were generated. Antisense RNA probes were generated the arrowheads in Fig. 2B). Stage 26 embryos (Fig. 2C and D) from each of these templates for in situ hybridization. and stage 31 embryos (Fig. 2E) both have quite novel Table 1. Data collected regarding each gene studied Gene ESR1 ESR2 ESR-5 Thylacine1 bowline ........................................................................................................................................................................................................................................ NIBB/clone number IMAGE: 5537441 IMAGE: 6955664 Xl207h08 XL204i05 Xl034g14 Unigene number Xl.8440 Xl.12067 Xl.14524 Xl.54 Xl.16077 Plasmid pCMV. SPORT 6 pCMV. SPORT 6.1 pBluescript sk- pBluescript sk- pBluescript sk- 5 clone site SalI EcoRV EcoRI EcoRI EcoRI 3 clone site NotI NotI XhoI XhoI XhoI RE for linearizing EcoRI XbaI EcoRI BgIII SacI Polymerase T7 T7 T7 T7 T7 Gene insert size (kb) 1.6 1.1 0.6 1.7 1.8 Transcript size (bp) 600 1100 (hydrolysed to 300) 600 800 1000 ......................................................................................................................................................................................................................................... 25 Research article Bioscience Horizons † Volume 2 † Number 1 † March 2009 ......................................................................................................................................................................................................................................... Figure 1. In situ hybridization showing the expression of Thylacine1 in X. laevis. (A) Stage 12 vegetal view (dorsal side up), (B) Stage 16 dorsal view, (C) Stage 26 lateral view and (D) Stage 31 lateral view. In (B–D), anterior is to the left. Black arrowheads point to somitomeres expressing Thylacine1 mRNA and the asterisk marks the ventral extension of Thylacine1 expression in S-III. expression patterns, significantly different to that described in the literature for similar stages; others describe seeing one to two stripes of expression in the posterior of the 26, 27 embryo, correlating to S-II and S-III, and nothing else. However, this is not reflected in the in situ hybridization Figure 2. In situ hybridization showing the expression of bowline in X. laevis. (A) Stage 12 vegetal view, (B) Stage 16 dorsal view, (C) Stage studies I carried out for bowline. Not only is their consider- 26 lateral view, (D) Stage 26 dorsal view and (E) Stage 30 lateral view. In ably strong expression in the stated somitomeres, but fairly (B–E), anterior is to the left. Arrowheads mark bowline expression in strong expression can also be seen in the somites. A gap of somitomeres. approximately three to four somitomeres/somites can be seen between the most anterior somitomere marked, and possible to see colour developing in the somites and the the most posterior somite marked by bowline expression. somitomeres at the same time, thus it was not background The reason for this gap in expression is not known as occurring due to prolonged exposure to the BM purple bowline expression in the somites has not previously been substrate. documented. This observed difference in bowline expression is surprising—the cDNA used to create the antisense probe ESR-5 Transcripts Are Found in the PSM and in One to was sequenced and found to be the same as that used by Three Somitomeres in X. laevis others. This study was repeated three times with the same result each time, suggesting that it was not an exper- Carrying out in situ hybridizations with an ESR-5 antisense imental procedure or similar causing this unexpected result. probe reveals that this gene is very strongly expressed in the In addition, while watching the colour development it was PSM and in the anterior half of S-II and S-III (Fig. 3). During ......................................................................................................................................................................................................................................... 26 Bioscience Horizons † Volume 2 † Number 1 † March 2009 Research article ......................................................................................................................................................................................................................................... gastrulation, ESR-5 expression is seen surrounding the blas- and further studies would be needed to decipher this exten- topore but is excluded from the dorsal lip (Fig. 3A). In sion of expression. addition to this, two stripes of expression can be seen above the blastopore corresponding to the newly formed somitomeres. Similarly, at stages 16, 26 and 31, strong Expression Analysis of ESR1 in X. laevis Highlights Its expression can be seen in the PSM and in one to three somi- Strong Neural Expression and Some Tailbud Expression tomeres (Fig. 3B – D, respectively). The dark colouring seen In situ hybridization using the antisense probe for ESR1 in the head is a common background noise, caused as cavities revealed a predominantly neural expression pattern. This in the head trap the antisense probe and this should not be neural expression is of no great significance to this study as considered as ESR-5 expression. The ventral extension it is not relevant to somitogenesis; however, there is some of expression in S-III can be seen here (Black asterisk, expression seen in the tailbud region of later stages. At Fig. 3D) similar to that seen in Thylacine1 expression stage 12, expression can be seen around the blastopore in (Fig. 1D). Here, the stripe appears parallel to the PSM high- the ventral region of the embryo, and also bilaterally lighting the possibility that this extension of mRNA (Fig. 4A); no expression is seen in the dorsal part of the expression could occur in younger somitomeres which have embryo. By stage 16, a clear neural expression can be seen, not yet restricted gene expression laterally, as older somito- with stripes of primary neuronal precursors evident on meres may have done. This is only one possibility though, either side of the neural plate and a more anterior neural structure shown (Fig. 4B). In addition to these neural struc- tures, strong expression can be seen in the posterior of the stage 16 embryos, apparently in the tailbud domain. By stages 26 and 31 (Fig. 4C and D, respectively), expression can be seen in many anterior neural structures, including the eye and various parts of the brain. It is also found along the entire length of the neural tube, as verified by trans- verse sections taken for both stages (Fig. 4E and F, respect- ively). Black arrowheads on Fig. 4C and D, point to ESR1 expression in the tailbud domain of stages 26 and 31 embryos, respectively. The gene expression patterns of all similar stage embryos were compared to look for any differ- ences that may indicate the gene was cycling. However, no significant differences were observed at any stage, indicating that ESR1 mRNA does not cycle during somitogenesis. ESR2 mRNA Is Seen in Many Neural Structures in Addition to the Tailbud Region in X. laevis At stage 12, expression can be seen in the mesoderm of the ventral half of the embryo, surrounding the blastopore (Fig. 5A). By stage 16, ESR2 expression can be seen in the posterior paraxial mesoderm of the embryo and also marking some primary neural precursors either side of the neural tube (Fig. 5B). Expression in a stage 26 embryo is pre- dominantly in the tailbud region, but can also be seen in some of the more anterior neural structures including the eye (Fig. 5C). At stage 31, ESR2 expression is seen in many anterior neural structures, including the eye and brain (Fig. 5D). In addition to this, expression can be seen in the neural tube, notochord and to a lesser extent in the Figure 3. In situ hybridization showing the expression of ESR-5 in somites. This was verified by studying a transverse section X. laevis. (A) Stage 12 vegetal view (dorsal side up), (B) Stage 16 lateral of a stage 31 embryo, which clearly shows ESR2 expression view, (C) Stage 26 lateral view and (D) Stage 31 lateral view. In (B–D), in these internal structures (Fig. 5E). Tailbud expression is anterior is to the left. Black arrowheads point to somitomeres expressing also apparent at this stage, as marked by the arrowhead in ESR-5 mRNA and the asterisk marks the ventral extension of ESR-5 expression in S-III. Fig. 5D. ......................................................................................................................................................................................................................................... 27 Research article Bioscience Horizons † Volume 2 † Number 1 † March 2009 ......................................................................................................................................................................................................................................... Figure 4. In situ hybridization showing the expression of ESR1 in X. laevis. (A) Stage 12 vegetal view (dorsal side up), (B) Stage 16 dorsal view, (C) Stage 26 lateral view and (D) Stage 31 lateral view. Transverse sections Figure 5. In situ hybridization showing the expression of ESR2 in X. laevis. can be seen for stage 26 (E) and stage 31 (F) embryos showing expression (A) Stage 12 vegetal view (dorsal side up), (B) Stage 16 dorsal view, in the neural tube. In (B–D), anterior is to the left. (C) Stage 26 lateral view and (D) Stage 31 lateral view. In addition, a trans- verse section for stage 31 can be seen (E) highlighting expression in the neural tube, the notochord and bilaterally in the somites. In (B–D), anterior is to the left. The black arrowhead in D points to tailbud expression. Variable Expression Patterns in Neurula and Early Tailbud Stage Embryos Highlights the Potential Cycling Nature of ESR2 variable patterns of gene expression, making this an exciting prospect for an oscillating gene. A considerable number of embryos were studied after in situ Figure 6 shows a series of stage 16 embryos aligned to hybridization to look for any sign of dynamic expression demonstrate a progressive mRNA expression pattern, similar which may indicate that ESR2 mRNA is cycling. There did to that seen for esr9. Figure 6A – C shows the first stage of not appear to be any differences in gene expression patterns the progression, with ESR2 expression in the PSM of these for stage 12 or 31 embryos, however, both stage 16 (Fig. 6) embryos, but not in any somitomeres. Figure 6A and B and stage 26 embryos (not shown) showed considerably ......................................................................................................................................................................................................................................... 28 Bioscience Horizons † Volume 2 † Number 1 † March 2009 Research article ......................................................................................................................................................................................................................................... Figure 6. In situ hybridization showing the dorsal view of stage 16 embryos with different ESR2 gene expression patterns. (A–C) ESR2 expression in the PSM, but no fully or partially separated somitomeres can be seen. (D–F) Expression in the PSM, and red arrowheads here point to partially separated somitomeres. (G–H) Expression in the PSM and in one fully separated somitomere (black arrowheads). (I–J) Expression in one fully separated somitomere (black arrowheads), and in the PSM with one somitomere partially separated (red arrowheads). (K–N) Expression in the PSM and in two fully separated somitomeres each (black arrowheads). In all pictures, anterior is to the left. shows considerably weaker expression in the PSM than Discussion Fig. 6C, but similar neural expression, so this may reflect an earlier stage of this expression cycle. Figure 6D – F each Expression of Thylacine1 and ESR-5 Can Be Seen in shows expression in the PSM and in one or two Somitomeres during Gastrulation somitomeres of each embryo, as marked by red arrowheads. One pair of somitomeres can be seen during gastrulation to The expression in these somitomeres appears to be express both Thylacine1 and ESR-5, as shown by black arrow- continuous with the PSM at the midline, while others (black heads in Figs 1A and 3A, respectively. There are no published arrows) do not. This may represent different stages of somito- reports of gene expression in segmented mesoderm being mere formation. In the next stage of this series (Fig. 6G and observed prior to neurula stages and so Thylacine1 and H), somitomeres exhibiting mRNA expression separately to ESR-5 expression in the late gastrula may be marking the the expression in the PSM can be seen on only one side of initial segmentation of mesoderm in the embryo. the embryo (as shown by black arrowheads), while Fig. 6I Interestingly, bowline expression is not restricted to somito- and J shows a combination of continuous (red arrowheads) meres at this same stage (Fig. 2A), even though bowline and separate (black arrowheads) expression in somitomeres mRNA is known to be expressed in the same somitomeres with relation to the PSM. Figure 6K – N shows the next as ESR-5 and Thylacine1. The reason for not seeing any stage in the process, with one somitomere either side of somitomere stripes at this stage may be as simple as there the midline displaying ESR2 mRNA expression separate to being widespread bowline expression circumblastoporally. that in the posterior PSM (shown by black arrowheads). Although this may be the case as there appears to be weak ......................................................................................................................................................................................................................................... 29 Research article Bioscience Horizons † Volume 2 † Number 1 † March 2009 ......................................................................................................................................................................................................................................... circumblastoporal expression (Fig. 2A), the colour seen is discriminate between the mesodermal and ectodermal faint and without defined margins, so was initially considered tissues (Fig. 5A). This expression in both the mesoderm to be background colour. One consideration to make is that and ectoderm makes the expression pattern of ESR2 some- 24 22 the bowline antisense probe used for this in situ hybridization what similar to that of both ESR1 (Fig. 4) and esr9. appeared less defined in comparison to other probes when ran The Dynamic Expression Pattern of ESR2 at Stages 16 on an agarose gel. This may cause the unexpected expression and 26 Correlates to that of a Known Cycling Gene, esr9 pattern by either creating the suspected circumblastoporal background noise or failing to detect any small amounts of The dynamic expression pattern of ESR2 seen in stage 16 bowline mRNA in the segmented mesoderm. Further investi- embryos (Fig. 6) and stage 26 embryos (not shown) gation is needed to clarify the expression of bowline mRNA at appears to show cyclic oscillations, similar to the way esr9 gastrula stage, and if there truly is no somitomere expression mRNA cycles —an exciting discovery as this makes ESR2 found, to determine why this is. the second potential oscillating gene in X. laevis. The cycle begins with expression restricted to the PSM, before the A Novel Expression Pattern Is Seen for bowline Which mesoderm forms somitomeres (Fig. 6A – C). These somito- May Be Linked to a Negative Feedback Loop of Gene meres initially show striped ESR2 expression continuous at Expression the midline with expression in the posterior PSM In previous experiments, bowline expression has only been seen (Fig. 6D – F). This expression then appears to separate at 26, 27 in the two most posterior somitomeres, S-II and S-III. the midline from that in the PSM, and as the somitomeres Expression in these somitomeres is apparent in my studies, mature they will eventually stop expressing ESR2 altogether but in addition to this, expression is also seen in the somites (Fig. 6G – M). This cycle will then start again with expression (Fig. 2). Interestingly, there is a gap the size of approximately restricted to the PSM (Fig. 6N). three somitomeres/somites between the most anterior somito- Many embryos were observed to be asymmetrical with mere and the most posterior somite expressing bowline regards to their expression of ESR2, including both stage mRNA. As bowline expression has not been documented in 16 (Fig. 6) and stage 26 embryos (not shown), but this asym- the somites before, the reason for this gap is also unknown. metry never seemed to exceed more than one somitomere in Kondow et al. have found that although the bowline either stage. This highlights the autonomy of PSM cells either mRNA is located in S-II and S-III, the functional protein is side of the midline, and indicates that oscillations between found in S-I and S0. This late translation of bowline mRNA either side of the midline may be regulated independently. may account for the gap seen between the somites and the Asymmetry in gene expression has also been observed in 22 29 somitomeres. They go on to explain that bowline expression esr9 and in hairy2a in X. laevis while mouse and chick is activated by Tbx6 and once the Bowline protein reaches a expression is symmetrical. threshold concentration Tbx6 is converted from a transcrip- tional activator to a transcriptional repressor by associating Conclusion and Further Studies with X-Grg4 (a corepressor member of the Groucho family) via Bowline. This new transcriptional repressor turns off This study has identified ESR2 as the second potential gene bowline mRNA expression and may be the mechanism in X. laevis to show periodic oscillations of gene expression responsible for repressing bowline expression in the older during somitogenesis. However, other experiments are and more anterior somitomeres, S-I and S0. required to show definitively whether or not ESR2 is cycling in the Xenopus PSM. Explanting the PSM of the Previously Unstudied ESR2 Shows Expression in Both embryo either side of the midline and fixing at varying Mesoderm and Ectoderm time points would reveal whether or not ESR2 mRNA oscil- Expression analysis of ESR2 has revealed expression in both lates autonomously, and thus whether or not ESR2 is a true mesodermal and ectodermal tissues. Neuronal expression is cyclic gene. Experiments similar or the same as this are very obvious in stages 16, 26 and 31 (Fig. 5B – D). Strong routinely carried out in other studies to identify cyclic 14, 16, 17, 19 expression in the neural tube, the brain and in the eye at genes. In addition, periodic oscillations of both stage 31 highlights a strong presence in ectodermal deriva- Hes1 in chick and ESR9 in X. laevis have been shown by tives, while expression in the notochord, somites and PSM application of cyclohexamine to be independent of protein 14, 22 demonstrate a strong mesodermal presence as well (Fig. 5D synthesis, and so replication of these experiments to and E). At stage 26, expression appears to be more restricted look at the effect of blocking protein synthesis on ESR2 to the mesoderm, with strong expression in the PSM and in a mRNA expression may also help elucidate whether or not few somites while neural expression falls mainly in the eye this gene is truly cycling. An in depth study into the structure (Fig. 5C). Primary neuronal precursors can be seen to of the ESR2 gene and its regulatory elements would be ben- express ESR2 at stage 16, as can the PSM (Fig. 5B), eficial to this research also by revealing activators and/or whereas circumblastoporal expression at stage 12 does not repressors of ESR2 expression, hopefully confirming its ......................................................................................................................................................................................................................................... 30 Bioscience Horizons † Volume 2 † Number 1 † March 2009 Research article ......................................................................................................................................................................................................................................... 11. Horikawa K, Ishimatsu K, Yoshimoto E et al. (2006) Noise-resistant and syn- role in the Notch pathway and possibly linking its expression chronized oscillation of the segmentation clock. Nature 441: 719–723. to that of genes known to be involved in the segmentation of 12. Wang J, Ding X. (2006) Cloning and analyzing of Xenopus Mespo promoter somitomeres, such as bowline and Thylacine1. In addition, in retinoic acid regulated Mespo expression. Acta Biochim Biophys Sin 38: the use of an antibody to detect protein localization for 759–764. ESR2 would be interesting in order to see if there is 13. Takahashi Y, Koizumi K, Takagi A et al. (2000) Mesp2 initiates somite seg- dynamic localization of the protein as well as the dynamic mentation through the Notch signalling pathway. Nat Genet 25: 390–396. mRNA expression. Unfortunately, these further studies 14. Palmeirim I, Henrique D, Ish-Horowicz D et al. (1997) Avian hairy gene were not carried out due to time constraints but would be expression identifies a molecular clock linked to vertebrate segmentation and somitogenesis. Cell 91: 639–648. a good starting point to further understand the process of 15. McGrew MJ, Dale JK, Fraboulet S et al. (1998) The lunatic fringe gene is a somitogenesis in X. laevis. target of the molecular clock linked to somite segmentation in avian embryos. Curr Biol 8: 979–982. Acknowledgements 16. Jouve C, Palmeirim I, Henrique D et al. (2000) Notch signalling is required for cyclic expression of the hairy-like gene HES1 in the presomitic mesoderm. I would like to thank my project director Dr Betsy Pownall Development 127: 1421–1429. for all her help, support and guidance throughout this 17. Leimeister C, Dale K, Fischer A et al. (2000) Oscillating expression of c-Hey2 project. I would also like to thank Dr Harv Isaacs, in the presomitic mesoderm suggests that the segmentation clock may use combinatorial signaling through multiple interacting bHLH factors. Dev Biol Dr Wendy Moore, Dr Laura Faas, Richard Maguire, Emily 227: 91–103. Guiral, Emily Winterbottom, Simon Ramsbottom, Julie 18. Forsberg H, Crozet F, Brown NA (1998) Waves of mouse Lunatic fringe Affleck and Sara Steane for their advice and support. expression, in four-hour cycles at two-hour intervals, precede somite bound- ary formation. Curr Biol 8: 1027–1030. 19. Bessho Y, Sakata R, Komatsu S et al. (2001) Dynamic expression and essential Funding functions of Hes7 in somite segmentation. Genes Dev 15: 2642–2647. Funding for this study was provided by the Biology 20. Holley SA, Geisler R, Nu¨ sslein-Volhard C (2000) Control of her1 expression Department, University of York. during zebrafish somitogenesis by a delta-dependent oscillator and an inde- pendent wave-front activity. Genes Dev 14: 1678–1690. 21. Sawada A, Fritz A, Jiang YJ et al. (2000) Zebrafish Mesp family genes, mesp-a References and mesp-b are segmentally expressed in the presomitic mesoderm, and Mesp-b confers the anterior identity to the developing somites. 1. Aoyama H, Asamoto K (1988) Determination of somite cells: independence Development 127: 1691–1702. of cell differentiation and morphogenesis. Development 104: 15–28. 22. Li Y, Fenger U, Niehrs C et al. (2003) Cyclic expression of esr9 gene in 2. Brand-Saberi B, Wilting J, Ebensperger C et al. (1996) The formation of Xenopus presomitic mesoderm. Differentiation 71: 83–89. somite compartments in the avian embryo. Int J Dev Biol 40: 411–420. 23. Wu JY, Wen L, Zhang WJ et al. (1996) The secreted product of Xenopus gene 3. Cinquin O (1995) Understanding the somitogenesis clock: what’s missing? lunatic Fringe, a vertebrate signaling molecule. Science 273: 355–358. Mech Dev 124: 501–517. 24. Lamar E, Kintner C (2005) The Notch targets Esr1 and Esr10 are differentially 4. Pourquie O (2003) Vertebrate somitogenesis: a novel paradigm for animal regulated in Xenopus neural precursors. Development 132: 3619–3630. segmentation? Int J Dev Biol 47: 597–603. 25. Sparrow DB, Jen WC, Kotecha S et al. (1998) Thylacine1 is expressed seg- 5. Meier S, Jacobson AG (1982) Experimental studies of the origin and mentally within the paraxial mesoderm of the Xenopus embryo and interacts expression of metameric pattern in the chick embryo. J Exp Zool 219: with the Notch pathway. Development 125: 2041–2051. 217–232. 26. Kondow A, Hitachi K, Ikegame T et al. (2006) Bowline, a novel protein loca- 6. Dubrulle J, McGrew MJ, Pourquie O (2001) FGF signaling controls somite lized to the presomitic mesoderm, interacts with Groucho/TLE in Xenopus. boundary position and regulates segmentation clock control of spatiotem- Int J Dev Biol 50: 473–479. poral Hox gene activation. Cell 106: 219–232. 27. Hitachi K, Kondow A, Danno H et al. (2008) Tbx6, Thylacine1, and E47 syner- 7. Cooke J, Zeeman EC (1976) A clock and wavefront model for control of the gistically activate bowline expression in Xenopus somitogenesis. Dev Biol number of repeated structures during animal morphogenesis. J Theor Biol 313: 816–828. 58: 455–476. 28. Kondow A, Hitachi K, Okabayashi K et al. (2007) Bowline mediates associ- 8. Dubrulle J, Pourquie O (2004) Coupling segmentation to axis formation. ation of the transcriptional corepressor XGrg-4 with Tbx6 during somitogen- Development 131: 5783–5793. esis in Xenopus. Biochem Biophys Res Commun 359: 959–964. 9. Moreno TA, Kintner C (2004) Regulation of segmental patterning by retinoic 29. Davis RL, Turner DL, Evans LM et al. (2001) Molecular targets of vertebrate acid signaling during Xenopus somitogenesis. Dev Cell 6: 205–218. segmentation: two mechanisms control segmental expression of Xenopus 10. Vermot J, Pourquie O (2005) Retinoic acid coordinates somitogenesis and hairy2 during somite formation. Dev Cell 1: 553–565. left-right patterning in vertebrate embryos. Nature 435: 215–220. ........................................................................................................................................................................................................................................ Submitted on 30 September 2008; accepted on 23 January 2009; advance access publication 17 February 2009 ......................................................................................................................................................................................................................................... http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioscience Horizons Oxford University Press

Enhancer of split-related-2 mRNA shows cyclic expression during somitogenesis in Xenopus laevis

Blewitt, Rachel
Bioscience Horizons , Volume 2 (1) – Mar 17, 2009
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Volume 2 † Number 1 † March 2009 10.1093/biohorizons/hzp006 ......................................................................................................................................................................................................................................... Research article Enhancer of split-related-2 mRNA shows cyclic expression during somitogenesis in Xenopus laevis Rachel Blewitt* Department of Biology, University of York, Heslington, York, YO10 5YW, UK. * Corresponding author: Molecular Haematology Unit, The Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK. Email: rachel.blewitt@imm.ox.ac.uk Supervisor: Dr Betsy Pownall, Department of Biology, University of York, Heslington, York YO105YW, UK. ........................................................................................................................................................................................................................................ Somitogenesis is responsible for production of the segmented body plan typical of vertebrate embryos. The somites are blocks of meso- derm, produced by this process, that give rise to the vertebrae and ribs, the dermis of the skin and all the skeletal muscle of the body. Many genes that regulate somitogenesis have been identified in chick and mouse, whereas considerably fewer are known in Xenopus laevis. The expression of Hairy/Enhancer of split-related genes is known to cycle during somitogenesis and provides a mechanism for the regular formation of somites. In this project, in situ hybridizations were carried out on bowline, Thylacine1, Enhancer of split-related-1 (ESR1), ESR2 and ESR-5 in order to study their expression in relation to somitogenesis. All genes were found to be expressed during somi- togenesis, even as early on as late gastrula stages in some cases. In addition, the expression of ESR2 is shown to be oscillating in the presegmented mesoderm of neurula and early-tailbud embryos. This study has identified ESR2 as the second known gene (after esr9) to show periodic oscillations of gene expression which can be considered as cycling during somitogenesis in X. laevis. Key words: somitogenesis, enhancer of split related, Xenopus laevis, cycling, somitomere. ........................................................................................................................................................................................................................................ to the dermis of the skin, the vertebrae and ribs and all the Introduction skeletal muscle of the body, respectively. Pairs of somites The segmented, or metameric, body plan characteristic of all condense from the anterior of the PSM at regular intervals vertebrates is apparent during embryonic development as in a strict anterior – posterior manner—a process known as blocks of mesodermal cells called somites. The generation somitogenesis. of these transient somites is a crucial step in development Three major phases of somitogenesis can be described: as they not only give rise to the segmented vertebrae of the first, the PSM is created as the paraxial mesoderm cells are adult, the dermis of the skin and all of the skeletal muscle produced and organized into two bars of mesenchymal of the body, but, moreover, somites also provide positional tissue during gastrulation, with one lying either side of the cues to the migrating neural crest cells. This means that the midline. This is followed by the establishment of a segmental somites are key players in establishing the overall metameric pattern and then finally the formation of somitic bound- body plan. aries. It is now widely accepted that segmental units called Lying bilaterally to the midline structures (the neural somitomeres exist in the PSM prior to the formation of tube and notochord), the somites originate from a region somites, and so the PSM can be thought of as two distinct in the posterior of the embryo known as the presegmented regions: the posterior tailbud domain and the more anterior mesoderm (PSM). Somites initially form as undetermined somitomeric domain, with the boundary between these two balls of epithelial cells initially exhibiting only antero- regions termed the determination front. posterior identity, and later, in response to signals from Cooke and Zeeman first proposed a model for the somi- surrounding tissues including the neural tube, notochord togenesis mechanism, named the clock and wavefront model. and dorsal ectoderm, the cells will differentiate into the The theory behind this model is that there is a molecular dermatome, scleratome and myotome which will give rise oscillator or ‘clock’ with gradients of signalling molecules ......................................................................................................................................................................................................................................... 2009 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 22 Bioscience Horizons † Volume 2 † Number 1 † March 2009 Research article ......................................................................................................................................................................................................................................... interacting to generate dynamic gene expression patterns new pair of somites. Many other cycling genes have now 4 15 16 within the PSM. More recently, Pourquie ´ explains that in been identified, including lunatic Fringe, Hes1 and 17 18 19 this model the PSM cells are thought to oscillate between a c-Hey2 in chick, Lunatic fringe and Hes7 in mice, 20, 21 permissive and a non-permissive state for somite formation, her1 in fish and enhancer of split-related-9 (esr9)in as controlled by a cell-autonomous temporal clock. The for- frogs. Hairy/enhancer of split (HES) bHLH transcription mation of a somite is thought to be triggered when permiss- factors are targets of Notch signalling, while Lunatic fringe ive anterior PSM cells are hit by a ‘wavefront’ of maturation, is a glycosyl transferase required for Notch signalling. which moves posteriorly along the axis of the embryo. These genes all exhibit similar behaviour to c-hairy1, indicat- The formation of a somite begins in PSM cells at the deter- ing the segmentation clock may be conserved among ver- mination front, where there is an intersection of the opposing tebrates. However, there is no obvious rule as to which fibroblast growth factor (FGF) posterior – anterior signal particular gene(s) in this family will cycle in each species, and the retinoic acid (RA) anterior – posterior signal gradi- for example, neither c-hairy1 or Lunatic fringe frog homo- ents. FGF8 is thought to produce a moving wavefront, logs cycle in the Xenopus PSM, indicating the conservation similar to that hypothesized by Cooke and Zeeman; the of cycling genes may not be as strong as previously overexpression of FGF8 can inhibit somitogenesis by thought. causing an anterior extension of posterior genes, indicating Although extensive research has been carried out on somi- that FGF8 may control the activation of the segmentation togenesis in chick and mouse, little is known about oscillat- programme by maintaining the posterior identity of PSM ing genes in Xenopus laevis. The only gene in X. laevis that cells. Similarly, RA is believed to regulate somite formation shows the dynamic cycling expression pattern associated as the altered expression of RA causes significant distortions with somitogenesis is esr9. To better understand the in the size and morphology of somites. These somite process of somitogenesis in frogs, I identified expressed changes are preceded by alterations in a basic helix – loop – sequence tags representing clones coding for ESR1, ESR2 helix (bHLH) transcription factor expression during segmen- and ESR-5 which were used to generate probes for in situ tation, which is thought to arise from the altered ability of hybridization. ESR-5 is a well characterized gene known to RA to inhibit the FGF signalling pathway at the determi- be expressed in the PSM and in the anterior half of the nation front. This is achieved by increasing the expression two most posterior somitomeres (S-II and S-III). ESR1 is a of MKP3, a protein known to dephosphorylate, and thus well-documented neural marker with expression also seen inactivate, ERK (a member of the MAPK pathway) in the tailbud, highlighting the possibility of its role in somi- which is essential for FGF signalling. Consequently, RA is togenesis. In contrast, there are no documented expression thought to play a key role in regulating gene expression pat- analyses of the ESR2 gene in X. laevis. In addition, the terns during segmentation. The Notch signalling pathway expression pattern of Thylacine1 and bowline mRNA were also has a crucial role in somitogenesis, through investigated, as they provide good molecular markers of Notch-dependent intercellular communication. This signal- somitomeres S-II and S-III. ling pathway is mediated through the Hairy/Enhancer of There are many different ways to order and number somi- split oscillator (the ‘clock’), and couples surrounding cells tomeres. In this report, somitomeres will be referred to in the 11 9 in order to produce an ordered, synchronized oscillation. same way Moreno and Kintner do, as S0 – S-III with S-III Mesp family genes, including Mesp2-like Thylacine1,are being the most posterior somitomere and S0 the most also bHLH transcription factors that appear to play a anterior. crucial role in the segmentation process in chick, mouse, fish and frog, as they are essential for the development of Methods the paraxial mesoderm. Acting upstream of the Notch cascade, they regulate somite boundary positioning at the Obtaining DNA determination front and establish the somite anterior – DNA sequences and clone numbers for required genes were posterior polarity. It is thought that RA signalling is able retrieved from the National Center for Biotechnology to regulate somitogenesis by controlling the Mesp family Information and clones ordered from the National Institute genes, evident as Mesp promoters are capable of responding for Basic Biology (NIBB), using both the clone number and to RA signalling. These cells at the determination front are DNA sequence. The plasmids were supplied, dried on then competent to respond to Notch pathway genes acting as paper and were extracted by soaking in water, vortexing segmentation cues and form somitomeres. and heating repeatedly, before storing at 2208C. Oscillatory waves of gene expression in the PSM was first observed in chick, where a gene coding for a bHLH tran- Escherichia coli Transformation scription factor c-hairy1, a chick homologue of the Drosophila pair-rule gene hairy, exhibited dynamic rhythmic Plasmids were introduced to Promega competent Escherichia waves of expression, repeating with the formation of each coli cells and shaken in Luria – Bertani (LB) broth before ......................................................................................................................................................................................................................................... 23 Research article Bioscience Horizons † Volume 2 † Number 1 † March 2009 ......................................................................................................................................................................................................................................... incubating on antibiotic plates and picking individual colo- 50 units of human chorionic gonadotropin (hCG), 48 – nies to select for transformed E. coli. These were then 72 h before the eggs are needed, then injecting a further shaken overnight in LB broth with ampicillin to allow 250 units of hCG in the night before the eggs are required. growth of the transformed E. coli ( protocol obtained from By gently squeezing the female with pressure on her http://www.promega.com/tbs/tb095/tb095.pdf ). A puri- abdomen and lower back, the eggs were expelled, and then fied plasmid solution was obtained using ‘QIAprep Spin the sperm solution was immediately pipetted over the eggs Miniprep’ according to the manufacturer’s protocol (which for fertilization. After 5 min, the embryos were flooded can be found online: http://www1.qiagen.com/HB/ with NAM/10 (10% NAM, 5% 0.1 HEPES) and left for QIAprepMiniprepKit_EN). The concentration of the final at least 40 min to allow for cortical rotation before a 2.5% plasmid solution was then determined using a NanoDrop cysteine solution ( pH 7.8) was used to de-jelly the eggs. spectrophotometer. These were then washed extensively with buffered water to remove all traces of cysteine, followed by one wash in Diagnostic DNA Digests NAM/10 before putting the embryos in trays lined with Diagnostic DNA digests using plasmid and gene sequences to 1% agarose and covering with NAM/10 for culturing. identify restriction enzyme (RE) sites were used to check the Fertilized embryos were recognized by their rigidity as the correct gene was in each plasmid, to determine the orien- vitelline membrane thickens, and any unfertilized eggs were tation of each gene in the plasmid and also the size of the discarded. insert. A linearizing RE was then identified for each. Fixing Making Probe Template Embryos were collected at the required stage after removing DNA template for the probe was generated by mixing 5 mg their vitelline membranes and were fixed in MEMFA (0.1 M DNA, 10% 10 buffer and 3% linearizing RE, and incubat- MOPS, 2 mM EDTA, 1 mM MgSO and 3.7% formal- ing at 378C for 90 min to digest the DNA before checking dehyde) for 60 min before being washed in 100% EtOH that all DNA has cut to completion by running on an and stored in fresh EtOH at 2208C. agarose gel. The digestion was then cleaned up using In situ Hybridization ‘QIAquick Gel Extraction kit’ according to manufacturer’s protocol, leaving a purified DNA template. Embryos were rehydrated in a graded series of EtOH washes with PBSAT (PBS, 0.1% Tween) and washed extensively in Making Antisense Probe PBSAT. They were permeabilized with Proteinase K treat- Antisense RNA probe was generated by mixing 20% tran- ment at a concentration of 10 mg/ml 20 – 35 min. After scription buffer, 5% 10 digoxigenin NTP mix (containing rinsing in 0.1% triethanolamine ( pH 7.8), acetic anhydride 10 mM of each ATP, CTP and GTP, 6.5 mM UTP and was added to make a 0.25% solution, and then again to 3.5 mM DIG-11-UTP), 10 mM dithiothreitol (DDT), 4% make a 0.5% solution. After refixing in 3.7% formal- RNasin, 6% appropriate polymerase and 50 mg/ml DNA dehyde/PBSAT, embryos were washed extensively in template, and incubating at 378C for 2 h to allow adequate PBSAT and then equilibrated in hybridization buffer (50% time for transcription, before checking the transcript on formamide, 5 SSC, 1 mg/ml total yeast RNA, 100 mg/ml agarose gel. DNA template was removed from the mixture heparin, 1 Denharts, 0.1% Tween, 0.1% Chaps and by adding RNase-free DNase before precipitating the probe 10 mM EDTA). After prehybridizing in fresh hybridization with 11% 5 M ammonium acetate and 72% ethanol buffer for 2 h at 608C, embryos were incubated overnight (EtOH) and leaving overnight at 2208C. The pellet was in probe solution ( probe plus hybridization buffer). spun down at 48C before washing in 70% EtOH, drying Embryos were rinsed twice in hybridization buffer at 608C and redissolving in dH O, to leave a purified antisense to remove any residual probe before a series of rinses were RNA probe solution. Probes were hydrolysed, if necessary performed, still at 608C, with 2 SSC (0.1% Tween) and by adding an RNase-free solution of 40 mM NaHCO / then 0.2% SSC (0.1% Tween) and then at room temperature 60 mM Na CO and leaving at 608C for 20 – 30 min. with maleic acid buffer (MAB: 100 mM MAB, 150 mM 2 3 NaCl, 0.1% Tween, pH 7.8). Following blocking in MAB, Fertilizations 2% Block (Blocking Reagent; Roche) and 20% heat-treated Mature male X. laevis were anaesthetized with 0.05% ben- lamb serum at room temperature for 2 h to prevent non- zocaine before removing the testes and storing them in specific antibody binding, embryos were rocked overnight normal amphibian media (NAM) (110 mM NaCl, 2 mM at 48C in MAB þ Block þ HT lamb serum þ 1/2000 . . KCl, 1 mM Ca(NO ) 4H O, 1 mM MgSO 7H O, dilution of sheep anti-digoxigenin antibody coupled to alka- 3 2 2 4 2 0.1 mM Na EDTA). One-fourth to one-eighth of a testicle line phosphatase. They were washed several times with MAB was macerated in a few drops of NAM to produce a sperm to remove any excess antibody and rinsed twice in alkaline solution. Ovulation was induced in females by injecting phosphatase buffer (100 mM Trizma, 50 mM MgCl , ......................................................................................................................................................................................................................................... 24 Bioscience Horizons † Volume 2 † Number 1 † March 2009 Research article ......................................................................................................................................................................................................................................... 100 mM NaCl, 0.1% Tween, pH9.5) in order to equilibrate Thylacine1 Expression Is Seen Exclusively in S-II and S-III embryos before application of the substrate. Embryos were In situ hybridization using the Thylacine1 antisense probe incubated in BM purple for as long as necessary to detect reveals the specific expression of this gene in the developing the antibody (between a few hours and a few days), as BM somitomeres. At stage 12, two very faint stripes can be seen purple substrate turns blue and precipitates in the presence above the blastopore, marking the early segmentation in the of the antibody. After the colour developed, embryos were gastrula embryo (arrowheads, Fig. 1A). By stage 16, washed twice in PBSAT to remove any residual BM purple Thylacine1 expression is much stronger and more apparent, and fixed overnight in 3.7% formaldehyde/PBSAT. marking three somitomeres in this case (Fig. 1B). The Embryos were later bleached in 5% H O /PBSAT rolling 2 2 marking of more than two somitomeres is not an unexpected under bright light until pigment was gone and then washed result as Sparrow et al. note that between one and three several times in PBSAT to remove all traces of H O before 2 2 stripes are usually observed, with two being the most fre- fixing again in 3.7% formaldehyde/PBSAT. quent. At stages 26 and 31 (Fig. 1C and D, respectively), two distinct lines can be seen in each, showing expression Embryo Photography of Thylacine1 in S-II and S-III. The more posterior of the two somitomeres seen at stage 31 shows an extension of Photographs of embryos were taken in PBSA on 1% agarose- Thylacine1 expression ventrally on the tailbud (marked by lined trays using Spot camera with a Leica MZFLIII stereo- a black asterisk on Fig. 1D) which has not previously been microscope, and Spot Advanced computer software. reported. Pictures were cropped and light levels adjusted using Adobe Photoshop. Expression Analysis of bowline in X. laevis Reveals a Novel Expression Pattern in the Somites and Somitomeres Results In a stage 12 embryo, in situ hybridization using an antisense Isolation of cDNAs probe for bowline reveals a faint circumblastoporal cDNAs coding for ESR1, ESR2 and ESR-5 were provided by expression pattern (Fig. 2A). This expression in a gastrula M.E. Pownall and are being used in other current studies stage embryo has not been described in any literature; in (unpublished data), while Thylacine1 and bowline cDNAs fact, the earliest noted expression pattern is the presence of were obtained from NIBB using their unique clone bilateral stripes in a stage 13 embryo. The fact that this numbers (Table 1). Information about the RE sites of each expression is circumblastoporal and does not show any gene and plasmid details were collected using their sequences specific stripe pattern as observed by Kondow et al. in ( plasmid sequences obtained from the I.M.A.G.E consor- stage 13 embryos raises the question of whether this is a tium; http://image.hudsonalpha.org/html/vectors.shtml) true representation of bowline mRNA expression in a gas- and used to determine both the orientation of the gene and trula embryo, or is background colour that has developed the best linearizing RE to generate a probe template due to the absence of true mRNA expression; further work (Table 1). From this information, diagnostic digests were is needed to decipher this. By stage 16, however, bilateral carried out and subsequently, linearized probe templates stripes can be clearly seen marking S-II and S-III (shown by were generated. Antisense RNA probes were generated the arrowheads in Fig. 2B). Stage 26 embryos (Fig. 2C and D) from each of these templates for in situ hybridization. and stage 31 embryos (Fig. 2E) both have quite novel Table 1. Data collected regarding each gene studied Gene ESR1 ESR2 ESR-5 Thylacine1 bowline ........................................................................................................................................................................................................................................ NIBB/clone number IMAGE: 5537441 IMAGE: 6955664 Xl207h08 XL204i05 Xl034g14 Unigene number Xl.8440 Xl.12067 Xl.14524 Xl.54 Xl.16077 Plasmid pCMV. SPORT 6 pCMV. SPORT 6.1 pBluescript sk- pBluescript sk- pBluescript sk- 5 clone site SalI EcoRV EcoRI EcoRI EcoRI 3 clone site NotI NotI XhoI XhoI XhoI RE for linearizing EcoRI XbaI EcoRI BgIII SacI Polymerase T7 T7 T7 T7 T7 Gene insert size (kb) 1.6 1.1 0.6 1.7 1.8 Transcript size (bp) 600 1100 (hydrolysed to 300) 600 800 1000 ......................................................................................................................................................................................................................................... 25 Research article Bioscience Horizons † Volume 2 † Number 1 † March 2009 ......................................................................................................................................................................................................................................... Figure 1. In situ hybridization showing the expression of Thylacine1 in X. laevis. (A) Stage 12 vegetal view (dorsal side up), (B) Stage 16 dorsal view, (C) Stage 26 lateral view and (D) Stage 31 lateral view. In (B–D), anterior is to the left. Black arrowheads point to somitomeres expressing Thylacine1 mRNA and the asterisk marks the ventral extension of Thylacine1 expression in S-III. expression patterns, significantly different to that described in the literature for similar stages; others describe seeing one to two stripes of expression in the posterior of the 26, 27 embryo, correlating to S-II and S-III, and nothing else. However, this is not reflected in the in situ hybridization Figure 2. In situ hybridization showing the expression of bowline in X. laevis. (A) Stage 12 vegetal view, (B) Stage 16 dorsal view, (C) Stage studies I carried out for bowline. Not only is their consider- 26 lateral view, (D) Stage 26 dorsal view and (E) Stage 30 lateral view. In ably strong expression in the stated somitomeres, but fairly (B–E), anterior is to the left. Arrowheads mark bowline expression in strong expression can also be seen in the somites. A gap of somitomeres. approximately three to four somitomeres/somites can be seen between the most anterior somitomere marked, and possible to see colour developing in the somites and the the most posterior somite marked by bowline expression. somitomeres at the same time, thus it was not background The reason for this gap in expression is not known as occurring due to prolonged exposure to the BM purple bowline expression in the somites has not previously been substrate. documented. This observed difference in bowline expression is surprising—the cDNA used to create the antisense probe ESR-5 Transcripts Are Found in the PSM and in One to was sequenced and found to be the same as that used by Three Somitomeres in X. laevis others. This study was repeated three times with the same result each time, suggesting that it was not an exper- Carrying out in situ hybridizations with an ESR-5 antisense imental procedure or similar causing this unexpected result. probe reveals that this gene is very strongly expressed in the In addition, while watching the colour development it was PSM and in the anterior half of S-II and S-III (Fig. 3). During ......................................................................................................................................................................................................................................... 26 Bioscience Horizons † Volume 2 † Number 1 † March 2009 Research article ......................................................................................................................................................................................................................................... gastrulation, ESR-5 expression is seen surrounding the blas- and further studies would be needed to decipher this exten- topore but is excluded from the dorsal lip (Fig. 3A). In sion of expression. addition to this, two stripes of expression can be seen above the blastopore corresponding to the newly formed somitomeres. Similarly, at stages 16, 26 and 31, strong Expression Analysis of ESR1 in X. laevis Highlights Its expression can be seen in the PSM and in one to three somi- Strong Neural Expression and Some Tailbud Expression tomeres (Fig. 3B – D, respectively). The dark colouring seen In situ hybridization using the antisense probe for ESR1 in the head is a common background noise, caused as cavities revealed a predominantly neural expression pattern. This in the head trap the antisense probe and this should not be neural expression is of no great significance to this study as considered as ESR-5 expression. The ventral extension it is not relevant to somitogenesis; however, there is some of expression in S-III can be seen here (Black asterisk, expression seen in the tailbud region of later stages. At Fig. 3D) similar to that seen in Thylacine1 expression stage 12, expression can be seen around the blastopore in (Fig. 1D). Here, the stripe appears parallel to the PSM high- the ventral region of the embryo, and also bilaterally lighting the possibility that this extension of mRNA (Fig. 4A); no expression is seen in the dorsal part of the expression could occur in younger somitomeres which have embryo. By stage 16, a clear neural expression can be seen, not yet restricted gene expression laterally, as older somito- with stripes of primary neuronal precursors evident on meres may have done. This is only one possibility though, either side of the neural plate and a more anterior neural structure shown (Fig. 4B). In addition to these neural struc- tures, strong expression can be seen in the posterior of the stage 16 embryos, apparently in the tailbud domain. By stages 26 and 31 (Fig. 4C and D, respectively), expression can be seen in many anterior neural structures, including the eye and various parts of the brain. It is also found along the entire length of the neural tube, as verified by trans- verse sections taken for both stages (Fig. 4E and F, respect- ively). Black arrowheads on Fig. 4C and D, point to ESR1 expression in the tailbud domain of stages 26 and 31 embryos, respectively. The gene expression patterns of all similar stage embryos were compared to look for any differ- ences that may indicate the gene was cycling. However, no significant differences were observed at any stage, indicating that ESR1 mRNA does not cycle during somitogenesis. ESR2 mRNA Is Seen in Many Neural Structures in Addition to the Tailbud Region in X. laevis At stage 12, expression can be seen in the mesoderm of the ventral half of the embryo, surrounding the blastopore (Fig. 5A). By stage 16, ESR2 expression can be seen in the posterior paraxial mesoderm of the embryo and also marking some primary neural precursors either side of the neural tube (Fig. 5B). Expression in a stage 26 embryo is pre- dominantly in the tailbud region, but can also be seen in some of the more anterior neural structures including the eye (Fig. 5C). At stage 31, ESR2 expression is seen in many anterior neural structures, including the eye and brain (Fig. 5D). In addition to this, expression can be seen in the neural tube, notochord and to a lesser extent in the Figure 3. In situ hybridization showing the expression of ESR-5 in somites. This was verified by studying a transverse section X. laevis. (A) Stage 12 vegetal view (dorsal side up), (B) Stage 16 lateral of a stage 31 embryo, which clearly shows ESR2 expression view, (C) Stage 26 lateral view and (D) Stage 31 lateral view. In (B–D), in these internal structures (Fig. 5E). Tailbud expression is anterior is to the left. Black arrowheads point to somitomeres expressing also apparent at this stage, as marked by the arrowhead in ESR-5 mRNA and the asterisk marks the ventral extension of ESR-5 expression in S-III. Fig. 5D. ......................................................................................................................................................................................................................................... 27 Research article Bioscience Horizons † Volume 2 † Number 1 † March 2009 ......................................................................................................................................................................................................................................... Figure 4. In situ hybridization showing the expression of ESR1 in X. laevis. (A) Stage 12 vegetal view (dorsal side up), (B) Stage 16 dorsal view, (C) Stage 26 lateral view and (D) Stage 31 lateral view. Transverse sections Figure 5. In situ hybridization showing the expression of ESR2 in X. laevis. can be seen for stage 26 (E) and stage 31 (F) embryos showing expression (A) Stage 12 vegetal view (dorsal side up), (B) Stage 16 dorsal view, in the neural tube. In (B–D), anterior is to the left. (C) Stage 26 lateral view and (D) Stage 31 lateral view. In addition, a trans- verse section for stage 31 can be seen (E) highlighting expression in the neural tube, the notochord and bilaterally in the somites. In (B–D), anterior is to the left. The black arrowhead in D points to tailbud expression. Variable Expression Patterns in Neurula and Early Tailbud Stage Embryos Highlights the Potential Cycling Nature of ESR2 variable patterns of gene expression, making this an exciting prospect for an oscillating gene. A considerable number of embryos were studied after in situ Figure 6 shows a series of stage 16 embryos aligned to hybridization to look for any sign of dynamic expression demonstrate a progressive mRNA expression pattern, similar which may indicate that ESR2 mRNA is cycling. There did to that seen for esr9. Figure 6A – C shows the first stage of not appear to be any differences in gene expression patterns the progression, with ESR2 expression in the PSM of these for stage 12 or 31 embryos, however, both stage 16 (Fig. 6) embryos, but not in any somitomeres. Figure 6A and B and stage 26 embryos (not shown) showed considerably ......................................................................................................................................................................................................................................... 28 Bioscience Horizons † Volume 2 † Number 1 † March 2009 Research article ......................................................................................................................................................................................................................................... Figure 6. In situ hybridization showing the dorsal view of stage 16 embryos with different ESR2 gene expression patterns. (A–C) ESR2 expression in the PSM, but no fully or partially separated somitomeres can be seen. (D–F) Expression in the PSM, and red arrowheads here point to partially separated somitomeres. (G–H) Expression in the PSM and in one fully separated somitomere (black arrowheads). (I–J) Expression in one fully separated somitomere (black arrowheads), and in the PSM with one somitomere partially separated (red arrowheads). (K–N) Expression in the PSM and in two fully separated somitomeres each (black arrowheads). In all pictures, anterior is to the left. shows considerably weaker expression in the PSM than Discussion Fig. 6C, but similar neural expression, so this may reflect an earlier stage of this expression cycle. Figure 6D – F each Expression of Thylacine1 and ESR-5 Can Be Seen in shows expression in the PSM and in one or two Somitomeres during Gastrulation somitomeres of each embryo, as marked by red arrowheads. One pair of somitomeres can be seen during gastrulation to The expression in these somitomeres appears to be express both Thylacine1 and ESR-5, as shown by black arrow- continuous with the PSM at the midline, while others (black heads in Figs 1A and 3A, respectively. There are no published arrows) do not. This may represent different stages of somito- reports of gene expression in segmented mesoderm being mere formation. In the next stage of this series (Fig. 6G and observed prior to neurula stages and so Thylacine1 and H), somitomeres exhibiting mRNA expression separately to ESR-5 expression in the late gastrula may be marking the the expression in the PSM can be seen on only one side of initial segmentation of mesoderm in the embryo. the embryo (as shown by black arrowheads), while Fig. 6I Interestingly, bowline expression is not restricted to somito- and J shows a combination of continuous (red arrowheads) meres at this same stage (Fig. 2A), even though bowline and separate (black arrowheads) expression in somitomeres mRNA is known to be expressed in the same somitomeres with relation to the PSM. Figure 6K – N shows the next as ESR-5 and Thylacine1. The reason for not seeing any stage in the process, with one somitomere either side of somitomere stripes at this stage may be as simple as there the midline displaying ESR2 mRNA expression separate to being widespread bowline expression circumblastoporally. that in the posterior PSM (shown by black arrowheads). Although this may be the case as there appears to be weak ......................................................................................................................................................................................................................................... 29 Research article Bioscience Horizons † Volume 2 † Number 1 † March 2009 ......................................................................................................................................................................................................................................... circumblastoporal expression (Fig. 2A), the colour seen is discriminate between the mesodermal and ectodermal faint and without defined margins, so was initially considered tissues (Fig. 5A). This expression in both the mesoderm to be background colour. One consideration to make is that and ectoderm makes the expression pattern of ESR2 some- 24 22 the bowline antisense probe used for this in situ hybridization what similar to that of both ESR1 (Fig. 4) and esr9. appeared less defined in comparison to other probes when ran The Dynamic Expression Pattern of ESR2 at Stages 16 on an agarose gel. This may cause the unexpected expression and 26 Correlates to that of a Known Cycling Gene, esr9 pattern by either creating the suspected circumblastoporal background noise or failing to detect any small amounts of The dynamic expression pattern of ESR2 seen in stage 16 bowline mRNA in the segmented mesoderm. Further investi- embryos (Fig. 6) and stage 26 embryos (not shown) gation is needed to clarify the expression of bowline mRNA at appears to show cyclic oscillations, similar to the way esr9 gastrula stage, and if there truly is no somitomere expression mRNA cycles —an exciting discovery as this makes ESR2 found, to determine why this is. the second potential oscillating gene in X. laevis. The cycle begins with expression restricted to the PSM, before the A Novel Expression Pattern Is Seen for bowline Which mesoderm forms somitomeres (Fig. 6A – C). These somito- May Be Linked to a Negative Feedback Loop of Gene meres initially show striped ESR2 expression continuous at Expression the midline with expression in the posterior PSM In previous experiments, bowline expression has only been seen (Fig. 6D – F). This expression then appears to separate at 26, 27 in the two most posterior somitomeres, S-II and S-III. the midline from that in the PSM, and as the somitomeres Expression in these somitomeres is apparent in my studies, mature they will eventually stop expressing ESR2 altogether but in addition to this, expression is also seen in the somites (Fig. 6G – M). This cycle will then start again with expression (Fig. 2). Interestingly, there is a gap the size of approximately restricted to the PSM (Fig. 6N). three somitomeres/somites between the most anterior somito- Many embryos were observed to be asymmetrical with mere and the most posterior somite expressing bowline regards to their expression of ESR2, including both stage mRNA. As bowline expression has not been documented in 16 (Fig. 6) and stage 26 embryos (not shown), but this asym- the somites before, the reason for this gap is also unknown. metry never seemed to exceed more than one somitomere in Kondow et al. have found that although the bowline either stage. This highlights the autonomy of PSM cells either mRNA is located in S-II and S-III, the functional protein is side of the midline, and indicates that oscillations between found in S-I and S0. This late translation of bowline mRNA either side of the midline may be regulated independently. may account for the gap seen between the somites and the Asymmetry in gene expression has also been observed in 22 29 somitomeres. They go on to explain that bowline expression esr9 and in hairy2a in X. laevis while mouse and chick is activated by Tbx6 and once the Bowline protein reaches a expression is symmetrical. threshold concentration Tbx6 is converted from a transcrip- tional activator to a transcriptional repressor by associating Conclusion and Further Studies with X-Grg4 (a corepressor member of the Groucho family) via Bowline. This new transcriptional repressor turns off This study has identified ESR2 as the second potential gene bowline mRNA expression and may be the mechanism in X. laevis to show periodic oscillations of gene expression responsible for repressing bowline expression in the older during somitogenesis. However, other experiments are and more anterior somitomeres, S-I and S0. required to show definitively whether or not ESR2 is cycling in the Xenopus PSM. Explanting the PSM of the Previously Unstudied ESR2 Shows Expression in Both embryo either side of the midline and fixing at varying Mesoderm and Ectoderm time points would reveal whether or not ESR2 mRNA oscil- Expression analysis of ESR2 has revealed expression in both lates autonomously, and thus whether or not ESR2 is a true mesodermal and ectodermal tissues. Neuronal expression is cyclic gene. Experiments similar or the same as this are very obvious in stages 16, 26 and 31 (Fig. 5B – D). Strong routinely carried out in other studies to identify cyclic 14, 16, 17, 19 expression in the neural tube, the brain and in the eye at genes. In addition, periodic oscillations of both stage 31 highlights a strong presence in ectodermal deriva- Hes1 in chick and ESR9 in X. laevis have been shown by tives, while expression in the notochord, somites and PSM application of cyclohexamine to be independent of protein 14, 22 demonstrate a strong mesodermal presence as well (Fig. 5D synthesis, and so replication of these experiments to and E). At stage 26, expression appears to be more restricted look at the effect of blocking protein synthesis on ESR2 to the mesoderm, with strong expression in the PSM and in a mRNA expression may also help elucidate whether or not few somites while neural expression falls mainly in the eye this gene is truly cycling. An in depth study into the structure (Fig. 5C). Primary neuronal precursors can be seen to of the ESR2 gene and its regulatory elements would be ben- express ESR2 at stage 16, as can the PSM (Fig. 5B), eficial to this research also by revealing activators and/or whereas circumblastoporal expression at stage 12 does not repressors of ESR2 expression, hopefully confirming its ......................................................................................................................................................................................................................................... 30 Bioscience Horizons † Volume 2 † Number 1 † March 2009 Research article ......................................................................................................................................................................................................................................... 11. Horikawa K, Ishimatsu K, Yoshimoto E et al. 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Journal

Bioscience Horizons – Oxford University Press

Published: Mar 17, 2009

Keywords: Key words somitogenesis enhancer of split related Xenopus laevis cycling somitomere

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