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OriginalArticle
The Study of the Transverse Sections of 7-10
Somite Chick Embryos by the Scanning Electron
Microscopy and Compared with the Classical
Light Microscopy
Kajee Pilakasiri, Ph.D*, Amphaphorn Pornkunnatham, M.S.*, Jantima Roongruangchai, D.D.S., Ph.D*, Kasem Koedpuch, B.Sc.*
*Department of Anatomy, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
ABSTRACT
Objective: To comparatively study the use of scanning electron microscopy and conventional light microscopy of the transverse
sections of the 7-10 somite staged chick embryos as model for the study of development of human embryo.
Methods: Conventional light microscopy and scanning electron microscopy had been applied as tools for the study of the
chick embryos.
Results: The results showed that scanning electron micrographs gave the clearer different views of chick embryo transverse
sections as compared with the conventional light microscopy.
Conclusion: From this study it was clearly shown that the three dimensional images obtained from scanning electron microscope
could give comprehensive view of chick embryo specimens. Hence this should be the good alternative way for Embryology study.
Keywords: Chick embryo, light microscopy, scanning electron microscopy, transverse section
Siriraj Med J 2012; 64 (Suppl 1): S86-S90
E-journal: http://www.sirirajmedj.com
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INTRODUCTION
he chick embryo has been used as an excellent
model for studying the early
development of human
embryo for a long time1-3. This may be because
of it is easily obtainable and the previous studies had
completely studied the light microscopic structure and also
compared the incubation time to the approximate age of
the human embryo. For instance, 18 hours incubation of
the chick embryo which is the primitive streak stage can
be compared1 to the 15-16 days of human embryo after
fertilization .
The yolk of the hen’s egg contains a single ovum
and is enormously expanded with stored food material.
When the egg cell is expelled from the ovary at the time
of ovulation it is enveloped by the3-5vitelline membrane,
secreted by the cytoplasm of the egg . As the egg moves
continuously down the oviduct, the viscid albumen, papery
shell membrane and calcareous shell are progressively
secreted by the epithelium lining the duct and are added
about the yolk as the accessory investment. During this
Correspondence to: Kajee Pilakasiri
E-mail: [email protected]
S 86
journey, which ends with the laying of the egg, a start
has been made toward the formation of a visible embryo.
Thus, before external incubation begins, the processes of
cleavage and ectodermal formation are completed. When
it is laid the embryonic area is represental by the familiar
whitish disc to be seen on the surface of the yolk.1 Thus
it is that, before external incubation begins, its several
components are the blastomeres, the consequence of the
process of cleavage, characterized by a tiny disc which1 is
separated from the yolk beneath by a cleft-like space.
When the egg is laid without further incubation,
there is no further development. On the commencement of
incubation, gastrulation begins. This state can be compared
to the human embryo of 15 days after fertilization. The
process is to produce the third germ layer, the mesoderm.
If the incubation time is prolonged to be about 25 hours,
the 3-5 somite embryo is obtained which can be compared
to the human embryo of about 17-18 days after fertilization. At this stage the notochord has induced the surface
ectoderm to form the neural fold, and the head fold begins.
If the incubation time is further to be about 27 hours, the
embryo of about 7-10 somite stage appears which can be
compared to the human embryo of about 21-22 days after
fertilization. At this stage the anterior neuropore is nearly
closed, the heart begins beating and the optic vesicle is
visible with the progressive enormous head fold.
Normally the chick embryo has been used for a long
time as the study models for human development. By
the use of the total mount of chick embryo stained with
camine, the total body of the embryo is revealed by light
microscope. This can be done because the embryo body is
thin. The serial sections of each stage are used to compare
the structures seen in the total mount. The observation of
the total mount and the serial sections of the embryos of
each stage make more understanding of the event than
reading or listening. In this study, another technique of
three dimensional image, the scanning electron microscopy
or SEM, is used in order to get the best image and best
understanding of the human development. Although the
development of the chick embryo is studied by the SEM
technique, the human development can be obtained since
each stage of chick embryos can be compared to human
embryos. This three dimensional images are very useful to
make a real understanding of the embryology. This study
is focus on the chick embryo of about 27 hour incubation
or about 7-10 somite stage which can be compared to the
21-22 day human embryo after fertilization.
the chick embryo cut across the head fold. Fig 1A is more
cephalic than Fig 1C. The optic vesicles are presented
in the cephalic region of Fig 1A. Figure 1C is sectioned
across the foregut level and the future mouth area with
the oral membrane (prochordal plate) where the ectoderm
and endoderm are tightly adhered are observed. Fig 1B
is the scanning electron micrograph of the head fold and
the proamnion beneath the head. Fig 1D is the scanning
electron micrograph just caudal to the head where the
ventral wall of the foregut is opened into the yolk sac, this
area is called the midgut. Fig 1A and B can be viewed
together to compare and more details of three dimension
image in Fig 1B shows the depth of the cylindrical shaped
embryo. Moreover, the head ectoderm, mesoderm, and
the neural tube with the optic vesicles are revealed three
dimensionally of the same image as in Fig 1A. In the Fig
1D at the area of the midgut, the neural folds are forming
and the anterior neuropore is still opened, since there is
no fusion of the most cephalic part of the neural folds.
MATERIALS AND METHODS
The obtained fertilized chick eggs were incubated at
in order to get the embryo
37oC for 27 hour incubation
at 7-10 somite stages1-2. Ten fertilized eggs were included
for the stage studied.
For scanning electron microscopy, each chick embryo
of each stage mentioned was processed as followed. After
cutting the egg shell at the embryonic pole off, the embryos were seen floating on the yolk sac. The embryos
were separated from the yolk by using a sharp tip scissors
cutting around the body of embryos. With the aid of a
spatula, the embryos were collected and put into 3%2 warm
normal saline for washing the yolk granules off . Then
they were fixed in 2.5%2 glutaraldehyde
in 0.1 M Sodium
cacodylate buffer pH7. at 4oC for 24 hours and washed
with sodium cacodylate buffer and then with distilled water.
Each embryo of each stage was cut transversely by a sharp
razor blade under the stereomicroscope at various levels of
the embryo depending on the important structure location
which corresponded to the structures examined by light
microscopy. Subsequently, each piece of the embryo was
dehydrated through a graded series of ethanol and dried
in a critical point drying apparatus. After mounting on
stubs, they were coated with gold and examined under a
scanning electron microscope operated at 15 kv.
Concerning the conventional light microscopy, the
achieved embryos as mentioned were fixed in Bouin’s fluid
for one hour at room temperature, washed with 70% ethanol
until the yellow color staining the embryos disappeared.
This might consume about one week duration with ethanol
changing everyday. The specimens were then stained with
Mayer’s carmine dye for half and hour at room temperature,
washed and dehydrated with graded series of ethanol to
absolute ethanol, cleared in two changes of Cedar wood
oil and washed in two changes of chloroform, processed
for routine paraffin section, sectioned serially with 14 µm
thick by microtome, routinely mounted on the glass slides Fig 1. The transverse sections at the head region of the 7-10
with Canada Balsam and covered with cover slips.
somite chick embryo. A and C, the light micrographs. Band D
RESULTS
The head region (Fig 1)
Fig 1A and C are the light microscopic images of
Siriraj Med J, Volume 64 (Suppl 1), January-February 2012
the scanning electron micrographs. Cl, coelom; Ed, ectoderm;
Fg, foregut; He, head ectoderm; Hm, head mesenchyme; Nc,
notochord; Ng, neural groove; Nt, neural tube; Ov, Optic vesicle;
Pa, proamnion; Pc, prochodal plate; Pm, paraxial mesoderm; Sm,
somatic mesoderm; Sp, splanchnic mesoderm.
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Fig 2. The light micrographs (A and C) and the scanning electron micrographs (B and D) of the transverse sections of the
7-10 somite chick embryo at the developing heart tube region.
Cj, cardiac jelly; Da, dorsal aorta; Dm, dorsal mesocardium;
Ed, ectoderm; Em, epimyocardium; Et, endocardial tube; Fg,
foregut; Nc, notochorcl; Nt, neural tube; Pc, pericardial eavity;
Vm, ventral mesocordium.
The mesoderm comprises the paraxial mesoderm in the
middle as well as the somatic and splanchnic mesoderms
laterally.The coelom is located between the somatic and
splanchnic mesederm.
The developing heart tube region (Fig 2)
The light micrographs of Fig 2 A and C show the
developing heart that is composed of a single endocardial
tube covered by the thickening splanchnic mesoderm of
two layers, the epimyocardium laterally and the cardiac
jelly medially. The heart tube is attached to the dorsal
body wall by the dorsal mesocardium while the ventral
by the ventral myocardium (Fig 1A). The ventral mesocardium at the more caudal sections is degenerated (Fig
1C). All of the structures mentioned above are developed
S 88
Fig 3. The light micrograph (A) and the scanning electron
micrographs (B and C) of the transverse sections of the 7-10
somite chick embryo at the somite level. Da, dorsal aorta; Ed,
ectoderm; En, endoderm; Im, intermediate mesoderm; Lm, lateral
mesodurm; Nc, notochord; Nf, neural fold; Ng, neural groove;
Nt, neural tube; So, somite.
from the splanchnic mesoderm. The Fig 2B and D are
the scanning electron micrographs, which more details of
three dimensional views of the heart in the same area as
Fig 1A and C are demonstrated.
The somite area (Fig 3)
The general level of the primitive segments is characterized by the greater specialization of the mesoderm,
the elevation of high neural folds and the presence of a
dorsal aorta on each side between the mesodermal somites
and the endoderm. The notochord is a sharply defined,
oval mass of cells which will be observed just below the
neural groove or the neural tube, it appears in all sections
of the series, except those through the tip of the head and
the primitive streak. The somites are somewhat triangular
in outline and is connected with the lateral mesoderm by a
short plate, the intermediate mesoderm. Three dimensional
images of Fig 3B and C reveal clearly of the shape of
the columnar cells of the neural tube as well as the cell
of the somites.
inside. Fig 1A reveals the detail of cellular morphology
in the two dimensions while Fig 4B and C reveal three
dimensions of the tissue to make more understanding of
the Embryology.
DISCUSSION
The chick embryos of 27 hour incubation become
about 7-10 somite embryos. Therefore, they can be call
7-10 somite stage which refers to as the human embryo
of about 22 days after fertilization. At this stage the head
fold with the endoderm of the yolk sac grows inside to
form a blind sac (the foregut). The neural tube at the
head end is still opened as the anterior neuropore. At a
little caudal to anterior neuropore, there are the expansion
of the neural tube forming the optic vesicles. Cephalic
to the anterior intestinal portal, the heart is forming from
the thickend splanchnic mesoderm. Caudal to the anterior
intestinal portal the dorsal mesoderm arranges themselves
to form the somites and the cells change their morphology
to be the columnar cells. Laterally there are the somatic
and splanchnic mesoderms covering the intraembryonic
coelom.
It should be accepted that the study of Embryology
requires the work in the laboratory room and the students
have to trace the serial section slides. This can make
them get the good imagination to draw the image of the
embryo by observing several serial sections. Another new
technique to get the image of three dimensions is the scanning electron microscopy or SEM. The images reveal the
detail of the depth and help to confirm the imagination.
For instance, at the heart region, it is quite difficult to
explain the morphology of the heart in three dimensional
aspect. This problem can be solved by the SEM. This
work studies the three dimension figures of the transverse
sections of 7-10 somite chick embryos. The results of the
study can reveal the development more clearly by studying
the three dimensional view which can be compared
with
6
and
3-5
the
study
of
the
whole
mount
chick
embryos.
Fig 4. The light micrograph (A) and the scanning electron
7
micrographs (B and C) of the transverse sections of the 7-10 somite chick embryos in the three dimensional studies.
somite chick embryo at the region caudal to the anterior intestinal
portal. Cl, coelom; Ed, ectoderm; En, endoderm; Mg, midgut;
Nf, neural fold; Nt, neural tube; Pm, paraxial mesoderm; Sm,
somatic mesoderm; Sp, splanchnic mesoderm; Vv, vetelline vein.
The region caudal to the anterior intestinal portal
(Fig 4)
The section is characterized by the nearly meeting
of the neural folds preparatory to closing the neural tube.
The endoderm is arched and coming closer to form the
foregut in the more cephalic sections. The vitelline vein
is present between the endoderm and fold of splanchnic mesoderm. The wide separation of the somatic and
splanchnic mesoderm consequences the increase in size
of the coelom. In this location the coelom later surrounds
the heart and becomes the pericardial cavity (Fig 4A).
Figure 4B and C show the three dimensions of
the ventral aspect of the embryo while Fig 4C is higher
magnification. At the cut surface there are the neural tube
in the middle and the paraxial mesoderm in lateral. The
most lateral mesoderm, the somatic mesoderm, and the
ectoderm lining above form the somatospleure while the
splanchnic mesoderm and the endoderm lining underneath
form the splanchnopleure. The lateral body folds cause
elevation of the body while the splanchnopleure curved
Siriraj Med J, Volume 64 (Suppl 1), January-February 2012
CONCLUSION
The study of Embryology requires much of the
imagination in order to build the real figure of the organs
or system of the embryos. This study tries to solve the
difficulty of the two dimensional technique of the light
micrography by using the three dimensional technique or
SEM. This technique reveals less detail about the cellular
morphology and organelles, but it gives more details about
the depth or the third dimension of the figures. If the SEM
technique is used together with the conventional serial
section technique of light microscopy, the whole body or
the whole organs in three dimensions are demonstrated.
ACKNOWLEDGMENTS
The authors would like to express sincere appreciation
to the EM staffs of the Department of Pathology, Faculty
of Medicine Siriraj Hospital for their kind assistance for
the use of the scanning electron microscope.
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