The Japanese Society of Developmental Biologists

Develop. Growth Differ. (2013) 55, 350–358

doi: 10.1111/dgd.12046

Original Article

b-adrenergic signaling promotes posteriorizationin Xenopus early development
Shoko Mori, 1 Yuki Moriyama, 1 Kumiko Yoshikawa, 2 Tomoyo Furukawa 1 andHiroki Kuroda 1,2 *1Graduate School of Science and Technology and 2Department of Education (Sciences), Shizuoka University, 836 Ohya
Suruga-ku, Shizuoka, 422-8529, Japan

Adrenaline (also known as Epinephrine) is a hormone, which works as major regulator of various biologicalevents such stages of vertebrate, the role of adrenaline for early embryogenesis has been as heart rate, bloodvessel and air passage diameters, and metabolic shifts. Although its specific receptors are expressing at theearly developmental stage those functions are poorly understood. Here, we show that loss-of-functional effects
of adrenergic receptor b-2 (Adrb2), which was known as the major receptor for adrenaline and highly expressedin embryonic stages, led posterior defects at the tadpole stage of Xenopus embryos, while embryos injectedwith Adrb2 mRNA or treated with adrenaline hormone adversely lost anterior structures. This posteriorization
effect by adrenaline hormone was dose-dependently increased but effectively rescued by microinjection of antisense morpholino oligomer for Adrb2 (Adrb2-MO). Combination of adrenaline treatments and microinjection of
Adrb2 mRNA maximized efficiency in its posteriorizing activity. Interestingly, both gain- and loss-of-functional
treatment for b-adrenergic signaling could not influence anterior neural fate induced by overexpression of Chordin mRNA in presumptive ectodermal region, meaning that it worked via mesoderm. Taken together with theseresults, we conclude that adrenaline is a novel regulator of anteroposterior axis formation in vertebrates.
Key words: adrenaline, anteroposterior patterning, fibroblast growth factor, posteriorization, Wnt.

Introduction
During blastula and gastrula stages the establishmentof anteroposterior (A–P) patterning of the amphibianembryo starts in response to signaling by several
groups of secreted molecules. Xenopus anterior neuralinduction first occurs at blastula stage by bone morphogenetic protein (BMP) antagonists Chordin and
Noggin, which are expressed at the dorsal-animal
region, and this low-BMP region called the blastula
Chordin- and Noggin-expressing (BCNE) center gives
rise to anterior neural tissue and is indispensable forbrain formation (Kuroda et al. 2004; Ishibashi et al.
2008). At gastrula stage cerberus, which is expressedin the anterior endoderm region and functions as multiantagonists to block transforming growth factor (TGF)-*Author to whom correspondence should be addressed.
Email: ehkurod@ipc.shizuoka.ac.jp
Received 6 December 2012; revised 5 January 2013;
accepted 15 January 2013.
ª 2013 The Authors
Development, Growth & Differentiation ª 2013 JapaneseSociety of Developmental Biologists

b molecules such as BMPs and nodals and canonicalWnt proteins (Niehrs 2010), is required for head formation (Bouwmeester et al. 1996). Retinoic acid and fibroblast growth factor (FGF) also functions as an important
regulatory signaling for A–P patterning, and blockade of
FGF signaling downregulates the expression of members of the RAR (retinoic acid receptor) signaling pathway, resulting in anteriorization of Xenopus embryos(Tannahill et al. 1992; Blumberg et al. 1997; Shiotsugu
et al. 2004). Interestingly, Shisa, which is strongly
expressed in the prospective head ectoderm and the
Spemann organizer of Xenopus gastrula embryos, andphysically interacts with immature forms of the Wnt
receptor Frizzled and the FGF receptor within the endoplasmic reticulum. As a result Shisa inhibits their posttranslational maturation and trafficking to the cellsurface of Wnt and FGF, and Shisa therefore promoteshead formation like cerberus (Yamamoto et al. 2005).
As a hormone and neurotransmitter, adrenaline acts
on nearly all body tissues, and its actions are strongly
dependent on tissue type and tissue expression ofadrenergic receptors. Adrenaline acts by binding to avariety of adrenergic receptors and is a nonselective
agonist of all adrenergic receptors, including the majorAdrenaline effects in development

subtypes a1, a2, b1, b2, and b3 (Cotecchia et al.
2012). b-adrenergic receptor (Adrb) and their associated guanine nucleotide regulatory protein (G protein)/adenylyl cyclase signal transduction pathways are central to the overall regulation of cardiant function (Moniotte et al. 2001; Wachter & Gilbert 2012). Interestingly,
in the early developmental stage of Xenopus embryos,
RNA coding b1 receptor is present in the mature
oocyte, decreases after fertilization up to stage six andthen gradually increases during gastrulation (Devic
et al. 1997), meaning that embryonic development islikely to be regulated by adrenaline. However, the roleof adrenaline for early developmental stages has neverbeen examined. This study is the first report of the roleof b-adrenergic signaling for early developmental stageof vertebrate. We show that adrenaline and its receptor
have posteriorizing activity during the gastrula stage.

Materials and methods
Embryo manipulations
Xenopus laevis eggs were obtained from females
injected with 400 units of human chorionic gonadotropin (HCG, Fuji Pharma, Japan), fertilized in vitro withminced testis, and then cultured in 0.19 Steinberg’s
solution (SS). Frog embryos were always cultured at
22 °C. The jelly coats were removed with thioglycolicacid solution (1% of thioglycolic acid in 0.19 SS, pH
8.0). Embryos were fixed with PBSFA for 2 h, dehydrated with ethanol, embedded in paraffin wax, sectioned at 8 μm, and stained with hematoxylin
and eosin (HE). The following primers were used forreverse transcription–polymerase chain reaction (RT–PCR); Adrb1-RT-fw, 5′-CACCCTTCCGCTACCAGAG
T-3′, Adrb1-RT-rv, 5′-AGGACGTGGCTATGGGAGAA-3′,
Adrb2-RT-fw, 5′-GTGGTCATGATCTTCGTCTA-3′, Adr
b2-RT-rv, 5′-TTGGAACCTGGTTTGCGTAAG-3′, PNM
T-RT-fw, 5′-GATGTTCATCAGCCCAATC-3′, PNMT-RT-rv, 5′-AAAACACCCCATTGACATC-3′, Six3-RT-fw, 5′GCAACTTCAGGGAGCTCTAC-3′, Six3-RT-rv, 5′-TAG
GGATCCTGCAAGTACCA-3′, Rx2a-RT-fw: 5′-TAGTCTTCCTCTGGACTCCT-3′, Rx2a-RT-rv, 5′-CCGAAAGACTGGATGTGTTC-3′, Engrailed-RT-fw, 5′-GGAGAGAAG
AAAAGTGACCTG-3′, Engrailed-RT-rv, 5′-GCCTCCTCTGCTCAGTCAAA-3′, HoxB9-RT-fw, 5′-TACAGTCAATTATCAGCCCCGA-3′, HoxB9-RT-rv, 5′-TGTAATGTT
GGGGTCCCAGTTT-3′, NCAM-RT-fw, 5′-GCCATTCGT
AAAGTGGAACCATA-3′, NCAM-RT-rv, 5′-CCAGTTTT
GGAGCACTAGGTT-3′, N-tubulin-RT-fw, 5′-CCATTCTCTGGGTGGTGGCA-3′, N-tubulin-RT-rv, 5′CTGTGAGGTAGCGTCCATGAC-3′, a-actin-RT-fw, 5′-CTGACTG
AACGTGGCTACTC-3′, a-actin-RT-rv, 5′-GTCAGCAATACCAGGGTACA-3′, ODC-RT-fw, 5′-GCAACTGATG

351

CATGATATTAAAGAAC-3′, ODC-RT-rv, 5′-GAACTTTT
ATTTGTAAAACTGGTCAA-3′.
Adrenaline treatments
Approximately 91.6 mg of adrenaline powder (#E4125,
Sigma, USA) was dissolved in 300 μL of acetic acid,and diluted by 0.19 SS up to a total 5 mL volume(100 mmol/L concentration in this period), and stored ina freezer as 500-μL aliquots (master solution). Mastersolutions were inactivated in a few weeks, so we always
spent all of them in a few days. Five hundred microlitersof master solution was diluted with 0.19 SS up to
50 mL volume and neutralized with 110 μL of 5N
NaOH solution (1 mmol/L concentration in this period),
and further dilutions were performed with 0.19 SS.
Adrenaline treatments were performed in Poly-Hema(12% of poly 2-hydroxyethyl methacrylate solution,#18894, Polysciences, USA)-coated 40 mm diameterplastic plates using 3 mL of diluted adrenaline solutions
under dark conditions. For Poly-Hema coating of plastic
plate, 500 μL of 4% of Poly-Hema solution diluted byethanol was spread on a plate, immediately removed,
and then dried out. It seemed that adrenaline activity
was time-dependently reduced at room temperature,so we started using it just before the designated time.
Cloning and mRNA synthesis and microinjection
Adrb2 gene was cloned by PCR and ligated to pCS2
plasmid. The following primers were used for cloning
from cDNA created from total RNA of stage 12
embryos; Adrb2-fw, 5′-AATTGAATTCATGGAACGGT
CG-3′ and Adrb2-rv, 5′-AATTCTCGAGCCTATAATAAGCAA-3′. To generate synthetic mRNA in vitro,pCS2-Adrb2 was linearized with NotI and transcribedwith SP6 RNA polymerase by mMESSAGE mMACHINE kit (#AM1340, Ambion, USA). mRNA and MO
microinjections into Xenopus embryo were performedat the 2-cell stage. The sequence of Adrb2-MO was
5′-GGCTGGCGCTGACCGACCGTTCCAT-3′. For creating five-mismatch mRNA of Adrb2, long-range PCR
was performed for pCS2-Adrb2 using KOD-plus NEO
DNA polymerase (#KOD-401, Toyobo, Japan) and thefollowing primers; 5′-ATGGAGCGGTCTGTTAGCGCTAGTCCTAA-3′ and 5′-GAATTCGAATCGATGGGATCCTGCAAAAAGAA-3′. The regions of mismatched nucleotides without changing amino acid sequence areshown in Figure 2A.
Western blot
Embryo lysate was prepared in PhosphoSafe Extraction Buffer (#71296, Novagen, USA), lipids removed byª 2013 The Authors
Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists

352

S. Mori et al.

extracting once with an equal volume of trichloroethylene (#208-02486, Wako, Japan), and proteins separated by 5–20% gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels(#197-15011, Wako, Japan). Western blots were performed using monoclonal rabbit antibodies against diphospho-ERK1/2 (1:2000, #4370, Cell Signaling, USA)and ERK1/2 (1:1000, #4695, Cell Signaling, USA).

Results
The receptor of adrenaline expresses from egg totadpoleIf adrenaline has functions at early embryogenesis, thereceptor of adrenaline should be expressed at theearly stage of embryos. It is actually reported that
Adrb1 mRNA is expressed from oocyte (Devic et al.
1997) but the details of function of it and expressionsof the other types of b-adrenergic receptors havenever been investigated. Therefore, in order to examine the expression pattern of Adrb1 and Adrb2, inembryos at various developmental stages, RT–PCR
was carried out. Then, we found that high amounts of
Adrb2 mRNA were expressed in all stages of Xenopus
embryos, while Adrb1 was only maternally expressed(Fig. 1). In addition, we also checked the expression
level of phenylethanolamine N-methyltransferase(PNMT), which is known as an enzyme to create
adrenaline. Interestingly, PNMT started expression atstage 9 and continued to express until tadpole stages(Fig. 1), suggesting that adrenaline hormone musthave some effect in early embryogenesis. We also
tried to learn the expression area of Adrb2 and PNMT,
but both genes seemed to express uniformly in all
stages (data not shown).

first 25 nucleotides of the open-reading frame (ORF)(Fig. 2A). We found that embryos lost tail structure by
injection of Adrb2-MO (Fig. 2B,C), indicating that thisreceptor may have a role of posteriorization. If thisloss-of-functional effect is true, gain-of-functional
experiment of b-adrenergic signaling should havesome sort of effects on A–P axis formation. Therefore,
we cloned Xenopus Adrb2 gene by PCR and created
its mRNA. As we expected, clear anterior defects werecaused by both microinjection of Adrb2 mRNA
(Fig. 2D). In order to rescue the effects of Adrb2-MO,
we created a modified version of Adrb2 that had fivemismatches in the first 25 of ORF region but still kept
the same information of amino acids sequence(Fig. 2A). This modified mRNA (5mis-RNA) could
induce posteriorization the same as the case of normalmRNA (Fig. 2D,E) and completely cancel the effect of
Adrb2-MO (Fig. 2G) although normal mRNA did not do
it efficiently (Fig 2F).

(A)

(B)

(C)

(D)

(E)

(F)

(G)

Adrenaline has posteriorizing activity
In order to directly know the function of Adrb2, we
designed Adrb2-MO, which specifically targeted theFig. 1. Expressions of Adrb1, Adrb2, and PNMT mRNA during
the Xenopus embryonic period. In all stages expression of Adrb2
were detected, while maternal or zygotical expressions wereobserved in Adrb1 and PNMT mRNAs, respectively. ODC wasused for loading control.

Fig. 2. Loss-of-functional effects by antisense morpholino oligomer for Adrb2 (Adrb2-MO) for Xenopus embryogenesis. (A)Experimental design using Adrb2-MO. Adrb2-MO was designed
to target the translation initiation site (blue bar). This MO does
theoretically not bind to artificially created mRNA containing fivemismatched nucleotides (5mis) shown below. Red and green letters indicate start codon and changed nucleotides, respectively,
without changing coding information. (B) Control embryo at stage
38. (C) Adrb2-MO injected embryos. 75% (6/8) of embryos lost
tail structures. (D) Adrb2 mRNA injected embryos. 89.5% (17/19)
of embryos lost eye structures. (E) 5mis mRNA injected
embryos.88.2% (15/17) of them lost eye structures. (F) Adrb2MO and Adrb2 mRNA coinjected embryos. 9.5% (2/21) ofembryos lost tail structures. (G) Adrb2-MO and 5mis mRNA coinjected embryos. No embryo lost tail structures, and 53.6% (15/28) of embryos looked almost normal. Scale bars represent
1 mm.

ª 2013 The Authors
Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists

Adrenaline effects in development

To evaluate the effects of b-adrenergic signaling during development, we then used treatments of chemical
reagent of adrenaline hormone on live Xenopus
embryos (Fig. 3A). The treatments were performed
from stage 8 (early gastrula) in dark conditions(Fig. 3A). The reason why we did not start treatmentsfrom earlier stages was because the effect of adrenaline reagent worked most effectively in this condition in
our preliminary experiments. This reagent might be significantly inactivated in room temperature as timeadvances. Probably that is why the effects of adrenaline on early embryogenesis have never been reported.
Interestingly, anterior structures such as eye andcement gland were clearly blocked in a dose-dependent manner of adrenaline hormone (Fig. 3B–G).
Chemical reagent of adrenaline hormone should be
dissolved in acetic acid, diluted to 1 mmol/L concentration, and then neutralized with NaOH. We confirmedthat these neutralized solutions did not make any
effects on early embryogenesis (Fig. 3B). Moreover,these effects by adrenaline hormone were completely
rescued by microinjection of Adrb2-MO (Fig. 3H) andamplified by Adrb2 mRNA (Fig. 3I). Then, we examinedthe effects of b-adrenergic signaling on molecular marker expressions of Rx2a and HoxB9, which were,respectively, anterior and posterior neural markers at
tailbud stage embryo (Fig. 4). Compared to the353

expression pattern of control embryos (Fig. 4A,B), single injection of Adrb2 showed slight inhibition of Rx2a
expression level at anterior region (Fig. 4C,D), andAdrb2-MO clearly reduced the extent of HoxB9
expression area at the posterior region (Fig. 4E,F).
Expectedly, treatments of adrenaline hormone strongly
reduced Rx2a expression but increased HoxB9
expression (Fig. 4G,H), while these effects by chemical
reagent were obviously rescued by microinjection of
Adrb2-MO (Fig. 4I,J). From these data, we confidently
conclude that b-adrenergic signaling had a role of posteriorization activity for early development in Xenopus.
Anterior neural fate induced by BMP antagonist is notchanged by b-adrenergic signaling
Chordin (Chd) is a large secreted protein expressed
specifically in the BCNE center of the blastula stageembryo and is required for anterior neural formation(Kuroda et al. 2004; Ishibashi et al. 2008). In the casesof zebrafish and Xenopus, loss of Chordin reducesanterior neural formation (Schulte-Merker et al. 1997;€ger et al. 2003). Chordin and Noggin douOelgeschla
ble-homozygous mutants of mice have severe defects
in the development of forebrain structure (Bachiller
et al. 2000). In order to check whether b-adrenergic
signaling is able to affect this anterior neuralization by

(A)

(B)

(C)

(D)

(E)

(F)

(G)

(H)

(I)

Fig. 3. Posteriorizing effects by adrenaline treatments. (A) Experimental procedures. Embryos injected with/without antisense morpholino
oligomer for Adrb2 (Adrb2-MO) or with/without Adrb2 were cultured. Animal cap regions (ACs) were cut at stage 8 and then treated withvarious concentrations of adrenaline solutions (0.05–0.5 mmol/L). (B) Control embryos at stage 38. (C) Embryos treated with acetic acidsolutions were neutralized with NaOH. All embryos (13/13) developed normally. (D–G) Embryos treated with 0.05, 0.1, 0.25, or
0.5 mmol/L of adrenaline solutions. 17.4% (4/23), 14.3% (4/28), 23.1% (6/26), or 27.6% (8/29) of embryos, lost eye structures. (H)Embryos treated with 0.5 mmol/L of adrenaline solution following microinjection of Adrb2-MO. 45.6% (5/11) of embryos developed normally. Compared to G, it indicated that posteriorizing effects by adrenaline hormone were clearly rescued by Adrb2-MO. (I) Embryos
treated with 0.05 mmol/L of adrenaline solution following microinjection of Adrb2 mRNA. 80.0% (8/10) lost eye structures. Compared toD, it indicated that posteriorizing effects by adrenaline hormone were clearly amplified by Adrb2 mRNA. Scale bars represent 1 mm.
ª 2013 The Authors
Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists

354

S. Mori et al.

(A)

(B)

(C)

(D)

(E)

(F)

(G)

(H)

(I)

Fig. 4. The effects of b-adrenergic signaling on anterior and posterior neuralmarker expressions. (A, C, E, G, I) Lateral views of embryos. (B, D, F, H, J)
Dorsal views of embryos. (A, B) Control
embryos. (C, D) Embryos injected with
Adrb2 mRNA. (E, F) Embryos injectedwith antisense morpholino oligomer for
Adrb2 (Adrb2-MO). (G, H) Embryos
treated with adrenaline solutions. (I, J)
Embryos treated with adrenaline solutions following microinjection of Adrb2MO at 2-cell stage. Scale bars represent 0.5 mm.

(J)

BMP antagonism, we then used a presumptive ectoderm region, which is generally called the animal cap
region (AC). AC were cut from blastula stage embryosthat were injected with mRNAs and/or MO at the
2-cell stage and cultured with/without adrenaline hormone until stage 26 to examine anterior neural markers Six3 and Rx2a, mid-hindbrain boundary marker
En2, posterior neural markers HoxB9, pan-neuralmarkers NCAM and N-tubulin, mesoderm marker aactin, and loading control ODC (Fig. 5A). The expression of both anterior neural and pan-neural markers
induced by Chd was not blocked by both gain-offunctional way of b-adrenergic signaling activity(Fig. 5B) and loss-of-functional way (Fig. 5C). These
results indicated that b-adrenergic signaling worked
through mesoderm and indirectly changed anterior
neural fate in embryos, so some mesodermal signals

should be taken into account. It has been reported
that Adrb2 stimulation can activate extracellular signalregulated kinase, ERK (Fig. 5D, Sivamani et al. 2007;
Yang et al. 2010). Therefore, we next focused on
phosphorylation of ERK. We simply put early stage of
Xenopus embryos into adrenaline solutions and cultured, resulting in the phosphorylation level of ERK
being remarkably increased when b-adrenergic signaling was upregulated (Fig. 5E).
Late effect of β-adrenergic signaling causes
gastrointestinal malformation
Adrenergic neurotransmitters, which is also called norepinephrine or noradrenaline, is involved in the formation of regularly-structured visceral morphogenesis of
Xenopus embryos (Toyoizumi et al. 1997). In ourª 2013 The Authors
Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists

Adrenaline effects in development

Fig. 5. The effects of b-adrenergic signaling on mesoderm-independent neural marker expressions. (A) The experimental procedure for B and C. Animal cap regions (ACs) were cut fromembryos injected with/without mRNAs and/or Adrb2-MO, cultured with/without 1 lM of adrenaline solution until stage 26, andused for reverse transcription–polymerase chain reaction (RT–
PCR) analyses. More than 0.01 mmol/L of adrenaline solution
killed AC cells for unfathomable reasons. (B, C) Inhibition ofanterior neural marker expression by up- or downregulation of badrenergic signaling. Anterior neural markers, Six3 and Rx2a,
mid-hindbrain boundary marker, En2, posterior neural (spinalcord) marker, HoxB9, and pan-neural markers, NCAM and Ntubulin, were not affected in both cases. a-actin and ODC wererespectively used for showing no contamination of mesoderm
and loading control. (D) Downstream model of Adrb2. (E) Western blotting analysis of phospho-ERK in adrenaline treatedembryos. Embryos were treated from 2-cell stage with 0.5 mmol/
L of adrenaline solution and fixed at stage 26.

(A)

(B)

the initiation period of the morphogenetic movement ofgastrointestinal formation in Xenopus development,
and fixed embryos at stage 45. Compared to control
embryos (Fig. 6A–D), neutralized solution, which was
the same solution used in (Fig. 3C), did not have any
effect (Fig. 6E–F). However, embryos treated with
0.5 mmol/L adrenaline hormone still keep all gastrointestinal organs, but all of them, especially liver andsmall intestine, were not well developed (Fig. 6I–L). In
the case of 1 mmol/L, although only a few organs
such as lung and pancreas were observed, separationof small and large intestine and bile duct formation
were not detected at all (Fig. 6M–P). Interestingly, inthese late-stage treatments, tail formation was obviously shortened and shrunken (Fig. 6 M,N). Heartbeat
is started just after stage 40, and the bloodstream carries many secreted molecules and hormones away.
Probably, many tissues and organs may alreadyrequire supply of adrenaline from the bloodstream for
growth. Gastrointestinal malformation can easily occur
if developmental timing is disordered by rapamycin
reagents, which is target of rapamycin (TOR) kinase
inhibitor (Moriyama et al. 2011), so it is quite possiblethat a similar mechanism is working in the case of
adrenaline treatment on late stage embryos.

(C)

(D)

355

(E)

Discussion
experiments shown above, it was very difficult to evaluate gastrointestinal formation because A–P effects
were too strong, and adrenaline hormone should becompletely inactivated before reaching the tadpole
stage. Therefore, we tried to start treatments of adrenaline hormone since stage 30, which is thought to be

In this work, we have investigated the functions ofb-adrenergic signaling on Xenopus early development
and defined that it has a posteriorizing activity via
mesodermal signal in the early stage. The neural ectoderm is patterned along its A–P axis. This patterning is
initiated by posteriorizing signals derived from prospective or definitive mesendodermal tissues (Nieuwkoopª 2013 The Authors
Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists

356

S. Mori et al.

(A)

(E)

(I)

(M)

(B)

(F)

(J)

(N)

(C)

(G)

(K)

(O)

(D)

(H)

(L)

(P)

Fig. 6. The late effect of b-adrenergic signaling on Xenopus embryogenesis. (A, E, I, M) dorsal, (B, F, J, N) lateral, and (C, G, K, O) ventral and enlarged view of embryos. (D, H, L, P) Histology of gastrointestinal regions. Sectioned sites were indicated in C, G, K, O by dotted lines. (A–D) Control embryo. (E–H) 100% (3/3) of embryos treated with acetic acid solution neutralized with NaOH. (I–L) 100% (3/3)
embryos treated with 0.5 mmol/L of adrenaline solutions. (M–P) 100% (4/4) of embryos treated with 1 mmol/L of adrenaline solutions.
bd, bile duct; i, intestine; li, large intestine; lu, lungs; pa, pancreas; si, small intestine; st, stomach. Scale bars represent 1 mm.

1952; Toivonen & Saxen 1968). Three candidate posteriorizing signals have been suggested: retinoic acid
(RA; Durston et al. 1989; Sive et al. 1990; Conlon
1995; Blumberg et al. 1997), fibroblast growth factors(Fgfs; Kengaku & Okamoto 1993;Cox & HemmatiBrivanlou 1995; Lamb & Harland 1995; Koshida et al.
1998), and Wnts (Kelly et al. 1995; McGrew et al.
1995; Fekany-Lee et al. 2000; Kazanskaya et al.
2000; Kiecker & Niehrs 2001; Yamaguchi 2001).
Although it is likely that factors in all three families participate in this process, the precise role of each in thetemporal and spatial aspects of neural patterning aswell as the molecular consequences of their action
have not been fully clarified, but posteriorizing activity
by b-adrenergic signaling is highly possible to berelated with at least one of these three candidate posteriorizing signals.

adrenaline treatment (Fig. 5). We think that it is quite
reasonable that strong ventralization should occur if
b-adrenergic signaling blocks BMP signaling activity,
but we did not observe it. This strongly suggests thatb-adrenergic signaling is not a simple posteriorizing
factor on neuroectoderm and has other activity toaffect the mesodermal region. It is likely to be caused
by upregulation of FGF signaling because phosphorylation of ERK can also be induced by FGF signaling(Kuroda et al. 2005). FGF is able to change the fateof ectoderm into mesoderm (Kimelman & Kirschner1987), but mesodermal marker was not detected in(Fig. 5B), and elongation or swelling phenotypes that areusually observed in mesoderm-induced AC explants
were not observed either (data not shown). This meansthat posteriorizing activity of b-adrenergic signaling canwork on the basis of presence in mesoderm.

Mechanism of posteriorization by b-adrenergic
signaling

Crosslink of b-adrenergic and FGF signaling

Anterior neural marker expressions induced by Chd
were not affected by both upregulation and downregulation of b-adrenergic signaling, but interestingly phosphorylation level of ERK was strikingly increased by

The catecholamines epinephrine (adrenaline) and norepinephrine (noradrenaline) are agonists for a family of
G-protein coupled receptors (GPCRs) known as
adrenergic receptors. There are three subfamiliesof adrenergic receptors: a1, a2 and b, and the bª 2013 The Authors
Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists

Adrenaline effects in development

subfamily contains three subtypes: b1, b2, and b3
located predominantly in heart, lung and adipose tissue, respectively (Ma & Huang 2002). Adrenergic
receptors are GPCRs that link to trimeric G-protein. Gproteins are typically composed of a, b and c subunits.
Each subunit exists in multiple isotypes with differential
specificity for effector signaling. Specificity of response
to catecholamines is mediated in part by the Ga subunits, but the Gb-Gc dimers are also involved in regulation. b-adrenergic receptors favor interaction withheterotrimeric G-proteins that contain the Ga-s andGa-i subunit. The Ga-s subunits activate various isoforms of adenylate (adenylyl) cyclases. Consequently,
b-adrenergic receptors typically elevate the level ofcyclic AMP (cAMP) an important mediator of cell signaling. Both Ga-s and Ga-i have been linked to thestimulation of Src family tyrosine kinases (Huang et al.
2004). Src tyrosine kinase is a major downstream factor of FGF receptor, and Src tyrosine kinase activatedby FGF receptor contributes to certain FGF-induced
biological responses via the Raf1-MEK-ERK pathways,
and it has strong posteriorizing activity (Kuroda et al.
2004). These suggest that the A–P patterning inembryonic body plan may be regulated by activationof Raf1-MEK-ERK via upstream crosstalk between badrenergic and FGF signaling in the cytoplasmic
region.

Acknowledgments
This research was supported by the grants-in-aid
received from the Shizuoka Research Institute.

References
Bachiller, D., Klingensmith, J., Kemp, C., Belo, J. A., Anderson,
R. M., May, S. R., McMahon, J. A., McMahon, A. P., Harland, R. M., Rossant, J. & De Robertis, E. M. 2000. The
organizer factors Chordin and Noggin are required formouse forebrain development. Nature 403, 658–661.
Blumberg, B., Bolado, J. Jr, Moreno, T. A., Kintner, C., Evans,
R. M. & Papalopulu, N. 1997. An essential role for retinoid
signaling in anteroposterior neural patterning. Development
124, 373–379.
Bouwmeester, T., Kim, S., Sasai, Y., Lu, B. & De Robertis, E. M.
1996. Cerberus is a head-inducing secreted factor
expressed in the anterior endoderm of Spemann’s organizer.
Nature 382, 595–601.
Conlon, R. A. 1995. Retinoic acid and pattern formation in vertebrates. Trends Genet. 11, 314–319.
Cotecchia, S., Stanasila, L. & Diviani, D. 2012. Protein-protein
interactions at the adrenergic receptors. Curr. Drug Targets
13, 15–27.
Cox, W. G. & Hemmati-Brivanlou, A. 1995. Caudalization of neural fate by tissue recombination and bFGF. Development
121, 4349–4358.

357

Devic, E., Paquereau, L., Steinberg, R., Caput, D. & Audigier, Y.
1997. Early expression of a beta1-adrenergic receptor andcatecholamines in Xenopus oocytes and embryos. FEBS
Lett. 417, 184–190.
Durston, A. J., Timmermans, J. P., Hage, W. J., Hendriks, H. F.,de Vries, N. J., Heideveld, M. & Nieuwkoop, P. D. 1989.
Retinoic acid causes an anteroposterior transformation in thedeveloping central nervous system. Nature 340, 140–144.
Fekany-Lee, K., Gonzalez, E., Miller-Bertoglio, V. & Solnica-Krezel, L. 2000. The homeobox gene bozozok promotes anterior neuroectoderm formation in zebrafish through negative
regulation of BMP2/4 and Wnt pathways. Development 127,
2333–2345.
Huang, J., Sun, Y. & Huang, X. Y. 2004. Distinct roles for Src
tyrosine kinase in beta2-adrenergic receptor signaling to
MAPK and in receptor internalization. J. Biol. Chem. 279,
21637–21642.
Ishibashi, H., Matsumura, N., Hanafusa, H., Matsumoto, K., De
Robertis, E. M. & Kuroda, H. 2008. Expression of Siamois
and Twin in the blastula Chordin/Noggin signaling center isrequired for brain formation in Xenopus laevis embryos.
Mech. Dev. 125, 58–66.
Kazanskaya, O., Glinka, A. & Niehrs, C. 2000. The role of Xenopus dickkopf1 in prechordal plate specification and neuralpatterning. Development 127, 4981–4992.
Kelly, G. M., Greenstein, P., Erezyilmaz, D. F. & Moon, R. T.
1995. Zebrafish wnt8 and wnt8b share a common activity
but are involved in distinct developmental pathways. Development 121, 1787–1799.
Kengaku, M. & Okamoto, H. 1993. Basic fibroblast growth factor
induces differentiation of neural tube and neural crest lineages of cultured ectoderm cells from Xenopus gastrula.
Development 119, 1067–1078.
Kiecker, C. & Niehrs, C. 2001. A morphogen gradient of Wnt/
beta-catenin signalling regulates anteroposterior neural patterning in Xenopus. Development 128, 4189–4201.
Kimelman, D. & Kirschner, M. 1987. Synergistic induction of
mesoderm by FGF and TGF-beta and the identification of anmRNA coding for FGF in the early Xenopus embryo. Cell 51,
869–877.
Koshida, S., Shinya, M., Mizuno, T., Kuroiwa, A. & Takeda, H.
1998. Initial anteroposterior pattern of the zebrafish central
nervous system is determined by differential competence ofthe epiblast. Development 125, 1957–1966.
Kuroda, H., Wessely, O. & De Robertis, E. M. 2004. Neuralinduction in Xenopus: requirement for ectodermal and endomesodermal signals via Chordin, Noggin, b-Catenin, andCerberus. PLoS Biol. 2, 623–634.
Lamb, T. M. & Harland, R. M. 1995. Fibroblast growth factor is adirect neural inducer, which combined with Noggin generates anterior-posterior neural pattern. Development 121,
3627–3636.
Ma, Y. C. & Huang, X. Y. 2002. Novel signaling pathway throughthe beta-adrenergic receptor. Trends Cardiovasc. Med. 12,
46–49.
McGrew, L. L., Lai, C. J. & Moon, R. T. 1995. Specification of
the anteroposterior neural axis through synergistic interactionof the Wnt signaling cascade with Noggin and follistatin.
Dev. Biol. 172, 337–342.
Moniotte, S., Kobzik, L., Feron, O., Trochu, J. N., Gauthier, C. &Balligand, J. L. 2001. Upregulation of beta(3)-adrenoceptors
and altered contractile response to inotropic amines inhuman failing myocardium. Circulation 103, 1649–1655.

ª 2013 The Authors
Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists

358

S. Mori et al.

Moriyama, Y., Ohata, Y., Mori, S., Matsukawa, S., Michiue, T.,
Asashima, M. & Kuroda, H. 2011. Rapamycin treatment
causes developmental delay, pigmentation defects, and gastrointestinal malformation on Xenopus embryogenesis. Biochem. Biophys. Res. Commun. 404, 974–978.
Niehrs, C. 2010. On growth and form: a Cartesian coordinate
system of Wnt and BMP signaling specifies bilaterian bodyaxes. Development 137, 845–857.
Nieuwkoop, P. D. 1952. Activation and organization of the central
nervous system in amphibians. Part III. Synthesis of a newworking hypothesis. J. Exp. Zool. 120, 83–108.
€ger, M., Kuroda, H., Reversade, B. & De Robertis, E.
Oelgeschla
M. 2003. Chordin is required for the Spemann organizertransplantation phenomenon in Xenopus embryos. Dev. Cell
4, 219–230.
Schulte-Merker, S., Lee, K. J., McMahon, A. P. & Hammerschmidt, M. 1997. The zebrafish organizer requires chordino.
Nature 387, 862–863.
Shiotsugu, J., Katsuyama, Y., Arima, K., Baxter, A., Koide, T.,
Song, J., Chandraratna, R. A. & Blumberg, B. 2004. Multiple
points of interaction between retinoic acid and FGF signaling
during embryonic axis formation. Development 131, 2653–
2667.
Sivamani, R. K., Lam, S. T. & Isseroff, R. R. 2007. Beta adrenergic receptors in keratinocytes. Dermatol. Clin. 25, 643–653.
Sive, H. L., Draper, B. W., Harland, R. M. & Weintraub, H. 1990.
Identification of a retinoic acid-sensitive period during pri-mary axis formation in Xenopus laevis. Genes Dev. 4, 932–
942.
Tannahill, D., Isaacs, H. V., Close, M. J., Peters, G. & Slack, J.
M. 1992. Developmental expression of the Xenopus int-2(FGF-3) gene: activation by mesodermal and neural induction. Development 115, 695–702.
Toivonen, S. & Saxen, L. 1968. Morphogenetic interaction of presumptive neural and mesodermal cells mixed in different
ratios. Science 159, 539–540.
Toyoizumi, R., Kobayashi, T., Kikukawa, A., Oba, J. & Takeuchi,
S. 1997. Adrenergic neurotransmitters and calcium ionophore-induced situs inversus viscerum in Xenopus laevis
embryos. Dev. Growth Differ. 39, 505–514.
Wachter, S. B. & Gilbert, E. M. 2012. Beta-adrenergic receptors,
from their discovery and characterization through their
manipulation to beneficial clinical application. Cardiology
122, 104–112.
Yamaguchi, T. P. 2001. Heads or tails: Wnts and anterior-posterior patterning. Curr. Biol. 11, 13–24.
Yamamoto, A., Nagano, T., Takehara, S., Hibi, M. & Aizawa, S.
2005. Shisa promotes head formation through the inhibitionof receptor protein maturation for the caudalizing factors,
Wnt and FGF. Cell 120, 223–235.
Yang, X., Zheng, J., Xiong, Y., Shen, H., Sun, L., Huang, Y.,
Sun, C., Li, Y. & He, J. 2010. Beta-2 adrenergic receptor
mediated ERK activation is regulated by interaction with
MAGI-3. FEBS Lett. 584, 2207–2212.

ª 2013 The Authors
Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists