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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 ธT 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. S 87 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. REFERENCES 1. Arey LB. Developmental Anatomy. A Textbook and Laboratory Manual of Embryology. 7th ed. WB Saunders, 1966. p.375–95. S 89 2. 3. 4. 5. S 90 Beynon PH. Cooper JE. Manual of Exotic Pets. British Smali Animal Veterinary Association. West Sussex, 1991. p.39–50. Carlson BM. Human Embryology and Developmental Biology. Mosby. USA, 1994. p.372–406. Langman J. Langman’s Medical Embryology, 7th ed. Williums & Wilkins USA, 1995. p.183–231. Moore KL. The Developing Human. Clinically Oriented Embryology, 6th ed. WB Saunders, 1998. p.341–403. 6. 7. Pilakasin K, Sangvichien S, Koedpuech K, Amonmattajit N. The study of each developmental stage of the whole mount chick embryo by the scanning electronmicroscope as compared with the classical light microscope. RTAMJ 1999. p.52:137-50. Pilakasiri K, Roongroungchai J, Koedpuech K, Amonmattajt N, Prapamontol U, Pilakasiri C. The study of the use of scanning electron microscopy as compared with conventional light microscopy to examine the transverse sections of the chick embryos at the primitive streak and the 3-5 somite stages. Siriraj Med J. 2007 Nov-Dec;59(Supp l2):170-4.