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M E C H A N I S M S O F D E V E L O P M E N T 1 2 6 ( 2 0 0 9 ) 1 9 8 – 2 1 1
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Body wall morphogenesis: Limb-genesis interferes with bodywall-genesis via its influence on the abaxial somitederivatives
Isabella Kurnia Liema,b, Hirohiko Aoyamaa,*
aDepartment of Anatomy and Developmental Biology, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi,
Minami-ku, Hiroshima 734-8551, JapanbDepartment of Anatomy, Faculty of Medicine, University of Indonesia, Jl. Salemba 6, Jakarta 10430, Indonesia
A R T I C L E I N F O
Article history:
Received 4 November 2007
Received in revised form
3 November 2008
Accepted 14 November 2008
Available online 24 November 2008
Keywords:
Abaxial
Primaxial
Body wall
Limb
Somite
Axial skeleton
Skeletal muscle
Morphogenesis
Body plan
Evolution
0925-4773/$ - see front matter � 2008 Elsevidoi:10.1016/j.mod.2008.11.003
* Corresponding author. Tel.: +81 82 257 51E-mail address: [email protected]
A B S T R A C T
The vertebrate body wall is regionalized into thoracic and lumbosacral/abdominal regions
that differ in their morphology and developmental origin. The thoracic body wall has ribs
and intercostal muscles, which develops from thoracic somites, whereas the abdominal
wall has abdominal muscles, which develops from lumbosacral somites without ribs cage.
To examine whether limb-genesis interferes with body wall-genesis, and to test the possi-
bility that limb generation leads to the regional differentiation, an ectopic limb was
induced in the thoracic region by transplanting prospective limb somatopleural mesoderm
of Japanese quail between the ectoderm and somatopleural mesoderm of the chick pro-
spective thoracic region. This ectopic limb generation induced the somitic cells to migrate
into the ectopic limb mesenchyme to become its muscles and caused the loss of distal tho-
racic body wall (sterno-distal rib and distal intercostal muscle), without causing any signif-
icant effect on the more proximal region (proximal rib, vertebro-distal rib and proximal
intercostal muscle). According to a new primaxial–abaxial classification, the proximal
region is classified as primaxial and the distal region, as well as limb, is classified as abax-
ial. We demonstrated that ectopic limb development interfered with body wall develop-
ment via its influence on the abaxial somite derivatives. The present study supports the
idea that the somitic cells give rise to the primaxial derivatives keeping their own identity
and fate, whereas they produce the abaxial derivatives responding to the lateral plate
mesoderm.
� 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
The vertebrae of early tetrapods possessed ribs in any body
segment; therefore, they did not have distinct body regional-
ization. However, during the course of evolution, as tetrapod
limbs became reoriented to hold the trunk well above the
ground, ribs became confined mostly in the thoracic region
er Ireland Ltd. All rights
10; fax: +81 82 257 5114.c.jp (H. Aoyama).
and thoraco-lumbosacral/abdominal regionalization became
visible, especially in birds and mammals (Feduccia, 1975;
Kent, 1983; Carroll, 1988). Cranio-caudally, the limb and the
ribs appear to be complementary to each other. Forelimbs
locate in the cervico-thoracic transitional region, followed
by rib in the thoracic region and hind limbs in the lumbosa-
cral region. In chick, these regions correspond to the somite
reserved.
M E C H A N I S M S O F D E V E L O P M E N T 1 2 6 ( 2 0 0 9 ) 1 9 8 – 2 1 1 199
level: 15–20 for forelimb; 19–26 for rib: and 26–32 for hind limb
(Fig. 1A; Chevallier, 1975, 1979; Burke, 2000; Huang et al, 2000a;
Christ et al., 2000).
Study on an avian embryo has revealed that body wall
components derived from embryonic mesoderm, i.e. somite
and somatopleural mesoderm. Somites give rise to the body
wall skeleton and muscles in both thoracic and abdominal
region, except the sternum, which derived from somatopleur-
al mesoderm (Fig. 1A; Chevallier, 1975, 1977, 1979; Christ et al,
1983; Burke, 2000; reviewed by Christ et al, 2007). Most of the
dermis and fascias also derived from somatopleural meso-
derm. Here, we defined rib-bearing body wall in the thoracic
region as the thoracic body wall and no rib-bearing body wall
in the lumbosacral region as the lumbosacral/abdominal body
wall (Fig. 1A). The origin of the abdominal wall muscles were
reported to overlap with the origin of the leg and pelvic girdle
muscles. Seno (1961) identified somites 26–27 and Chevallier
(1979) identified somites 27–29 as the origin of abdominal
muscles. They also give rise to the muscle of leg and pelvic
girdle (somites 26–32; Chevallier, 1979).
When limb somatopleural mesoderm was grafted into the
thoracic region, the myogenic cells of the thoracic somite
migrated into the limb mesenchyme to become the limb
muscles (Hayashi and Ozawa, 1995). Therefore, it was sug-
gested that limbs contain a potent signaling mechanism to
Fig. 1 – Developmental origin and primaxial–abaxial classificatio
of the thoracic wall, which shows the cartilages and the interco
Nowicki, 2003) and the first layer of the abdominal wall, which s
muscle (eom; cut). Dashes lines are cartilages that are derived fro
lateral view of eight-day-old chick embryo, right side. The ribs
(Aoyama et al., 2005): proximal rib (pr), vertebro-distal rib (vdr) a
intercostal muscles, follows that of Yasuda (2002): uncinate pro
sternum (st), ensiform process of sternum (ep), ilium (il), pubis (p
classification follows that of Burke and Nowicki (2003).
reprogram the thoracic somite (Alvares et al., 2003). Besides,
according to Burke and her colleagues, cells from somites that
cross the lateral somitic frontier (abaxial cells, Nowicki et al.,
2003) will conform to the morphological pattern appropriate
to the abaxial region to the host, because their gene expres-
sion and morphological fate is apparently controlled by the
lateral plate (Nowicki and Burke, 2000; Burke and Nowicki,
2003). In another study, Cohn and colleagues (1995) reported
that malformation in ribs was observed when they induced
an additional limb in the thoracic region with the FGF2 bead.
These studies aroused in us the question of whether in the
course of phylogenesis, limb development causes the tetra-
pod to lose its cervical ribs and lumbosacral ribs. On this
study, we tried to reproduce the regionalization of the axial
skeleton in ontogenesis by inducing an ectopic limb in the
thoracic region. The ectopic limb was induced by transplant-
ing limb somatopleural mesoderm of quail embryo into chick
embryo at their thoracic level. By somite-labeling experiment,
we confirmed that the ectopic limb induced somitic cells to
migrate into the ectopic limb mesenchyme, where they be-
came the ectopic limb muscles. They did not develop into
the body wall muscles. The ectopic limb-genesis in the thorax
resulted in deficiencies in the ventral body wall components,
such as the sterno-distal rib, distal intercostal muscles, ster-
num and abdominal muscles at the ectopical limb level.
n of the chick body wall and limbs. Figure is the second layer
stal muscles, proximal (pim) and distal (dim) (Burke and
hows the internal oblique muscle (iom) and external oblique
m somatopleural mesoderm. This figure was drawn from the
consist of three compartments based on their development
nd sterno-distal rib (sdr). The other terminology, except the
cess of the rib (puc), vertebrae (vt), scapula (sc), coracoid (cr),
b), ischium (is). The primaxial (bluish) and abaxial (reddish)
200 M E C H A N I S M S O F D E V E L O P M E N T 1 2 6 ( 2 0 0 9 ) 1 9 8 – 2 1 1
These missing components, as well as the limb, are classified
as abaxial (Burke and Nowicki, 2003). Whereas we found no
significant changes in the development of dorso-lateral body
wall, i.e. proximal ribs, vertebro-distal ribs and proximal
intercostal muscles, which is classified as the primaxial
(Burke and Nowicki, 2003). We will discuss the interference
of ectopic limb development on body wall development in
the context of the new classification of somite derivatives,
primaxial–abaxial (Fig. 1B), proposed by Burke and Nowicki
(2003).
2. Results
2.1. Body wall morphogenesis in the embryo with anectopic limb in its thorax
We induced an ectopic limb in the thoracic region by trans-
planting somatopleural mesoderm of the limb forming region
into the thoracic region (Fig. 2A) and examined its influence
on body wall development, both skeletal and muscular ele-
ments. The skeletal element was observed on whole mount
preparation stained with cartilage marker, alcian blue
(Fig. 2B–D and Table 1) and the muscular element was
observed histologically using eosin staining and a muscle
marker, anti-desmin antibody immunostaining alternatively
(Figs. 3–5 and Table 2). We also identified the developmental
origin of the ectopic limb component using two types of
Fig. 2 – Skeletal development of chimeras with an ectopic limb
transplantation. (B–D) Lateral view of eight-day-old embryos sta
ectopic leg and (C) is chimera with an ectopic wing. The ectopic
sterno-distal ribs (red arrows). (D) Control chimera shows no ect
ectopic ischium; xpb, ectopic pubis; xf, ectopic femur; xsc, ectop
anti-quail antibodies, QCPN for the nuclei and QCR1 for the
cartilage cells (Figs. 3–5 and Table 2).
We did not remove the host somatopleural mesoderm and
replace it with the graft. Instead, we transplanted the quail
somatopleural mesoderm between the surface ectoderm
and the host somatopleural mesoderm, which degenerates
within three days as a result of this type of transplantation
(see Section 2.2, last paragraph and Section 3.1, last para-
graph). In total 32 embryos were operated on, of which 26
(81%) survived. Among the surviving chimeras, fourteen had
leg somatopleural mesoderm transplants, six had wing
somatopleural mesoderm transplants and six had thoracic
somatopleural mesoderm transplants as the control experi-
ment. Two of the 14 samples with leg somatopleural meso-
derm transplants were discarded because no ectopic limb
was seen.
2.1.1. Abaxial skeleton deficiencies of chimeras with anectopic limb
Transplantation of leg somatopleural mesoderm pro-
duced an ectopic leg in the thoracic region (12 of 14 oper-
ated embryos or 86%) and caused abnormalities in the
ventral skeletal body wall element (n = 12, Table 1). The
ectopic legs were complete (with both pelvic girdle and
leg bones, n = 8) or truncated (n = 4) and located at two to
four level of thoracic vertebrae I–VII. Ribs and sternum
development on the experimental side were disturbed. All
in the thoracic region. (A) Schematic drawing of the
ined with alcian blue, right side. (B) Is chimera with an
leg and wing development were accompanied by the lost of
opic limb and normal rib development. xil, ectopic ilium; xis,
ic scapula; xh, ectopic humerus.
Table 1 – Skeleton of the chimeric embryos with wing, leg and thoracic somatopleural mesoderm transplantation.
Graft Ectopiclimb
Body wall Host limb
Type Number Vertebralcolumn
Rib Sternum Pectoralgirdle
Wing Pelvicgirdle
Leg
Proximal Vertebro-distal Sterno-distal
Wing 6 (+) Wing N N N (�) T N N N 5N/1AbN
Leg 12 (+) Leg N N (+) CD (�) 3N/9T N N N N
Thoracic 6 (�) N N N 4N/2CD N N N N N
(+), the presence of skeletal element; (�), the absence of skeletal elements. Abbreviations: N, normal; AbN, abnormal; CD, changed its direction;
T, truncated. Number in front of these letters indicates the number of cases.
Fig. 3 – Histology of proximal body wall development of chimeras with an ectopic leg or wing in the thoracic region. Figures
are double immunostaining of QCPN-QCR1 (brown) followed by alcian blue (blue)-eosin (pink) counter staining, except (G),
whole mount chimera with an ectopic leg stained with alcian blue and (I–L) (Des), which depict double immunostaining of
QCPN (brown)-anti-desmin (red) with alcian blue (blue) counter staining. (A–F) Experimental side (right side body). (A 0–F 0)
Control side (left side body). (G) Indicates the frontal section plane of (A–E) (red lines). (A) Proximal intercostal muscles
developed normally (red arrow) as shown in the control (A 0, red arrow). The cutaneous muscle (A 0, cm) was absent. (B–D) Along
with the directional changes of vertebro-distal rib (vdr), distal part of the proximal intercostal muscles developed disorderly.
They elongated (B, red arrow) compared with the control (B 0, red arrow) and broke finally (C and D red arrow). Between ribs with
a narrower space (D, rib 5–6; E, rib 3–4), they were compact (D and E, green arrow). (E) The base of the ectopic limb was
separated with the body cavity only by connective tissue (red arrow). The external oblique muscle (E 0, eom) was absent. (F) The
vertebro-distal rib and proximal intercostal muscle developed normally in the chimera with an ectopic wing (red and green
arrows). (H–K) Are four consecutive sections, starting from section (D) plane, showing detailed alteration of the intercostal
nerve development (black arrows). (H) Is an enlargement of dotted line frame in (D). (L) Is an enlargement of dotted line frame
in (J). The rib cartilages (blue without brown color) and the ectopic limb muscles (xlm, red with unstained nucleus) derived
from chick cells. All the ectopic limb cartilage derived from quail cells (A–F, blue with brown color; H, green arrows), except the
ectopic pubic cartilage (E, xpb). red bar 1 mm; black bar 500 lm; green bar 200 lm; blue bar 50 lm.
M E C H A N I S M S O F D E V E L O P M E N T 1 2 6 ( 2 0 0 9 ) 1 9 8 – 2 1 1 201
the embryos lost their three to five sterno-distal ribs
(Fig. 2B). Most of their sternum (9 of 12 cases) lost its ensi-
form process and had a shorter body. On the other hand,
the proximal rib and vertebro-distal ribs developed almost
Fig. 4 – Histology of distal body wall development of chimeras with an ectopic leg and wing in the thoracic region. Figures are
double immunostaining of QCPN-QCR1 (brown) followed by alcian blue (blue)-eosin (pink) counter staining, except (D 0, D and I),
which depict double immunostaining of anti-desmin (red)-QCPN (brown) with alcian blue (blue) counter staining and (G and H)
Whole mount chimeras stained with alcian blue. (A–F) Experimental side (right side body). (A 0–F 0) Control side (left side body).
(G and H) Indicate frontal section planes of the chimeras (G for A–C and A 0–C 0; H for D–F and D 0–F 0). Sterno-distal ribs (A 0, B 0, D 0,
E 0, sdr), distal intercostal muscles (D 0, black arrow), cutaneous muscle (A 0, D 0, cm), external oblique muscles (A 0, D 0, black stars)
and ensiform process of sternum (B 0, E 0, ep) did not develop in the experimental side (A, B, D, E). The major part of the pectoral
muscle (p) was not lost, but the distal part of the thoracobrachial part (p 0) did not develop (A, D, parentheses). In the chimera
with an ectopic wing, the pectoral muscle duplicated (dp). Ectopic ischium (B, C, xis), ectopic coracoid (E, xcr) and ectopic
sternum (F, xst) derived from quail cells, but ectopic pubis was from chick cells (C, xpb). (I) Is an adjacent section to D. It is an
enlargement of the corresponding region to the dotted line frame (J) in (D), showing the chick derived-ectopic limb muscle (red
with unstained chick nucleus, black arrows) surrounded by quail derived-connective tissue cells (QCPN positive brown nuclei
cells, green arrows). (J) Is an adjacent section to D. It is an enlargement of the corresponding region to the dotted line frame (J)
in (D), showing the quail derived-ectopic humerus cartilage (alcian blue positive tissue with QCPN and QCR1 positive brown
color, red arrows). red bar 1 mm; black bar 500 lm; yellow bar 50 lm; green bar 20 lm.
202 M E C H A N I S M S O F D E V E L O P M E N T 1 2 6 ( 2 0 0 9 ) 1 9 8 – 2 1 1
normally except that the vertebro-distal ribs deformed to
avoid the ectopic leg (red arrows, Fig. 2B). This directional
change was suggested to be caused by a developmental
space limitation rather than a direct effect of the develop-
ing ectopic leg. A wide ectopic pelvis might intercept the
rib precursor cells.
Transplantation of wing somatopleural mesoderm pro-
duced almost the same results as that of the leg somatopleural
mesoderm transplant (Table 1). A complete ectopic wing (5 of 6
cases) with their shoulder girdle (6 cases) developed at the level
of thoracic vertebrae II–VI, and varied from two to four levels of
vertebrae. All the embryos lost their three to five sterno-distal
ribs (Fig. 2C) and had truncated sternum. However, in contrast
to the case of leg somatopleural mesoderm transplantation, a
wide ectopic scapula did not form, probably because scapula
is largely somitic origin (Huang et al., 2000a), and significant
changes in the vertebro-distal ribs were not seen.
Homotopic transplantation of thoracic somatopleural
mesoderm produced no ectopic limb and their ribs were al-
most normal (Table 1 and Fig. 2D). Only in two of six cases
was there a slight disorientation of one (rib VI) or two ribs
(ribs VI and VII) observed.
Fig. 5 – Body wall development in the control chimera (homotopic thoracic somatopleural mesoderm transplantation). (D) Is
lateral view of the embryo shown in frontal section planes (A–C). (E and F) Are enlargements of the black frames in (B). The
experimental side (A–C) has almost the same figure as the control side (A 0–C 0). (B, C, E) Sterno-distal rib (sdr) and pectoral
muscle (p, black arrows) derived from chick cells. Ensiform process of the sternum (ep) and caudal part of sternal body (C, green
arrow) derived from quail cells. (F) Quail cells also gave rise to the dermis (d), cells of connective tissue and some endothelials.
Epidermis derived from chick cells (epd). v, vessel; ram, rectus abdominis muscle; red arrows distal intercostal muscle; yellow
arrow cranial part of sternal body; red bar 1 mm; black bar 500 lm; green bar 50 lm; yellow bar 10 lm.
M E C H A N I S M S O F D E V E L O P M E N T 1 2 6 ( 2 0 0 9 ) 1 9 8 – 2 1 1 203
Thus, the ectopic limb forming in the thoracic region
caused loss of sterno-distal ribs (see Fig. 6 for the summary),
which along the limbs are classified as abaxial (Burke and
Nowicki, 2003).
Table 2 – Summary of the histological observation of the chimmesoderm transplantation.
Graft Ectopic limb Body wall
Type Number Cartilage Muscle Thoracic
Ribcartilage
Intercostal mus
Proximal Dis
Wing 5 Q C C C (�)
Leg 9 Q* C C C (�)
Thoracic 4 (�) (�) C C C
Origin of tissues are indicated by C (host, chick) and Q (graft, somatopleu
tissues; (*), except for the pubic bone, which derived from chick cell. A
Number in front of these letters indicates the number of cases.
2.1.2. Abaxial muscle deficiencies in the chimeras with anectopic limb
Histological examination of the chimera with an ectopic
limb revealed a disruption of the development of the body
eric embryos with wing, leg and thoracic somatopleural
PectoralmuscleAbdominal
cle Externaloblique
Internaloblique
Transversusabdominis
Rectusabdoministal
(�) C C 3C/2(�) C (5def, 4dp)
(�) C C 6C/3(�) C (1N/8def)
C C C C C
ral mesoderm, quail). (+), the presence of tissue; (�), the absence of
bbreviations: def, deficient; dp, with duplication of pectoral muscle.
Fig. 6 – Schematic diagrams summarizing body wall morphogenesis of chimera with an ectopic limb on its thorax. At the
ectopic limb level, the sterno-distal rib and distal intercostal muscles, thoracobrachial part of pectoral muscle (black arrows)
and external oblique muscles did not develop. There was a duplication of pectoral muscle (dp) caudal to the proper
thoracobrachial part in the ectopic wing experiment. Blue color cartilages were derived from host chick cells; while, green
color cartilages of ectopic limb (except the extra pubic) were derived from quail cells.
204 M E C H A N I S M S O F D E V E L O P M E N T 1 2 6 ( 2 0 0 9 ) 1 9 8 – 2 1 1
wall muscles, which is accompanied by a malformation of the
skeletal system (Table 2 and see Fig. 6 for the summary). On
the operation side, all the embryos were lacking their distal
intercostal muscles and the external oblique muscle in addi-
tion to the missing of the sterno-distal rib (Figs. 3 and 4). Most
of the proximal intercostal muscles developed normally
(Fig. 3A and F), though, in the chimera with an ectopic leg,
we found disordered development in the distal part of the
proximal intercostal muscles that inserted in the ribs dislo-
cated because of the ectopic hind limb. These intercostal
muscles elongated and finally broke between ribs with wider
space (red arrows, Fig. 3B–D). Between ribs with narrower
space, they were compact (green arrow, Fig. 3D and E). The
external oblique muscle was recognizable cranial, caudal
and in some cases ventral to the ectopic limb, but was not
found in the area where ectopic limb attached. The internal
oblique, transversus abdominis and rectus abdominis mus-
cles usually developed normally; however, in a few cases,
their muscle masses decreased (not shown). In addition, the
embryos were also lacking their cutaneous muscles (Figs.
3A, B, E and 4A, D). Thus, there were no body wall muscles
at the base of the ectopic limb, which was separated from
the body cavity only by connective tissue that covered the
body cavity (red arrow, Fig. 3E).
The pectoral muscle development patterns in the chimera
with an ectopic leg were different from that in the chimera
with an ectopic wing. Along with the ectopic leg develop-
ment, the distal part of the thoracobrachial part of the pector-
al muscle was lost (Fig. 4A). On the other hand, along with the
ectopic wing development, the loss of this muscle was fol-
lowed by the development of extra pectoral muscle. This ex-
tra pectoral muscle developed caudally to the proper
thoracobrachial part of pectoral muscle (Fig. 4D–F, see Fig. 6
for the summary).
Ectopic limb development also influenced the develop-
ment of intercostal nerves. They grew into the ectopic limb
area (red stars, Fig. 3A–C), joined each other to make a plexus,
and finally became the ectopic limb nerve (black arrows,
Fig. 3H–L).
2.1.3. Developmental origin of ectopic limb componentsWe confirmed that most of the ectopic limb cartilages and
their surrounding connective tissue were graft/quail-derived
cells (Figs. 3 and 4, see Fig. 6 for the summary), except the
pubic bone and its surrounding connective tissue. They were
composed of host chick cells (xpb, Figs. 3E and 4C). The ribs
and intercostal muscles were comprised of host chick cells,
and so were their surrounding connective tissues (Fig. 3A–F).
The ectopic limb muscles were host-derived cells, while the
surrounding connective tissue cells and tendons were quail
somatopleural mesoderm-derived cells (Figs. 3L and 4I). The
graft also gave rise to the dermis and endothelial cells. In
M E C H A N I S M S O F D E V E L O P M E N T 1 2 6 ( 2 0 0 9 ) 1 9 8 – 2 1 1 205
the control chimeras homotopically transplanted with the
somatopleural mesoderm, the graft gave rise to the dermis,
endothelial cells, connective tissue and sternum (Fig. 5).
Thus, the ectopic limb muscles were derived from the host
tissue, which were likely the somites because of their normal
developmental fate (Fig. 1A). To confirm that this was the
case, we examined the migration pattern of thoracic somite
cells in the chimera with a graft of leg somatopleural
mesoderm.
2.2. Thoracic somite cells migration in the chimera with agraft of leg somatopleural mesoderm
Transplantations of quail leg somatopleural mesoderm
(28–30th somite level) between ectoderm and somatopleural
mesoderm of chick prospective thoracic region (22–26th
somite level) were performed on two-day-old chick embryos
(19–25 somite stage or HH stage 13–15) and quail embryos
(24–29 somite stage of HH stage 15–16) in ovo. We followed
somitic cell migration in the chimera everyday during four
days after the transplantation using two methods, vital stain-
ing with fluorescent dyes and immunohistochemistry to
identify quail cells derived from the graft. We used 1,
1 0-dioctadecyl-3,3,3 0,3 0tetramethylindocarbocyanine perchlo-
rate (DiI)–3,3 0-dioctadecyloxacarbocyanine perchlorate (DiO)
for vital labeling (Fig. 7A–D). It was hard to follow the cells by
DiI–DiO labeling more than one day because their fluores-
cence became too weak to detect. Therefore, we took the
advantage of quail nuclei specific monoclonal antibody, QCPN
(Hybridoma bank) immunohistochemistry and 4 0,6-diamidi-
no-2-phenylindole, dihydrochloride (DAPI) counter staining
(Fig. 7E–G) to follow the graft and the host cells, respectively.
By this method, QCPN positive cells were not stained with
DAPI (Nowicki et al., 2003). A total of 45 surgeries resulted in
survival in 35 cases (78%), all of which had an ectopic limb.
Seven of the 35 surviving embryos were discarded because
of weak DiI labeling or unmatched position of DiI injected-so-
mite with the ectopic limb.
One day after the surgery (HH stage 19–21), in 93% (13 from
14 cases) of the chimeras, we found migration of thoracic so-
mite cells (labeled with DiI) into the limb bud derived from
quail somatopleural mesoderm (labeled with DiO, Fig. 7B–D).
Using QCPN with DAPI counter staining, we confirmed this re-
sult (4 of 5 cases or 80%). Some chick cells (blue fluorescence
of DAPI) were found in the dark field (QCPN positive, Fig. 7E–F)
of the limb bud. Two (HH stage 23–24; n = 4; not shown) and
three days after surgery (HH stage 26–27; n = 2; Fig. 7G), blue
fluorescence chick cells were seen in the ventral and dorsal
part of the limb bud. Thus, using both techniques, we con-
firmed that thoracic somite cells migrated into the ectopic
limb bud.
To examine the fate of these immigrating somite cells, we
stained the adjacent sections to those mentioned above with
QCPN and anti-desmin, and alcian blue counter staining.
Anti-desmin and alcian blue were used to identify muscle
cells and cartilage cells, respectively. Both markers were not
detected until three days after the surgery. Three days after
the surgery, alcian blue positive cartilage was found in the
center of the limb bud, where there were no DAPI positive
cells (Fig. 7G and H), suggesting these cartilage cells were de-
rived form quail somatopleural mesoderm. This was con-
firmed by immunostaining using QCPN, which revealed
quail specific large heterochromatin in the nuclei (Fig. 7J).
Dorsal and ventral to the central cartilage of the limb bud,
where DAPI positive cells immigrated (white arrows,
Fig. 7G), anti-desmin positive cells were found (black arrows,
Fig. 7H). They were long like myotubes and had QCPN nega-
tive nuclei (black arrows, Fig. 7I). Anti-desmin negative cells
around the positive cells were QCPN positive quail cells (green
arrows, Fig. 7I).
Four days after the surgery, cartilages and muscles fur-
ther differentiated (Fig. 7K). It was now obvious that the
anti-desmin positive muscle cells were multinuclear cells
of host chick origin (black arrows, Fig. 7L), while their sur-
rounding non-muscle cells were of quail origin (green ar-
rows, Fig. 7L).
These findings suggested that the host (chick) thoracic so-
mite cells immigrated into the limb bud, and then differenti-
ated into muscle cells. By contrast, we found few QCPN
negative, anti-desmin positive cells at the ventral of the ecto-
pic limb (Fig. 7K, right parentheses; Fig. 7M). On the opposite
corresponding site (on the control side), there were the rib
cartilages and intercostal muscles throughout the body wall
(Fig. 7K, left parentheses; Fig. 7N). Thus, the thoracic somite
cells that originally formed the body wall cartilage and mus-
cles did not follow their original fate, but some of them mi-
grated into the graft somatopleural mesoderm to form limb
muscles (see Fig. 8 for the summary).
These continuous observations also confirmed that the
host somatopleural mesoderm gradually decreased, espe-
cially on the face of the graft. One day (Fig. 7E and F) and
two days after the surgery (not shown), the chimeras had
two layers of somatopleural mesoderm, the host (QCPN neg-
ative, DAPI positive) and the graft (QCPN positive, DAPI nega-
tive). Three days after the operation, the host somatopleural
mesoderm that faces the graft was finally lost, replaced by
the graft somatopleural mesoderm (red arrow, Fig. 7G and H).
3. Discussion
3.1. Ectopic limb induction changes the developmental fateof thoracic somite
The trunk of tetrapods is suggested to have a latent
limb-growing ability (reviewed by Tamura et al., 2001). Stud-
ies on the avian embryos revealed that the entire dorsal
and lateral region of the trunk could produce additional
limbs. These additional limbs were generated by implanta-
tion of the prospective limb mesoderm into the prospective
flank region (Dhouailly and Kieny, 1972), or by application of
FGFs, such as FGF1, FGF2, FGF4, FGF8 or FGF10 (Cohn et al.,
1995; Crossley et al., 1996; Ohuchi et al., 1997; Yonei-Tamura
et al., 1999; see Ohuchi and Noji, 1999 for a review). The
additional limb induced by FGFs was found to induce
migration of the flank somitic cells to give rise to its mus-
cles (Heymann et al., 1996; for a review; see Ohuchi and
Noji, 1999). The same findings were also reported when
the additional limb was induced by replacing the thoracic
mesoderm with the limb mesoderm (Hayashi and Ozawa,
1995; Alvares et al., 2003).
206 M E C H A N I S M S O F D E V E L O P M E N T 1 2 6 ( 2 0 0 9 ) 1 9 8 – 2 1 1
Above previous studies have been concentrated on the
development of the additional limb in the thorax, and scarce
attention was given to the body wall development. The pres-
ent study was focused on the interaction between the tho-
racic somite and the limb somatopleural mesoderm. Here,
we presented detail data from a precise observation of the
body wall morphogenesis, especially the development of the
ribs, intercostal muscles and abdominal muscles due to the
development of an ectopic limb in the thoracic region. The ec-
topic limb development in the thorax caused the loss of ster-
no-distal ribs, distal intercostal muscles and part of the
abdominal body wall muscles (Fig. 6). By labeling the somites
after the transplantation, we followed the migration of the
thoracic somite cells at the ectopic limb level and found that
the somitic cells migrated into the graft derived-extra limb
bud mesenchyme and became ectopic limb muscles. These
migrating somitic cells did not develop into the body wall
muscles. Thus, the lost of distal thoracic body wall cartilage
and muscles in the embryo with the ectopic limb was results
of the fate alteration of the migrating somitic cells, from the
precursor of body wall muscle and maybe the cartilage to be-
come the precursor of limb muscles.
Studies on the avian embryo revealed that the mesoder-
mal components of the limbs and the girdles derived from
somatopleural mesoderm, except the muscles and the scapu-
lar blade that derived from somite (reviewed by Christ et al.,
2007). Our study showed that the ectopic limb cartilages were
graft/quail-derived cells and the ectopic limb muscles were
host/chick-derived cells. These confirmed that the former de-
rived from the somatopleural mesoderm and the latter de-
rived from the somite. However, one of the ectopic pelvic
girdle bones, the pubic bone was composed of host chick
cells. Since the pelvic girdle elements showed differential
gene expressions and regulation (Malashichev et al., 2005), it
is possible that the pubic bone derived from a different origin,
Fig. 7 – Thoracic somite cells migration in the chimera with a g
vital labeling. (B–D) One day after the surgery, cross-section of
cells (red, white arrow) migrated into the DiO-labeled graft (gre
immunostaining on one day after the surgery shows successfu
mesoderm (brown colored) between the chick thoracic ectoderm
There was a clear border and no cells mixing between the graft
F). Medial region of the graft well contacted with the somite (v
staining showed that chick nuclei fluoresced, whereas quail nu
fluorescent-chick cells were seen in the dark field-limb bud (w
cells were found dorsal and ventral to the central dark field-lim
anti-desmin followed by alcian blue counter staining confirme
blue (ct, cartilage) and DAPI positive cells were stained by anti-
the limb bud was lost (red arrow, see also G) and replaced wit
line frame in (H). (I) QCPN positive cells (green arrows) surrounde
were stained positively with anti-desmin (red, black arrows). (J
connective tissue cells were QCPN positive (brown colored nuc
cells differentiated further. (L) Enlargement of (L) black frame i
arrows), surrounded by quail connective tissue cells (green arro
almost all of the area ventral to the limb bud tissue consisted
anti-desmin positive cells were seen on the endothelial of the
Enlargement of N black frame in K shows that in the control s
50 lm; green bar 100 lm; red parentheses, distal body wall.
i.e. somite. Nevertheless, it is also possible that the graft (leg
somatopleural mesoderm of the quail) induced the host (tho-
racic somatopleural mesoderm of the chick) to develop the
ectopic pubic bone. Detail cell lineage experiment to reveal
this issue is under way in our laboratory.
To replace the host somatopleural mesoderm with the
graft somatopleural mesoderm, we transplanted the addi-
tional somatopleural mesoderm to the host somatopleural
mesoderm instead of cutting it off and transplanting the
graft. When we simply made a slit between the thoracic som-
ites and somatopleural mesoderm without doing any other
treatment, the embryos lost their sternal ribs in spite of the
fact that the slit became invisible within 24 h (unpublished
data). It has been shown that without maintenance signals
from the ectoderm, somatopleural mesoderm degenerated
(Funayama et al., 1999; Sudo et al., 2001). The homotopic
transplantation of thoracic somatopleural mesoderm (the
control chimeras) showed that the graft somatopleural meso-
derm could replace the host without causing any significant
effects on the ribs development (Figs. 2D and 5). Thus, at first,
the chimera had two layers of somatopleural mesoderm.
Then, however, only the graft survived. The host somatople-
ural mesoderm degenerated and was replaced by the graft fi-
nally. Therefore, on the wing or leg somatopleural mesoderm
transplantation, besides inducing an ectopic limb, the graft
acts as a barrier between the host surface ectoderm and
somatopleural mesoderm (Fig. 8A), which makes the host
somatopleural mesoderm degenerate.
3.2. Primaxial and abaxial
Anatomically, somite derivatives are classified as epaxial
and hypaxial. This classification groups the muscles based
on their positional relationship to the horizontal septum
(for fishes and some amphibians) and the innervations of
raft of leg somatopleural mesoderm. (A) Schema of DiI–DiO
the chimera at the transplantation level. DiI-labeled somitic
en), which has become an ectopic limb bud. (E) QCPN
l transplantation of the quail prospective leg somatopleural
(ect) and somatopleural mesoderm (uncolored, red arrow).
and host somatopleural mesoderm (red dotted line, see also
ll, ventro-lateral lip of dermomyotome). (F) DAPI counter
clei stained positively with QCPN did not (dark field). Blue
hite arrow). (G) Three days after the surgery, DAPI positive
b bud (white arrows). (H) Double immunostaining QCPN and
d that the central dark field-limb bud was stained by alcian
desmin (black arrows). Host somatopleural mesoderm faced
h the developing graft. (I and J) Are enlargements of dotted
d the QCPN negative cells, which began to differentiate and
) All alcian blue stained-cartilage cells and the surrounding
lei). (K) Four days after the surgery, cartilages and muscle
n (K) shows well differentiated chick muscle cells (black
ws). (M) Enlargement of (M) black frame in (K) shows that
of QCPN negative, anti-desmin negative cells, except false
vessel (blue arrows), macrophage and white blood cells. (N)
ide the muscles developed well (black arrows). black bar
c
M E C H A N I S M S O F D E V E L O P M E N T 1 2 6 ( 2 0 0 9 ) 1 9 8 – 2 1 1 207
the resident muscles (adult positional and functional criteria).
Epaxial muscles are innervated by the dorsal rami of the
spinal nerves and lie above the horizontal septum, whereas
hypaxial muscles are innervated by the ventral rami of the
spinal nerves and lie below the horizontal septum (reviewed
by Sporle, 2001). This classifies the intercostal muscles (prox-
Fig. 8 – Schematic diagrams summarizing the interference of
the ectopic limb-genesis on the body wall-genesis. (A) At first,
the chimera had two layers of somatopleural mesoderm.
While the graft was maintained by the ectoderm (via BMP2
and/or BMP7; Funayama et al., 1999), the host somatopleural
mesoderm, gradually degenerated. The inductive signal from
the developing limb somatopleural mesoderm (BMP4 and SF/
HGF; Alvares et al., 2003) then controls the migration of the
thoracic somite cells, called the abaxial somitic cells. (B) The
migrated cells gave rise into the limb muscles, but did not
develop into the body wall muscles. (C) Finally, the thoracic
body wall lost its distal or abaxial muscles and cartilages. The
proximal or primaxial muscles and cartilages were not
affected.
208 M E C H A N I S M S O F D E V E L O P M E N T 1 2 6 ( 2 0 0 9 ) 1 9 8 – 2 1 1
imal and distal muscles) and the abdominal muscles, in the
same category as the limb muscle, as hypaxial. In the avian,
this classification has also been supported molecularly
(Cheng et al., 2004). Recently, Burke and Nowicki (2003) pro-
posed a new classification, primaxial and abaxial. This classifi-
cation is defined by embryonic criteria. Primaxial refers to
areas that are comprised of somitic cells only, and abaxial
to areas that contain somitic cells migrating and mixing with
lateral plate cells. The boundary between these domains is
called the lateral somitic frontier (Nowicki et al., 2003). This
classifies not only the muscle elements, but also the skeletal
elements (Fig. 1B). The vertebrae, vertebral rib (proximal and
vertebro-distal ribs) and proximal intercostal muscle are clas-
sified as primaxial, whereas the sterno-costal rib, distal inter-
costal muscle, sternum and abdominal muscles are classified
in the same category as those of limb skeleton and muscles,
as abaxial.
Although the primaxial and abaxial domains are different
from the epaxial and hypaxial regions, these two types of
classification do not contradict. The embryological, primaxi-
al–abaxial classification is more appropriate and precise for
discussing developmental phenomena (Burke and Nowicki,
2003). In this study, the interference of body wall development
by the ectopic limb development is difficult to explain using
the epaxial–hypaxial classification, because both the affected
and non-affected muscles are classified as hypaxial. On the
contrary, according to the primaxial–abaxial classification,
the ectopic limb development disrupted the development of
the abaxial derivatives, such as sterno-distal rib, sternum,
distal intercostal muscle, abdominal muscles, pectoral mus-
cle and body wall cutaneous muscles. On the other hand,
the primaxial derivatives, such as vertebrae, proximal rib, ver-
tebro-distal rib and proximal intercostal muscles, were not
significantly influenced (Figs. 6 and 8).
Somitic cells that cross the lateral somitic frontier (abax-
ial somitic cells) in the thoracic region include both muscle
(distal intercostal muscle) and cartilage (sterno-distal rib)
precursors (Nowicki et al., 2003). However, grafting experi-
ments of limb somatopleural mesoderm showed that an ec-
topic limb could generate myogenic cell migration of the
thoracic somite (Hayashi and Ozawa, 1995). These cells
were identified as ‘migratory muscle precursors of the hyp-
axial somite’ (Alvares et al., 2003). Our transplantation and
labeling experiment confirmed their results, suggesting that
the migratory somitic cells should be called ‘abaxial muscle
precursors’, because they are somitic cells that migrated
into the lateral plate mesoderm, rather than remaining
primaxial and forming proximal–hypaxial intercostal
muscle.
When the host somatopleural mesoderm was replaced by
the limb somatopleural mesoderm, the permissive inductive
signals for sterno-distal rib formation from the somatopleural
mesoderm (Chevallier, 1975; Sudo et al., 2001) was lost, which
may cause the loss of sterno-distal rib in the thoracic body
wall (Fig. 8). Responding to the ectopic limb development,
the cartilage precursor cells of sterno-distal rib may change
their fate to form the limb muscle, although they might die
at the loss of thoracic somatopleural mesoderm. If the fate
changed, it is a change from the ‘abaxial cartilage’ to the
‘abaxial muscle’.
M E C H A N I S M S O F D E V E L O P M E N T 1 2 6 ( 2 0 0 9 ) 1 9 8 – 2 1 1 209
In summary, our results demonstrated that the ectopic
limb development interferes with the development of the
abaxial somite derivatives, rather than the primaxial deriva-
tives in the thoracic body wall. As has been proposed by Now-
icki and Burke (2000), we also inferred that primaxial somitic
cells have a tendency to keep their own identity and fate,
whereas abaxial somitic cells have a tendency to change in
response to the lateral plate mesoderm.
3.3. Limb-genesis and thoraco-abdominal regionalizationin the course of phylogenesis
The thoracic wall possesses ribs and muscle, which devel-
ops in a segmental fashion and derives from thoracic somites
(somites 19–26; Seno, 1961; Chevallier, 1979). On the contrary,
the abdominal wall does not possess ribs, and the appearance
of the muscles does not show a distinct segmental pattern.
Based on morphological observations (Seno, 1961; Chevallier,
1979), the intercostal muscles undergo a transverse lengthen-
ing in the lateral and ventral directions (belt-like distribution),
whereas, the abdominal muscles not only have a transverse
dorso-ventral extension, but also an extended antero-posteri-
orly (fan-like distribution). Our experiment on regional differ-
entiation of somite fate (Yamaguchi and Aoyama,
unpublished data) also suggested that the abdominal body
wall is not segmented in developmental origin as it appears,
because it is mainly derived from somite 27. Somites 26 and
28 only gave rise to the rostal and caudal periphery of the
abdominal muscles, respectively, and somite 29 exclusively
gave rise to the hind limb muscles. Therefore, most of the
lumbosacral somites form neither the rib nor the body wall
muscle, or in other words, most of the lumbosacral somites
form the hind limb muscles, but not the ventro-lateral body
wall.
These data together with our present findings brought us
to a speculation that the loss of ribs in the abdominal wall
during the course of phylogenesis is a result of the loss of
the abaxial components of body wall induced by the develop-
ment of the lower limb, as well as minimization of the prim-
axial components of body wall. First, sterno-distal ribs and
distal intercostal muscles in the lumbosacral/abdominal re-
gion are lost because of the limb emergence as in our experi-
ment. Then, vertebro-distal part of ribs and their associated
proximal intercostal muscles are lost, although the mecha-
nism is still unknown. The role of the surface ectoderm
may be important, because only the region dependent on
the ectoderm, i.e. distal part of rib (Hirao and Aoyama, 2004;
Aoyama et al., 2005), is lost. Finally, the body wall lacking
muscles is then occupied by migrating somitic cells from
non-limb forming somites immediately cranial to the limb
forming somite to form abdominal muscles. As a result, the
body wall is regionalized into a thoracic body wall that pos-
sesses both muscles and rib, and a lumbosacral/abdominal
body wall that possesses muscles but no rib.
In the cervical region, mammals and birds have no ribs
and no ventro-lateral body wall muscles. Murakami and
Nakamura (1991) showed that cervical somites formed nei-
ther the ribs nor the intercostal muscles after an ectopic
transplantation of the cervical somites into the thoracic re-
gion. In addition, orthotopic transplant of the cervical somite
done by Nowicki et al (2003) showed that the majority of cer-
vical muscles lied in the primaxial domain or there were al-
most no abaxial muscles in the cervical region. The only
abaxial muscle, the cucullaris muscle, originates from the
first and second somites that migrate posteriorly through
the lateral plate (Huang et al., 1997, 2000b). The cervical som-
ites, thus, have no potential to form ventro-lateral body wall
skeletal and muscular elements. In chick, muscles of the wing
and pectoral girdle derive from somites 12–20 (Chevallier,
1979; Burke, 2000). This means that most of the upper limb
muscles derive from caudal cervical somites. The present
study showed that the ectopic wing formation in the thoracic
region also caused the loss of abaxial components of the ribs
and muscles. Here also is an example that somites have an
alternative developmental fate of body wall or limb in their
abaxial derivatives. As well as in the case of the abdominal
body wall, the evolution of upper limb formation might cause
the loss of cervical ribs, although for more rostral cervical re-
gion than the brachial level other mechanisms have to be
considered. For example, the rostral cervical segment might
be formed by duplications of the brachial segments.
4. Experimental procedures
4.1. Embryos
Fertilized eggs of the white Leghorn chicken (Gallus gallus
domesticus) and the Japanese quail (Coturnix coturnix japonica)
were obtained from a local farm and were incubated at
38 �C in a humidified incubator. Embryos were staged accord-
ing to Hamburger and Hamilton (1951).
4.2. Transplantation
Surgery was performed on two-day-old chick embryos (19–
23 somite stage or HH stage 13/14) in ovo (Fig. 2A). To develop
an extra limb in the thoracic region, somatopleural meso-
derm of the prospective leg (28–30th somite level), wing (17–
19th somite level) or thorax (23–25th somite level, as control)
of quail embryos (25–27 somite stage or HH stage 15) were
transplanted between ectoderm and somatopleural meso-
derm of the chick prospective thoracic region (22–25th somite
level). First, the chick embryos were prepared and somites
were counted for staging as described previously (Aoyama
and Asamoto, 1988). The window of the eggshell was sealed
with Sekisui OPP tape and reincubated until grafting. Second,
the quail embryos were removed from the yolk in Tyrode’s
solution and their somites were counted staging. Quail right
somatopleura at the prospective leg, wing or thorax region
were cut out using a surgery scalpel and incubated in
500 IU/mL dispase in Tyrode’s solution supplemented with
10% fecal calf serum (FCS) for 15 min at room temperature.
After that, the somatopleural mesoderm was separated from
other tissues using two tungsten needles in the Tyrode’s solu-
tion and immediately transferred into 10% FCS/Tyrode’s solu-
tion at room temperature. The graft orientation was marked
by making it in a trapezoid form. Finally, the right side of
the host (chick) surface ectoderm at the 20–27th somite level
was separated from underlying somatopleural mesoderm,
and the graft was inserted between ectoderm and somatople-
210 M E C H A N I S M S O F D E V E L O P M E N T 1 2 6 ( 2 0 0 9 ) 1 9 8 – 2 1 1
ural mesoderm keeping its original orientation using a micro
scalpel made from a sewing needle. After the surgery, the egg-
shell window was sealed again and the egg was incubated for
a further six days.
4.3. Skeletal preparation
Six days after the surgery (HH stage 35), embryos were
fixed with Carnoy fixative overnight and washed with 70%
ethanol overnight twice. After evisceration, embryos were
stained with alcian blue 8GX/70% ethanol/1% HCl overnight
(Simons and Van Horn, 1971), dehydrated through an ethanol
series and cleared with methyl salicylate.
4.4. Histology
After skeletal observation, embryos were treated with
absolute ethanol, xylene and embedded in paraffin to pre-
pare 7 lm serially frontal sections. Adjacent sections were
stained in two different ways. They were subjected to immu-
nohistochemistry using two types of quail specific monoclo-
nal antibodies, QCPN (Hybridoma bank) and QCR1 (Aoyama
et al., 1992), followed by alcian blue 8GX/H2O/0.3% HCl and
1% eosin/H2O counter staining. Alternatively, they were
stained with QCPN and anti-desmin (diluted 1:40; Sigma,
St. Louis, MO) and alcian Blue 8GX/H2O/0.3% HCl counter
staining.
For QCPN and QCR1, a horseradish peroxidase conjugated
goat anti-mouse IgG (H + L; diluted 1:300; Bio-Rad, Hercules,
CA) was used as the secondary antibody. 3,3 0-Diaminobenzi-
dine tetrahydrochloride (DAB) was used as a chromogen,
yielding a brown signal. For the muscle specific marker,
anti-desmin, an alkaline phosphatase-conjugated polyclonal
goat anti-rabbit IgG (diluted 1:50; DakoCytomation, Dako,
Denmark) was used as the secondary antibody. Fuchsin
(Dako) was used as a substrate-chromogen to reveal a red
signal.
4.5. Tracing somitic cells
4.5.1. DiI and DiO vital labelingFive microliters of 0.5% 1,1 0-dioctadecyl-3,3,3 0,3 0-tetram-
ethylindocarbocyanine perchlorate (Dil, Molecular Probes,
Carlsbad, CA) in absolute ethanol was diluted to 45 lL of
0.3 M sucrose in warm conditions (45 �C) and vortexed. One
hundred microliters of 0.25% 3,3 0-dioctadecyloxacarbocya-
nine perchlorate (DiO, Molecular Probes) in DMSO was diluted
to 400 lL of 0.3 M sucrose in warm conditions (45 �C), vortexed
and passed through a 0.45 lm Millipore filter to remove dye
aggregates.
Before transplantation, the graft was immersed in DiO
solution for 1 min and washed with 10% FCS/Tyrode’s solu-
tion. After transplantation, chick embryos were reincubated
to reach the 24–25 somite stage (HH stage 15). Then, one,
two or three of the 22nd to 24th somites were injected with
DiI and the embryos were further incubated. Thus, green fluo-
rescence of DiO-labeled transplanted somatopleural meso-
derm, and red fluorescent of DiI (Fig. 7A) labeled the somite.
After incubation for about 24 h, the whole mount embryos
(at HH stage 19–21) were observed under fluorescence stereo-
microscope (Leica MZ FLIII, Heerbrugg, Germany) and were
fixed with 4% paraformaldehyde in phosphate buffer saline
(PBS) overnight. After that, they were washed in PBS, soaked
in 10% sucrose/PBS overnight, soaked in 20% and 30%
sucrose/PBS until they sunk, embedded in embedding med-
ium for frozen tissue specimens, Tissue-Tek OCT compound
(Sakura, Torrance, CA), and frozen with liquid nitrogen
immediately.
After whole mount embryo observation, cryosections of
14 lm thick were performed transversely and the sections
were immediately observed under stereomicroscope (Zeiss
Axioxkop 2, Jena, Germany). Migration was assessed accord-
ing to Hayashi and Ozawa (1995). Sections containing 0–4 cells
migrating out of the somite were scored as (�), those contain-
ing 5–9 cells as (+/�), those containing 10–29 cells as (+) and
those containing more than 29 cells as (++).
4.5.2. QCPN and DAPI counter stainingOne (HH stage 19–21), two (HH stage 23–24), three (HH
stage 26–27) and four days (HH stage 28–29) after the trans-
plantation, embryos were fixed with Carnoy fixative over-
night, dehydrated with 70% ethanol overnight twice, and
then with 90% and absolute ethanol for 1 h each. After stain-
ing with 0.1% light green/ethanol to visualize the specimen in
paraffin block, they were rinsed with absolute ethanol, trea-
ted with xylene and embedded in paraffin to prepare 7 lm
transverse sections.
QCPN immunostaining was done first using procedure as
describe above, followed by counter staining of 0.5 lL/mL
DAPI in PBS. After being washed with PBS, they were immedi-
ately observed under stereomicroscope (Zeiss Axioskop 2,
Jena, Germany). This labeling facilitated detection of scat-
tered chick cells within quail tissue, since chick nuclei fluo-
resced after DAPI stain, whereas quail cells were not
fluorescent after QCPN immunostaining (Nowicki et al.,
2003). The adjacent sections were stained with double immu-
nostaining for QCPN and anti-desmin, followed by alcian blue
counter staining.
Acknowledgments
We thank to Noriko Kato for guiding the transplantation tech-
nique, Nobuyuki Sakamoto for somite-labeling techniques
and Etsuko Suzaki for immunostaining techniques. This re-
search was supported by Grants-in-Aid for Scientific Research
from MEXT Japan and from JSPS.
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