An in vitro model of region-specific rib formation in chick axial skeleton: Intercellular interaction between somite and lateral plate cells

概要

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An in vitro model of region-specific rib formation in chick axial skeleton: intercellular
interaction between somite and lateral plate cells
Kaoru Matsutania, Koji Ikegamia, Hirohiko Aoyamaa,b,*
a

Department of Anatomy and Developmental Biology, Graduate School of Biomedical

Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
b

Present address; Department of Medical Science and Technology, Faculty of Health

Sciences, Hiroshima International University, 555-36 Kurosegakuendai,
Higashihiroshima city, Hiroshima 739-2695, Japan.
*Corresponding author
Department of Medical Science and Technology, Faculty of Health Sciences, Hiroshima
International University, 555-36 Kurosegakuendai, Higashihiroshima city, Hiroshima
739-2695, Japan.; Email: aoyamah@hiroshima-u.ac.jp; Phone No: +81.823.70.4633;
Fax No: +81.823.70.4542

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Abstract
The axial skeleton is divided into different regions based on its morphological
features.
region.

In particular, in birds and mammals, ribs are present only in the thoracic
The axial skeleton is derived from a series of somites.

In the thoracic region

of the axial skeleton, descendants of somites coherently penetrate into the somatic
mesoderm to form ribs.

In regions other than the thoracic, descendants of somites do

not penetrate the somatic lateral plate mesoderm.

We performed live-cell time-lapse

imaging to investigate the difference in the migration of a somite cell after contact with
the somatic lateral plate mesoderm obtained from different regions of anterior–posterior
axis in vitro on cytophilic narrow paths.

We found that a thoracic somite cell

continues to migrate after contact with the thoracic somatic lateral plate mesoderm,
whereas it ceases migration after contact with the lumbar somatic lateral plate
mesoderm.

This suggests that cell–cell interaction works as an important guidance cue

that regulates migration of somite cells.

We surmise that the thoracic somatic lateral

plate mesoderm exhibits region-specific competence to allow penetration of somite cells,
whereas the lumbosacral somatic lateral plate mesoderm repels somite cells by contact
inhibition of locomotion.

The differences in the behavior of the somatic lateral plate

mesoderm toward somite cells may confirm the distinction between different regions of
the axial skeleton.
Keywords
Chick, Somite, Lateral plate, Intercellular interaction, Axial skeleton,
Morphogenesis

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1. Introduction
Cell migration is considered one of the fundamental processes in embryo
morphogenesis (Le Douarin, 1984; Reig et al., 2014).

Cell migration behavior can

change by some signals, and depending on the source of the signal and the way it is
received, signaling mechanisms are classified into chemotaxis, haptotaxis, contact
guidance, contact inhibition of locomotion (CIL), and cell–cell adhesion (Jotereau and
Le Douarin, 1982; Hernandez-Fleming et al., 2017; Carter, 1965; de la Loza et al.,
2017; Laumonnerie et al., 2015; Kridsada et al., 2018; Abercrombie and Heaysman,
1954; Carmona-Fontaine et al., 2008; Villar-Cervino et al., 2013; Davis et al., 2015;
Takeichi, 2014; Mayor and Etienne-Manneville, 2016; Scarpa and Mayor, 2016; Cousin,
2017).

Migratory behavior of cells may be regulated by combinations of these

guidance cues.
The axial skeleton of the trunk comprises a series of metamerically arranged
vertebrae.

The rib is such a characteristic thoracic structure that distinguishes the

thorax from other regions of the axial skeleton in birds and mammals.
skeleton is derived from somites.

The axial

Although thoracic somites have the potency to form

ribs (Kieny et al., 1972), its exertion depends on the neighboring tissues around somites.
In birds, a rib is composed of three compartments, viz., proximal rib, vertebro-distal rib,
and sterno-distal rib, according to developmental dependencies of the adjacent tissues
(Aoyama et al., 2005).

The proximal rib is derived from a caudal half of somites under

the influence of the ventral neural tube and notochord; the distal rib is derived from a
caudal and rostral half of two adjacent somites (and ventrolateral lip of myotome or its
periphery) under the influence of the surface ectoderm.

Furthermore, the sterno-distal

rib develops in relation to penetration into the somatic lateral plate mesoderm (LP)
(Kato and Aoyama, 1998; Nowicki and Burke, 2000; Aoyama and Asamoto, 2000;
Huang et al., 2000; Sudo et al., 2001; Aoyama et al., 2005).
It has been reported that interaction between thoracic somites and thoracic LP is
important for the development of the sterno-distal rib.

When the thoracic segmental

plate is ectopically transplanted into the cervical region, the ribs derived from the
explants are shorter than normal ribs (Kieny et al., 1972; Shearman and Burke, 2009).
A blockage between thoracic somites and LP causes the absence of sterno-distal ribs

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(Sudo et al., 2001).

Liem and Aoyama (2009) reported that when LP of the limb-

forming region is transplanted into the thoracic region, sterno-distal ribs are truncated or
absent with an ectopically formed wing or leg (Liem and Aoyama, 2009).

However,

when the thoracic segmental plate is transplanted into the lumbosacral region, the
grafted mesoderm forms ectopic ribs that have been much shorter than normal ribs (our
unpublished data).
Here, we established an in vitro co-culture system to investigate somite cell
behavior interacting with LP cells.

We found that thoracic somite cells change their

direction of migration after contact with lumbar LP (LP-lu) cells but not after contact
with thoracic LP (LP-th) cells.
2. Results
2.1. Co-culture experiment on a two-dimensional (2D) substrate: behavior of thoracic
somite cells on contact with LP-th or LP-lu cells
The isolated tissues were co-cultured in a liquid culture medium in a conventional
glass base dish.

The co-cultured tissues were as follows: thoracic somites (24th–25th

somites) with LP-th (LP at 23rd–25th somite level) (Fig. 1A) and thoracic somites (21st–
22nd somites) with LP-lu (LP at 30th–31st somite level) (Fig. 1B).

Position of the

nucleus in a time-lapse image was recorded every 10 min, and the direction of migration
was evaluated.

The direction of migration was compared between the tissues for 100

min before and after contact with a co-cultured cell (Fig. 1C and D).
We tested the null hypothesis that the direction of thoracic somite cell migration is
unchanged before and after contact.

The null hypothesis was rejected with two

combinations, thoracic somites with LP-th and with LP-lu. There were significant
differences between the direction of migration before and after contact (Fig. 1C and D).
However, changes in the direction of migration were different among the co-culture
combinations when comparing two components, i.e., a component leaving from the
explant to the opposite explant and a component returning to the explant from the
opposite explant, along a line between the explants.

Before contact, 85.5% of thoracic

somite cells migrated forward, and after contact with LP-th, 60.6% of somite cells
migrated forward (Fig. 1C).

However, before contact with LP-lu, 71.2% of thoracic

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somite cells migrated forward, and after contact, 53.1% of somite cells migrated
backward (Fig. 1D).
Quantitative analysis was difficult in this assay because the cells migrated in
various directions with changing migration speed.

Moreover, a cell may successively

or simultaneously contact several cells.
Therefore, we performed another assay on a one-dimensional (1D) substrate
wherein the cells migrated along narrow paths (Fig. 2A).

This system allowed us to

analyze the migratory behavior of a cell making contact with another cell in terms of
only their speed.
2.2. Co-culture experiment on a 1D substrate: thoracic or lumbar somite derivatives
with LP-th or LP-lu cells
We co-cultured the somite mesoderm with LP on CytoGraph in which the
cytophilic area is restricted (10-µm wide paths) (Fig. 2A).

The cells migrated away

from the explants on the cytophilic paths and made contact with the cell migrating from
the opposite side after 12–18 h (Fig. 2B and C).

The cell had to migrate either forward

or backward because it was confined to move on a cytophilic path.

The velocity of a

cell represents both the direction and speed of cell migration, i.e., positive and negative
velocity values indicate that the cell migrated forward and backward, respectively.
observed various patterns of migration.

We

For example, a cell continuously migrated

forward by pushing aside other cells, a cell changed its direction of migration to
backward, and a cell almost stopped migrating on contact.

To determine the effect of

intercellular interaction between somite and LP cells on contact, we tracked the points
of leading and trailing edges of the cells (Fig. 2D) using consecutive images and
calculated the migration velocity (µm/min).

In Fig. 3, each line represents a cell

whose leading and trailing edges (Fig. 2D) were tracked; the lines show migration 60
min before and after contact with co-cultured cells.

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2.3. Behavior of thoracic sclerotome (SC-th) cells co-cultured with LP-th or LP-lu cells
Before contact, there was no significant difference in the migration velocity of
SC-th and dermomyotome (DM) cells.

When SC-th cells were co-cultured with LP-th

or LP-lu cells, their average migrating velocity at the leading edge was 1.36 ± 0.23
µm/min (n = 11) and 1.56 ± 0.37 µm/min (n = 11), respectively (Fig. 4A).

However,

after contact with LP-th or LP-lu cells, the velocity decreased to 0.46 ± 0.15 µm/min (n
= 11) and −0.55 ± 0.23 µm/min (n = 11), respectively, wherein the negative value
represents backward migration (Fig. 4A).

Although contact with LP-th cells decreased

the velocity of SC-th cell migration by 34%, SC-th cell continuously migrated forward.
However, contact with LP-lu cells altered SC-th cell movement to the opposite direction.
Cells on the CytoGraph did not necessarily migrate at a constant velocity, and the
velocity varied from cell to cell.

Figs. 4B and C show the relative frequency of cells

(percentage) migrating at a certain velocity.
of the leading edge.

Here, the velocity was represented as that

Velocity data were classified into eight classes at intervals of 1

µm/min (Fig. 4B and C).

Bar graph on the right side represents forward movement

and that on the left side represents backward movement.
Based on these data, we analyzed the migratory behavior of somite cells before
and after contact with LP cells.
Most SC-th cells (66.7%) continuously migrated forward after contact with LP-th
cells, although their speed became relatively low (Fig. 4B).

Thirty minutes before

contact, 24.1% SC-th cells migrated at the speed of 0–1 µm/min, whereas the
percentage of cells increased to 48.5% after contact (Fig. 4B).

In contrast, most SC-th

cells (66.7%) migrated backward after contact with LP-lu cells (Fig. 4C).

Moreover,

more than 10% of the cells migrated backward at a velocity of more than 3 µm/min 30–
60 min after contact (Fig. 4C).

Thus, after contacting LP cells, the direction of

migration of SC-th cells depended on the rostro-caudal level of LP cells.

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2.4. Behavior of LP-th and LP-lu cells co-cultured with SC-th
Most LP-th cells migrated forward (88.7%) at a high speed (average velocity of
1.58 ± 0.39 µm/min; n = 11) before contact with SC-th cells (Fig. 4D and E).
and F show the proportions of cells migrating at different velocities.
velocity of a cell, the movement of its leading edge was measured.

Figs. 4E

To calculate the
Velocity data were

classified into eight classes, with intervals of 1 µm/min (Fig. 4E and F).

Within 30

min after contact, although LP-th cells still predominantly migrated forward (60.6%;
Fig. 4E), more than 30% of LP-th cells changed their direction of migration.
tendency was more conspicuous 30–60 min after contact.

This

However, the velocity of

LP-lu cells did not significantly change before and after contact with SC-th cells (Fig.
4D and F).
2.5. Behavior of thoracic DM (DM-th) cells co-cultured with LP-th and LP-lu cells
Most DM-th cells migrated forward 30 min before contact with LP-th cells
(78.8%; average velocity of 1.02 ± 0.35 µm/min; n = 11), whereas most DM-th cells
migrated in the opposite direction after contact (75.8%; average velocity of −0.56 ±
0.25 µm/min; n = 11) (Fig. 5A and B).

We observed that LP-th cells with their leading

edge in contact with the trailing edge of DM-th cells elongated backward in six of 11
cases (Fig. 3C).

After contact with LP-th cells, DM-th cells exhibited stagnation in its

leading edge and extension of the trailing edge, suggesting that these cells were to
migrate backward but were unable to move due to cell–cell adhesion.
Within 30 min before contact with LP-lu cells, more than 70% of DM-th cells
migrated forward (average velocity of 1.37 ± 0.40 µm/min; n = 11), whereas within 30
min after contact, approximately 50% of the cells migrated backward (average velocity
of −0.59 ± 0.33 µm/min; n = 11) (Fig. 5A and C).

In particular, more than 20% of

cells migrated backward at a velocity of more than 2 µm/min.

On contact with LP-lu

cells, DM-th cells exhibited a sudden regression in the leading edge and backward
extension of the trailing edge in five of 11 cases (Fig. 3D).

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2.6. Behavior of LP-th and LP-lu cells co-cultured with DM-th cells
Before contact with DM-th cells, LP-th cells migrated forward at a high speed
(average velocity of 1.91 ± 0.26 µm/min; n = 11), whereas their average velocity
decreased significantly to 0.43 ± 0.25 µm/min (n = 11) after contact (Fig. 5D). This
was primarily because of the decrease in cell population migrating at a velocity of more
than 3 µm/min and increase in cells migrating backward (Fig. 5E). Nevertheless, LPth cells predominantly and continuously migrated forward after contact with DM-th
cells.
LP-lu cells migrated forward at a high speed (average velocity of 1.20 ± 0.29
µm/min; n = 11) before contact with DM-th cells (Fig. 5D).

After contact with DM-th

cells, the average velocity of LP-lu cell leading edges was −0.03 ± 0.15 µm/min (n =
11) (Fig. 5D).

These cells did not cease to migrate, but approximately half of the cells

began to migrate backward (Fig. 5F).

Leading edges migrating forward at a velocity

of more than 2 µm/min decreased, whereas leading edges of cells migrating backward at
low speed (less than 1 µm/min) increased on contact with DM-th cells (Fig. 5F).
2.7. Behavior of lumbar DM (DM-lu) cells co-cultured with LP-lu cells
DM-lu cells migrated forward at a high speed (average velocity of 1.46 ± 0.29
µm/min; n = 11) before contact with LP-lu cells (Fig. 6A).

Thirty minutes before

contact with LP-lu cells, more than half of DM-lu cells migrated forward at a velocity
of more than 2 µm/min, whereas approximately 50% of cells migrated backward after
contact (Fig. 6B).

Only in the combination of this co-culture, the leading edges of

DM-lu cells migrated only slightly after contact (average velocity of 0.14 ± 0.20
µm/min; n = 11) (Fig. 3E, 6B).
2.8. Behavior of LP-lu cells co-cultured with DM-lu cells
LP-lu cells migrated forward (average velocity of 0.49 ± 0.23 µm/min; n = 11)
before contact with DM-lu cells (Fig. 6C).

Approximately 70% of LP-lu cells

migrated forward within 30 min before contact (Fig. 6D).

After contact with DM-lu

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cells, approximately 70% of LP-lu cells migrated backward, which decreased to
approximately 40% at 30 min after contact (Fig. 6D).

LP-lu cells migrated backward,

but on contact with another LP cell behind it, the cells changed the direction of
migration again in seven of 11 cases.
2.9. Behavior of SC-lu cells
We tried to perform experiments using SC-lu cells.

However, SC-lu cells hardly

migrated to make contact with other cells (data not shown).
2.10. Alteration in somite cell size before and after contact with LP cells
Fig. 7 shows the cell length between the leading and trailing edges of cells after
contact compared to that before contact in each examination.

After contact with LP-lu

cells, the length of both SC-th and DM-th cells reduced remarkably.

On contact with

LP-th cells, the length of SC-th and DM-th cells did not virtually change before and
after contact.

The length of DM-lu cells also did not change on contact with LP-lu

cells.
2.11. Grouping velocity distribution patterns by hierarchical clustering analysis
As shown in Figs. 4–6, somite and LP cells migrated at varying velocity
distribution patterns based on the combination of their co-culture (Fig. 4B, C, E, F; 5B,
C, E, F; 6B, D).

Clustering analysis revealed that velocity distribution patterns were

divided into three groups (Fig. 8A), and distinctive features were observed in all
velocity distribution patterns (Fig. 8B).

CL1 (cluster one) was characterized by

forward migration at more than 1 µm/min.
velocity.

In CL2, the cells migrated forward at low

In CL3, the proportion of backward migration was high.

SC and DM cells predominantly migrated forward before contact (Fig. 8C, 2nd
line; CL1).

The direction of migration of somite cells changed to backward after

contact with LP cells (Fig. 8C, 3rd, 5th, 7th, and 9th columns; CL3), except for SC-th cells

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that continuously migrated forward (CL2) after contact with LP-th cells (Fig. 8C, 1st
column).
Although migration of DM-th cells after contact with LP-lu cells was grouped
under CL2 (Fig. 8C, 7th column), 16.7% of DM-th cells migrated backward at a velocity
of more than 3 µm/min (Fig. 4C), indicating that a considerable number of DM-th cells
migrated backward, unlike other cells grouped in CL2 (Fig. 8A, heatmap).
On contact with SC-th cells, LP-th cells changed the direction of migration (Fig.
8C, 2nd column).

Before contact, LP-th cells migrated forward at a high velocity

(CL1), but the cells migrated backward after contact (CL3).

On the contrary, LP-lu

cells always migrated forward (CL1 and CL2) before and after contact with SC-th cells
(Fig. 8C, 4th column).

After contact with DM-lu cells, LP-lu cells transiently changed

the direction of migration to backward (CL3) and then again to forward at low velocity
(CL2) (Fig. 8C, 10th column).
3. Discussion
A major portion of the axial skeleton of vertebrates is derived from somites.
They form various bones along the rostro-caudal axis, from the occipital bone to the
coccygeal bone.

Of these somites, thoracic somites particularly form long ribs in

addition to the thoracic vertebrae.

As shown by Kieny et al. (1972), although thoracic

somites specifically form the ribs, their length depends on the neighboring conditions
(Kieny et al., 1972).

When the thoracic segmental plate is ectopically transplanted into

the cervical region, the transplants form vertebral ribs but no sternal ribs.
For rib formation, thoracic somite cells penetrate into the somatic lateral plate
mesoderm.

When the thoracic somite mesoderm is transplanted into the lumbar

region, it does not penetrate into the lumbar somatic mesoderm and forms short ribs
(Matsumori et al., unpublished data).

We hypothesized that the intercellular

interaction between somite and LP cells could control cell behavior to form the axial
skeleton.
Here, we present the migratory behavior of somite and somatic LP cells in an
in vitro co-culture system and showed that SC-th cells tended to continuously migrate
forward after contact with LP-th cells, whereas they migrated in the reverse direction

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after contact with LP-lu cells.
Embryonic cells isolated in vitro may change their developmental fate, which
would misinterpret our findings.
its surrounding tissues.

In fact, the epithelial somite develops depending on

The surface ectoderm induces the DM and notochord, and the

floor plate induces the SC.

Even if each somitic primordium is combined with an

ectopic tissue, it develops according to the tissue in contact (Aoyama and Asamoto,
1988; Ordahl and Le Douarin, 1992; Christ et al., 1992; Pourquié et al., 1993; Aoyama,
1993; Christ and Ordahl, 1995; Williams and Ordahl, 1997; Dockter and Ordahl, 2000;
Monsoro-Burq, 2005).

However, as we have shown, the caudal third somite (stage III

somite) begins to differentiate and at least some part of it cannot change its fate after
being rotated dorsoventrally (Aoyama and Asamoto, 1988).

The presumptive

dermomyotomal region and a part of the presumptive sclerotomal region of the somite
differentiate along their original fate under heterotopic circumstances.

Furthermore,

except for stage I–III somites, isolated somites differentiate into cartilage tissue and
muscle fibers without induction of other tissues in vitro (Ellison et al., 1969a, 1969b;
Kenny-Mobbs and Thorogood, 1987; Buffinger and Stockdale, 1994; Stern and
Hauschka, 1995).

We did not use stage I–III somites in the present study.

Thus, the

somite cells that we investigated in vitro were considered to retain their fate and
represent their behavior in vivo.
In the present study, we primarily used the 1D migratory system instead of the
2D system because of simplicity of analysis of cell migratory behavior.

It has been

reported that there is no considerable difference in neural crest cell behavior and
migratory abilities between the 1D and 2D systems, although speed and directionality
were slightly less in the 1D system than those in the 2D system (Scarpa et al., 2013,
2016).

The velocity of SC-th cells co-cultured with LP-th cells was 1.36 ± 0.23

µm/min (n = 11) (Fig. 4A), of DM-th cells co-cultured with LP-th cells was 0.94 ± 0.32
µm/min (n = 11) (Fig. 5A), and of LP-th cells co-cultured with SC-th cells was 1.58 ±
0.39 µm/min (n = 11) (Fig. 4D).

Our results almost agree with the velocities of SC

(1.095 µm/min), DM (1.085 µm/min), and LP (1.925 µm/min) cells reported by Bellairs
et al. (1980) at the interval of 12 s (Bellairs et al., 1980).
We found that SC cells changed the direction of migration just after contact
with LP-lu cells, suggesting that the effect of intercellular interaction is not based on

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long-range mechanisms, such as chemotaxis, haptotaxis, or contact guidance, but on
cell–cell contact.

After contact with LP-th cells, SC-th cells, which form the rib

anlagen, continued migrating in the same direction as that before contact (Fig. 8C, 1st
column).

However, SC-th cells migrated in the opposite direction after contact with

LP-lu cells (Fig. 8C, 3rd column). Abercrombie and Heaysman (1954) reported that
contact inhibition results in restriction of the velocity of cells and changes the direction
of cell migration (Abercrombie and Heaysman, 1954).
that cell–cell contact affects the behavior of cells.

A series of studies has reported

Eph family ligands guide the

migration of neural crest cells and motor axon from the neural tube (Wang and
Anderson, 1997).

Drosophila macrophages undergo contact repulsion and disperse

laterally to form a “three-lined” organization pattern from a linear cellular array at the
ventral midline (Stramer et al., 2010; Davis et al., 2012; Davis et al., 2015).

Neural

crest cells migrate in an N-cadherin/CIL-dependent mechanism (Erickson, 1985;
Carmona-Fontaine et al., 2008; Theveneau et al., 2010; Scarpa et al., 2015).

In these

studies, the behaviors of cells were analyzed in vitro and were confirmed in vivo.
To form the ribs, SC-th cells start to migrate ventrolaterally while
proliferating, and then penetrate into the somatopleure.

When SC-th cells contact LP-

th cells, they continued to migrate forward at a velocity of 0.46 ± 0.15 µm/min (n = 11;
Fig. 4A).

After migrating for 7.5 days at this velocity, the migrated distance would be

4.97 mm, which is almost the same length as the 4th vertebral rib, 4.95 ± 0.18 mm (n =
10) at HH-stage 36 (approximately 10 days after incubation).

This finding suggests

that cell migration speed is a significant factor to determine the length of a rib, which
may alternatively be affected by cell proliferation rate.
However, SC-th cells reversed the direction of migration after contact with
LP-lu cells (Fig. 8C, 3rd column). At 0.55 ± 0.23 µm/min (n = 11), SC-th cells
migrated away from LP-lu cells after contact (Fig. 4A).
represent the somite cell behavior in vivo.

This phenomenon may

Ectopic transplantation of leg somatopleural

mesoderm in the thoracic region alters the fate of somite descendant cells and causes the
loss of sterno-distal ribs (Liem and Aoyama, 2009).

Furthermore, we found that

thoracic somite cells did not enter into the tissue derived from the lumbar lateral plate
when the thoracic somite mesoderm was ectopically transplanted in the lumbar region
(Matsumori et al., unpublished data).

In normal development, SC-th cells can contact

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LP-lu cells only at the thoraco–lumbar boundary.

This repulsive nature of intercellular

interaction observed in the present study would help to avoid malformation caused by
penetration of rib-forming thoracic somite cells into the lumbar lateral plate by accident.
Burke and Nowicki (2003) proposed that the body wall should be classified
into two categories, viz., primaxial and abaxial regions, according to the relationship
between the somite and lateral plate mesoderm in the course of development (Burke and
Nowicki, 2003).

The domain comprising only somite cells is defined as the primaxial

region, and the domain of the lateral plate with somite and lateral plate cells is defined
as the abaxial region.

They named the interface between the primaxial and the abaxial

region as the lateral somitic frontier.

At the thoracic level, the cluster of somite cell

population elongates laterally into the lateral plate mesoderm to form the somitic bud
(Christ et al., 1983).

Furthermore, some somite cells migrate across the lateral somitic

frontier and mix with LP cells (Nowicki et al., 2003). These somite cells constitute the
abaxial region.

We have previously reported that the rib in birds is composed of three

compartments according to developmental dependencies on adjacent tissues (Aoyama et
al., 2005).

The proximal and vertebro-distal ribs correspond to the primaxial region,

whereas the sterno-distal rib corresponds to the abaxial region (Burke and Nowicki,
2003).

In the present study, although we could not distinguish primaxial and abaxial

cells in vitro, both categories of cells have such common nature that they penetrate the
lateral plate mesoderm at the thoracic level but not at the lumbar level.

Consequently,

in either case, our findings are consistent with the thoracic somite behavior in vivo.
We expected that DM-th cells would continuously migrate forward after
contact with LP-th cells and migrate backward after contact with LP-lu cells, similar to
that observed for SC-th cells.

However, in our in vitro system, the migratory behavior

of DM cells, which include the muscle anlagen, was different from that of SC-th cells,
which include the rib anlagen.

After contact with either LP-th or LP-lu cells, DM cells

reversed the direction of migration (Fig. 8C, 5th, 7th, and 9th column).
In vivo, DM-th cells migrate adhering to each other to form intercostal
muscles, whereas DM-lu cells migrate individually to form the limb muscles
(Chevallier, 1979; Jacob et al., 1979).
analyzed in vitro.

In the present study, the cells were individually

DM-th cells may be able to invade the tissue that repels because

they migrate as a mass.

Rovasio et al. (1983) showed that neural crest cells exhibited

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oriented migration when they were cultured at high density, whereas they migrated
randomly at a low density (Rovasio et al., 1983).

Although DM is an epithelial tissue,

a part of the somite undergoes epithelial–mesenchymal transition to form SC (Chal and
Pourquié, 2017).

After migration, SC-derived cells aggregate and finally form the

cartilage, suggesting that SC cells migrate individually in vivo.

The migratory

behavior of SC-derived cells in vitro shown in this study may reflect the pattern
observed in vivo.
This study showed that the behaviors of rib-forming SC-th cells were
significantly different depending on whether the cells made contact with LP-th or LP-lu
cells.

SC-th cells migrated backward after contact with LP-lu cells.

This

phenomenon was considered to be caused by contact of thoracic somite cells with LP-lu
cells.

In normal development, chicken ribs grow laterally/ventrally, and then the

sterno-distal ribs form pointing cranially but never enter the caudal–lumbar region.
This may be explained by the repulsive intercellular interaction, which prevents SC-th
cells from entering the lumbar somatic lateral plate.

Thus, the cell–cell interaction

presented here would assure that the rib forms only in the thoracic region.
4. Experimental procedures
4.1. Preparation of embryos
Fertilized eggs of White Leghorn chick were purchased from a local farm and
incubated at 38°C in a humidified incubator.
4.2. Isolation of explants from embryos
In the present study, “thoracic” indicated the 22nd–25th somite level, including the
3rd–6th pairs of rib anlagen, and possessed both vertebral and sternal ribs.

“Lumbar”

indicated the 29th–31st somite level, where somites form the 3rd–6th lumbosacral
vertebrae.

A part of the embryo with the desired embryonic tissues was dissected and

treated with 500 IU/mL dispase (Godo Shusei, Japan) in culture medium at 37°C for
15–30 min, followed by isolation of each tissue using sharpened tungsten needles in

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Tyrode’s saline.

For culturing the cells, we used a 1:1 mix of of Dulbecco’s modified

Eagle’s medium (DMEM; Sigma-Aldrich) and Ham’s F-10 (F10; Sigma-Aldrich),
supplemented with 10% fetal calf serum (FCS) and 100 µg/mL kanamycin sulfate.
piece of SC was separated from DM.

A

Although DM was separated carefully from SC,

some SC cells may remain in the excised DM.

Because SC is a mesenchymal tissue, it

was difficult to remove it without destruction.

Lateral half of DM was obtained by

sagittally cutting DM in the middle.
of somatic and splanchnic LP.

To isolate the somatic LP, it was cut at the border

SC-th and thoracic lumbar thoracic somatic lateral

plate (LP-th) were dissected from HH-stage (Hamburger and Hamilton 1951) 17–18
(29–37 somite stage) chick embryo.

DM-th and LP-lu were dissected from HH-stage

17–20 (29–41 somite stage) chick embryo.
4.3. Fluorescence staining
To trace cells in vitro, each explant was labeled with DiI or DiO (Invitrogen).
The stock dye solutions (0.05%) were added to the culture medium at a final
concentration of 0.01%.

The culture medium consisted of DMEM:F10 = 1:1

supplemented with 10% FCS and 100 µg/mL kanamycin sulfate. The isolated tissues
were incubated in the dye solution at 37°C in 5% CO2/air for 20 min, followed by
rinsing five times with Tyrode’s saline to remove unconjugated dye.
4.4. Co-culture experiment on a 2D substrate
Explants of somites and its derivatives were co-cultured with explants of LP in a
glass-base dish (3911-035 Iwaki, Japan) with the culture medium at 37°C under 5%
CO2/air. A pair of explants was placed with a spacing of 1 mm between the explants.
The explants settled and proliferated on the substrate.

Cells migrated away from the

explants and made contact with each other halfway between the explants.
4.5. Co-culture experiment on a 1D substrate

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The explants were co-cultured in a semisolid medium on CytoGraph (DNP,
Japan) type L10S300 with 10-µm wide hydrophilic paths intervening with 300-µm wide
hydrophobic areas.

The semisolid medium was composed of DMEM:F10 = 1:1, 15%

FCS, 100 µg/mL kanamycin sulfate, and 1.7% methylcellulose 4000 (Chameleon
Reagent).

The hydrophobic areas were constructed by coating with

tetraethyleneglycol layer, and hydrophilic paths were uncovered glass surface (Okochi
et al. 2009).

The substrate was treated with 3% bovine serum albumin (Sigma-

Aldrich) for blocking excess cell adhesion before culture.
4.6. Time-lapse imaging
The cultured cells were observed under a phase-contrast microscope (IX71,
Olympus, Japan) with a 4× or 10× objective and photographed every 1 min for 48 h
with a camera (Eos Kiss X6i, Canon, Japan) controlled by EOS Utility (Canon, Japan).
4.7. Cell tracking
We analyzed cell migration using consecutive photographs captured every 10 min
for 60 min before and after contact between the co-culture cells.

The cultured cells

migrated away from the explants; co-cultured cells approached each other, and finally
made contact. In this study, we named the preceding tip of the migrating cell as the
leading edge and the rear tip as the trailing edge.

These names were defined according

to the initial direction of cell migration and we did not rename when the cells changed
their direction of migration.

On consecutive photographs, we manually marked the

leading and trailing edges (Fig. 2D), and their positions were recorded in X–Y
coordinates of pixels using ImageJ Multi-point Analyze Tool (NIH, USA).

Then, we

calculated the migration velocity (µm/min) from the distance between the marked
points using Numbers (Apple) and Excel (Microsoft).
4.8. Statistical analysis
Statistical analysis was performed using R (R Development Core Team,

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Vienna, Austria). The migration velocity was compared by Student’s t-test (P > 0.05,
by F test) or the Wilcoxon rank sum test (P ≤ 0.05, by F test).

Comparison of the

direction of migration was performed using the Mardia–Watson–Wheeler test for
homogeneity using R.

We divided each 60-min period before and after contact into

two periods of 30 min each.

Velocity distribution patterns in each period were

grouped by hierarchical clustering analysis using the Ward’s method. The Ward’s
method is an agglomerative method based on a sum of squares criterion (Murtagh and
Legendre, 2014).

Observations in each cluster were agglomerated to minimize the

extra sum of squares at each clustering step.

Distance obtained by criterion of the

Ward’s method represented the height of dendrograms.
Acknowledgments
A part of this work was supported by JSPS KAKENHI Grant Numbers
JP26460254, JP23590219.
Conflicts of interest
The authors declare no conflict of interest.
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Figure captions
Fig. 1

Behavior of somite cell before and after contacting lateral plate cells on a

conventional two-dimensional substrate.

(A and B) Phase-contrast consecutive images.

(A) Thoracic somite cells (white arrowhead) did not change the direction of migration
for several minutes after contact with LP-th cells.

(B) Thoracic somite cells (blue

arrow) changed the direction of migration immediately after contact with LP-lu cells.
(C and D) Diagrams show the direction of somite cell migration.

The direction of

somite cell migration was defined by an angle to the reference line from the somite to
the lateral plate explants.
degree.

The direction to the lateral plate explant is indicated as zero

The cells were tracked manually.

written by Karsten D Bjerre (2002).

Polar plot was graphed using the code

The direction of somite cell migration for 100

min before and after first contact with lateral plate cells was compared by the Mardia–
Watson–Wheeler test for homogeneity using R.

(C) Thoracic somites were co-

cultured with LP-th cells, and 13 of them were recorded before they made contact.
Before contact, most thoracic somite cells migrated toward the thoracic lateral plate
explant.

After contact, 4 of the 13 cells became obscure, although somite cells more or

less changed the direction of migration, most cells appeared to migrate along nearly the
same direction as that before contact.
lu cells.

(D) Thoracic somites were co-cultured with LP-

After contact, 3 of the 12 cells became obscure, most somite cells appeared to

change the direction of migration.
made contact with LP cells.

They migrated toward LP-lu explant before they

Abbreviations: LP, somatic lateral plate; -th, thoracic; -lu,

lumbar.
Fig. 2

Cell migration on a one-dimensional substrate, the CytoGraph.

(A) SC and

LP fragments were isolated from chick embryos and explanted on the opposite side of
the cytophilic pathways [upper three pathways in (A) indicated with blue lines], which
were 10-µm wide with 300-µm cytophobic intervals.

SC (magenta arrow) and LP

cells (green arrow) migrated away from the explants and continued to migrate along the
cytophilic pathways.

(B and C) Consecutive photos of SC cells and LP cells migrating

from the opposite ends of the cytophilic pathways.

Photos were captured every 10 min

for 60 minutes before (negative values) and after (positive values) cells made contact

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with each other.

(B) SC-th and LP-th cells were co-cultured.

migration after contact with LP-th cells.

SC-th cells stopped

(C) SC-th and LP-lu cells were co-cultured.

SC-th cells migrated in the reverse direction after contact with LP-lu cells.
Definition of the leading and the trailing edge in the migrating cell.

(D)

Abbreviations: SC,

sclerotome; LP, somatic lateral plate; -th, thoracic; -lu, lumbar.
Fig. 3

Position of two co-cultured cells migrating from the opposite side on a one-

dimensional substrate.

Each line represents a cell with leading and trailing edges (x-

axis), which were determined according to the contact point.
represent the same cell in each figure.

(A) SC-th and LP-th cells were co-cultured five

times (N = 5), and 11 pairs of cells were recorded (n = 11).
(N = 5; n = 11).

Lines of the same color
(B) SC-th and LP-lu cells

(C) DM-th and LP-th cells (N = 3; n = 11).

cells (N = 5; n = 11).

(D) DM-th and LP-lu

(E) DM-lu and LP-lu cells (N = 5; n = 11).

We eliminated the

data from cells when their trailing edge was too obscure to determine.

Abbreviations:

SC, sclerotome; LP, somatic lateral plate; DM, dermomyotome; -th, thoracic; -lu,
lumbar.
Fig. 4

Change in the migration velocity of SC-th and LP-th or LP-lu cells before and

after contact with each other on the one-dimensional substrate.

Velocity was

represented as that of the leading edge of migrating cells and calculated every 10 min.
(A and D) Changes in average velocity of the cell 60 min before and after contact with
its counterpart.

(B, C, E, F) Horizontal stacked bar charts represent the percentage of

cumulative cell numbers migrating at the velocity indicated above the chart. (A) SC-th
cells continued to migrate at a lower velocity after contact with LP-th cells (n = 11) but
changed the direction of migration after contact with LP-lu cells (n = 11). (B) Forward
migrating SC-th cells decreased slightly to approximately 60% after contact with LP-th
cells.

(C) After contact with LP-lu cells, backward migration of SC-th cells increased

notably. (D) LP-th cells changed the direction of migration after contact with SC-th
cells, whereas LP-lu cells continued to migrate after contact with SC-th cells.

(E)

After contact with SC-th cells, backward migration of LP-th cells increased notably.
(F) Forward migration of LP-lu cells decreased slightly after contact with SC-th cells.
Abbreviations: SC, sclerotome; LP, somatic lateral plate; -th, thoracic; -lu, lumbar; n.s.,

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no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001.

Data are represented

as means ± SE.
Fig. 5

Change in migration velocity of DM-th and LP-th or LP-lu cells before and

after contact with each other on the one-dimensional substrate.

The velocity was

represented as that of the leading edge of migrating cells and was calculated every 10
min.

(A and D) Changes in average velocity of the cell 60 min before and after

contact with its counterpart.

(B, C, E, F) Horizontal stacked bar charts represent the

percentage of cumulative cell numbers migrating at the velocity indicated above the
chart. (A) DM-th cells changed the direction of migration after contact with LP-th
cells (n = 11), whereas the velocity of migration appeared to be very low after contact
with LP-lu cells (n = 10).

(B) Most DM-th cells changed the migration direction to

backward after contact with LP-th cells.

(C) More than half DM-th cells continued

forward migration, whereas a certain number of cells began backward migration at a
relatively high speed after contact with LP-lu cells. (D) LP-th cells continued to
migrate at a lower velocity after contact with DM-th cells, whereas LP-lu cells appeared
to cease migration after contact with DM-th cells.

(E) After contact with DM-th cells,

forward migration of LP-th cells decreased gradually.
continued to migrate forward (0–30 min).
migrate backward.

Just after contact, they

After 30–60 min, some cells began to

(F) Forward migration of LP-lu cells decreased to approximately

50% after contact with DM-th cells, which resulted in very low average velocity as
shown in D.

Abbreviations: DM, dermomyotome; LP, somatic lateral plate; -th,

thoracic; -lu, lumbar; n.s., no significant difference; *P < 0.05; **P < 0.01; ***P <
0.001.

Data are represented as means ± SE.

Fig. 6

Change in migration velocity of DM-lu and LP-lu cells before and after contact

with each other on the one-dimensional substrate.

Velocity was represented as that of

the leading edge of migrating cells and calculated every 10 min.

(A and C) Change in

the average velocity of cells 60 min before and after contact with its counterpart.

(B

and D) Horizontal stacked bar charts represent the percentage of cumulative cell
numbers migrating at the velocity indicated above the chart.

(A) DM-lu cells appeared

to continue migration at a lower velocity after contact with LP-lu cells (n = 11).

(B)

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Just after contact with LP-lu cells, a certain number of DM-lu cells began to migrate
backward at a relatively high speed (more than 3 µm/min).

The percentage of DM-lu

cells migrating forward decreased once to approximately 50% 0–30 min after contact,
and then it increased slightly in the following 30–60 min.

(C) LP-lu cells changed the

direction of migration after contact with DM-lu cells (n = 11).

(D) The percentage of

LP-lu cells migrating forward decreased once to approximately 30% 0–30 min after
contact with DM-lu cells, and then it increased to approximately 60% in the following
30–60 min after contact.

Just after contact (0–30 min), more than 70% of cells

migrated backward, although most cells migrated at a relatively low velocity (0–1
µm/min). Abbreviations: DM, dermomyotome; LP, somatic lateral plate; -th, thoracic;
-lu, lumbar; n.s., no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001.

Data

are represented as means ± SE.
Fig. 7

The ratio of length of somite cells after contact with SC cells before contact.

Cell length was measured from the leading edge to the trailing edge.

To calculate the

ratio of cell length, each cell length after the contact was divided by the average cell
length of the cell before contact.

The ratio of length of both SC-th and DM-th cells

was significantly shorter when they made contact with LP-lu cells than that when they
made contact with LP-th cells.

Abbreviation; -th, thoracic; -lu, lumbar; SC,

sclerotome; DM, dermomyotome; LP, lateral plate.
cells made contact with LP-lu cells, n = 10.
contact with LP-lu cells.
Fig. 8

the Ward’s method.
numbers.

DM-th

n = 11, except for when DM-th cells made

n.s., no significant difference; *P < 0.01.

Cluster analysis of migratory behavior.

distribution of the cell.

Error bars denote SE.

(A) Cluster dendrogram of velocity

Height indicates criterion value in our agglomeration adapting

Heatmap represents the relative frequencies of cumulative cell

White color indicates the maximum value.

Clustering cells are indicated

under the heatmap with co-cultured cells and with measured time period. (B)
Difference in the velocity distribution pattern between clusters.

In each cluster, we

calculated the average of the relative frequencies of cells (percentage) migrating at each
velocity indicated above the chart.
pattern are indicated by the cluster.

(C) The time-course of changes in cell migration
Same clusters are indicated with the same

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background color.

For example, the migratory behavior of SC-th cells co-cultured

with LP-th cells always belonged to CL1 or CL2, whereas those co-cultured with LP-lu
cells changed from CL1 to CL3 after contact with LP-lu cells.

Abbreviation; SC,

sclerotome; DM, dermomyotome; LP, lateral plate; th, thoracic; lu, lumbar; B60, before
60–30 min; B30, before 30–0 min; A30, after 0–30 min; A60, after 30–60 min.