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Anatomy of development
Anatomy of development A. Carretero, J. Ruberte, M. Navarro, S. Lope and A. Pujol
The purpose of this chapter is not to present an exhaustive overview of the mouse embryo morphology. There are currently several monographs describing specifically and in great detail the morphological changes that occur during ontogenetic development of the mouse (see the bibliography). The intention of the authors is, therefore, to present only some of the key stages of development. Special interest is given to correlating morphological changes of the embryo and the placenta with the images obtained using ultrasound technology and nuclear magnetic resonance.
■ ■ ONTOGENIC DEVELOPMENT The ontogenic development of an individual, also called embryonic development, corresponds to a continuous succession of morphological and functional changes, which do not end with birth. Many important developmental changes occur after birth, such as the invasion of blood
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vessels in the retina, which is not vascularized when the animal is born. Various chronological systems have been proposed to describe the ontogenetic development in the mouse. In this chapter, embryological (E) age in days is used, which is equivalent to days post-conception (dpc). This method considers that the morning of the first day of gestation (E0.5) is the morning of the day when the vaginal plug is found. This convention assumes that, in mice housed in standard conditions, with a cycle of 12 hours of light and 12 hours dark, ovulation and fertilization occur in the middle of the night. However, it is important to keep in mind that ovulation can last over an hour and that this factor may explain, at least partially, the degree of variability observed in the development of individuals from the same litter and between litters. Alternatively, the chronological method of Theiler (1989), which is based on the acquisition of specific morphological features during development, does not have this disadvantage. The average length of gestation in mice is 19.5 days.
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The prenatal stages of development in the mouse are: fertilization, segmentation, gastrulation, the embryonic period, and the fetal period. The stages of segmentation and gastrulation together are also known as the germinal period, with germ being the common term to refer to the individual at this particular time of development. Fertilization of the oocyte in the mouse occurs in the ampulla of the uterine tube. The fertilization process is complex and consists of several phases, including contact with the spermatazoa, oocyte activation, acrosome reaction, and penetration, amongst others. From the moment that one spermatozoon enters the oocite, the new entity formed is known as a zygote (Fig. 10-1). About 4-5 hours after oocyte activation, it is already possible to observe the male and female pronuclei in the cytoplasm of the zygote, with the male pronucleus sometimes appearing up to double the size of the female pronucleus (Fig. 10-1). The zygote is surrounded by the zona pellucida and the perivitelline space, which is located between the zygote and the zona pellucida. In the perivitelline space it is also possible to see the second polar body and the remains of the first polar body (Fig. 10-1). Development essentially begins with the segmentation period, where the zygote undergoes a series of mitotic divisions, which result in the formation of blastomeres. The first division occurs about 16-18 hours after fertilization, giving rise to a germ composed of 2 cells, the two-celled egg (Fig. 10-2). After two synchronous divisions, about 36 hours after the first division, a germ of 8 cells is formed, the eight-celled egg (Fig. 10-3). From that moment the divisions become increasingly asynchronous, and the morula is formed. The most important process of segmentation is compaction, during which the morula is condensed and the apparent individuality of blastomeres is lost. After compaction (E3-4), fluid begins to accumulates in the intercellular spaces of the morula (Fig. 10-4). This liquid eventually forms a unique cavity, the blastocoel, which will expand to form the blastocyst (Fig. 10-4). The expansion of the blastocyst, in combination with the action of proteolytic enzymes of the uterus, facilitates the breakdown of the zona pellucida and the release (eclosion) of the blastocyst (Fig. 10-4). This process is a prerequisite for implantation (E4.5). Within the blastocyst are distinguished two cell populations: the inner cell mass, which will give rise to the embryo, and the trophoblast or trophectoderm, which surrounds the inner cell mass (polar trophoblast) and the blastocoele (mural trophoblast). From the trophoblast are derived the extraembryonic organs, including the fetal portion of the placenta (Fig. 10-4). The blastocyst develops to give the gastrula (E7.5), which elongates to form a distinct structure known as the «egg cylinder» (Fig. 10-5). To form the «egg cylinder», the cells of the inner cell mass multiply and grow in the opposite direction to the implantation site. In addition, the primitive endoderm cells, which originate as a delamination of the inner cell mass, migrate to cover the mural trophoblast. When the migration of endodermal cells is complete, the blastocoel becomes the yolk sac cavity.
The cells that remain in the inner cell mass are the primitive ectoderm, which cavitates to form the proamniotic cavity. In turn, this cavity becomes split by the amniotic folds, forming the ectoplacentaria cavity, which is located below the ectoplacental cone and the definitive amniotic cavity. A third cavity, the exocoelomic cavity, subsequently appears from a cavitation in the caudal amniotic fold. Therefore, the mouse gastrula consists of three fluid-filled chambers: the ectoplacentaria cavity, exocoelomic cavity, and amniotic cavity (Fig. 10-5). During gastrulation, ectoderm cells migrate through the primitive groove to overlie the endoderm and thereby form the mesoderm, which is the third layer of cells that forms the gastrula (Fig. 10-5). The ectoderm, mesoderm and endoderm are the essential cell source necessary for the formation of the organs. The first of the embryonic organs that appears is the neural tube, which is the precursor of the central nervous system. The process by which the neural tube is formed is called neurulation and it is a precise chronological boundary that delimitates the germinal period and the embryonic period. During the embryonic period, the embryo undergoes a process of rotation («embryo turning»), whereby the endoderm located in the outer surface of the embryo ends forming the primitive gut and the ectoderm, which was originally located internally, eventually becomes the epidermis. As a representative of this embryonic period, an embryo of twelve and a half days of gestation (E12.5) is presented (Fig. 10-6 to 10-8). During the fetal period the fetus undergoes extensive growth and tissular differentiation, whereby the primary organs growth considerably to form the definitive fully functional organs. The morphological landmark for the beginning of the fetal period is the apparition of the humerus bone marrow. In the mouse, this arbitrary convention decided on by embryologists occurs between the fourteenth and fifteenth days of gestation (E14-15). The fetal period ends with delivery, although ontogeny last longer. As a representative of this fetal period a fetus of fifteen days of gestation (E15) is presented (Figs. 10-9 to 10-14).
■ ■ PLACENTA Mammalian development requires implantation or nidation of the embryo in the endometrium and the formation of a placenta (Fig. 10-14), which physically connects the embryo to their mother. Establishing this connection is a priority for the survival of the embryo and to ensure this it requires that the extra-embryonic tissues differentiate much faster than the embryo itself. The extra-embryonic tissues are the precursors of the placenta and are specialized in different functions, including invading the uterine epithelium, mediating the interaction between the fetal and maternal environment, and protecting and nourishing the embryo. The cells that invade the endometrium are the trophoblast cells of the blastocyst (Fig. 10-4). When the trophoblast is lined by primitive endoderm, it becomes what is known as the extraembryonic endoderm, which itself gives rise to
10. Anatomy of development
two sheets, the parietal endoderm and the visceral endoderm. The primary yolk sac cavity is located between these two sheets of endoderm (Fig. 10-15). Visceral endoderm cells are cylindrical, with a large number of vacuoles and numerous villi, and they are joined by tight junctions. Initially, parietal endoderm cells appear flattened and elongated and similar in appearance to fibroblasts. Subsequently, they become rounded and are no longer in contact together with the trophoblast basement membrane. In the mouse embryo the parietal endoderm cells synthesize and secrete large quantities of extracellular matrix components such as laminin, collagen IV, proteoglycans, etc. These extracellular proteins form the Reichert’s membrane, which is a thick sheet that is located between the parietal endoderm cells and the trophoblast (Fig. 10-15). Trophoblast cells become primary giant cells when they enter into contact with the uterine lining (E4.5), where their role is to erode and dissolve the endometrium. Trophoblast cells, which are in contact with the inner cell mass, form a compact cellular cap, the ectoplacental cone (Fig. 10-15). The ectoplacental cone will form the placental layers, the labyrinth, spongiotrophoblast, and secondary giant cells (Fig. 10-16). The maternal part of the placenta, the decidua, is formed by the proliferation and differentiation of endometrial cells, in a process known as decidualization. Consequently, this process results in the thickening of the uterine wall. Trophoblast giant cells, which are in contact with the decidua, express several angiogenic factors (such as VEGF, proliferin) and vasodilators (such as nitric oxide, adrenomedullin). These factors increase uterine vascularization at the implantation site, which leads to increased blood flow, an essential process for embryo survival. The radial arteries, branches of the uterine and ovarian arte-
ries (Fig. 9-10), divide into the myometrium, to give rise to the spiral arteries, which enter the decidua. Often, the spiral arteries are larger in diameter than the radial arteries from which they originate. Once they have crossed the decidua, the spiral arteries merge to form between 1 and 4 arterial channels, which cross the labyrinth (Fig. 10-18). Surrounding these channels are cells rich in cytokeratin. The endothelium of the maternal blood vessels undergoes a transition as it approaches the fetal part of the placenta, where the endothelial cells are replaced by trophoblast cells. Thus trophoblast cells are in contact with maternal blood (hemochorial placenta). The spongiotrophoblast and the secondary giant cells are fetal components of the placenta and they are in direct contact with the maternal decidua. They consist of different types of trophoblast cells, which are a source of steroid hormones and signaling factors essential for embryo development. These cells are classified into three groups: (1) spongiotrophoblast cells, (2) glycogenic cells, and (3) secondary giant cells. The giant cells are usually situated at the boundary between the decidua and spongiotrophoblast, but can also be seen associated with the spiral arteries and sinusoids of the labyrinth. The labyrinth is the largest area of the placenta and it is where exchange of gas and nutrients between mother and embryo takes place. Consequently, within the labyrinth both maternal and embryonic blood vessels can be found. The process of vascular growth and branching in the labyrinth is maintained until birth (Fig. 10-18). Placenta matures around the fourteenth day of gestation (E14) (Figs. 10-17 and 10-18), when the labyrinth zone represents more than half of the total size of the placenta and giant cells tend to disappear.
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Figure 10-1. Zygote (E0.5). A) Differential interference contrast microscopy image. B and C) Confocal laser microscopy images obtained from the same specimen. Transmission mode and mitochondria labeled with MitoTracker® (green), respectively.
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Figure 10-2. Two-celled egg (E1-1.5). A) Differential interference contrast microscopy image. B and C) Confocal laser microscopy images obtained from the same specimen. Transmission mode and mitochondria labeled with MitoTracker® (green), respectively. 1: Blastomere; 2: Cell nucleus; 3: Second polar body; 4: Pellucid zone; 5: Perivitelline space.
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Figure 10-3. Four-celled egg (E2-2.5). A) Differential interference contrast microscopy image obtained from the same specimen. B and C) Confocal laser microscopy images. Transmission mode and mitochondria labeled with MitoTracker® (green), respectively.
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Figure 10-4. Morula and blastocyst. Differential interference contrast microscopy images. A) Morula in cavitation phase (E3) (early blastocyst). B) Expanded blastocyst without zona pellucida (E4). 1: Pellucid zone; 2: Blastocoel in expansion; 3: Definitive blastocoel; 4: Polar trophoblast; 5: Mural trophoblast; 6: Inner cell mass.
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Figure 10-5. Egg cylinder (E7.5). A) Histological section of the gravid uterus. Immunodetection of a-SMA revealed with DAB (brown). Harris’ hematoxylin counterstain (20X). B) Magnification of egg cylinder. Longitudinal section, hematoxylin-eosin stain (100X). C and C’) Magnification of egg cylinder. Transverse sections. Hematoxylin-eosin stain (200X). D and E) Magnetic resonance images. Tranvese and dorsal sections, respectively. F) Ultrasound image. 1: Egg cilynder; 2: Ectoplacental cone; 3: Myometrium; 4: Stratum submucosum (myometrium); 5: Stratum subserosum (myometrium); 6: Decidua (endometrium); 7: Amniotic cavity; 8: Exocoelomic cavity; 9: Ectoplacental cavity; 10: Chorion; 11: Embryonic ectoderm; 12: Embryonic mesoderm; 13: Visceral endoderm; 14: Amnion; 15: Yolk sac cavity; 16: Parietal endoderm and Reichert’s membrane; 17: Lumbar vertebra; 18: Wing of ilium; 19: Epaxial muscles; 20: Cecum; 21: Horn of uterus.
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Figure 10-6. Uterus of twelve and half days of gestation. A) The abdominal muscles were removed. B) The intestines were displaced cranially to observe the uterine topography. C and E) Magnetic resonance images. Horizontal and transverse sections, respectively. D) Isolated gravid uterus. 1: Right lateral lobe of liver; 2: Left lateral lobe of liver; 3: Stomach; 4: Duodenum; 5: Jejunum; 6: Cecum; 7: Right horn of uterus; 8: Left horn of uterus; 9: Right ovary; 10: Left ovary; 11: Lumbar vertebra; 12: Free border; 13: Mesometrial border; 14: Wing of ilium; 15: Epaxial muscles.
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Dorsal Lateral Figure 10-7. Embryo and yolk sac of twelve and half days of gestation (E12.5). A) Embryo within the horn of uterus. B) Histological section of the horn of gravid uterus. Immunodetection of a-SMA revealed with DAB (brown). Harris’ hematoxylin counterstain (20X). C) Isolated embryo within the yolk sac and placenta. D) Histological section of the embryo and placenta. Hematoxylin-eosin stain (20X). E) Magnetic resonance image. F) Ultrasound image. 1: Myometrium; 2: Placenta; 3: Labyrinth; 4: Spongiotrophoblast; 5: Decidua; 6: Embryo; 7: Amnion; 8: Yolk sac cavity; 9: Yolk sac; 10: Umbilical cord.
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Figure 10-8. Embryo of twelve and half days of gestation (E12.5). A) Embryo within the amnion. B) Sagittal histological section of isolated embryo. Hematoxylin-eosin stain (20X). C) Magnetic resonance image. Sagittal section. D and E) Magnetic resonance images. Transverse sections. 1: Telencephalon; 2: Diencephalon; 3: Mesencephalon; 4: Rombencephalon; 5: Spinal cord; 6: Nasal cavity; 7: Vomeronasal organ; 8: Internal ear; 9: Mandibular process (first branchial arch); 10: Ventricle; 11: Atrium; 12: Lung; 13: Liver; 14: Umbilical cord; 15:Amnion; 16: Yolk sac cavity; 17: Forelimb bud; 18: Hindlimb bud; 19: Placenta.
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6 5 Figure 10-9. Uterus of fifteen days of gestation. A) The gravid uterus was removed from the abdomen. B and C) Magnetic resonance images. Horizontal and transverse sections, respectively. 1: Caudal vena cava; 2: Right ovarian vein; 3: Uterine vein; 4: Radial veins; 5: Fetuses; 6: Placenta; 7: Descending colon; 8: Stomach; 9: Cecum; 10: Lumbar vertebra; 11: Wing of ilium; 12: Epaxial muscles.
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E 22 17 Figure 10-10. Fetus of fifteen days of gestation (E15). A) Fetus inside of the horn of uterus. B) Fetus inside of yolk sac. C) Sagittal histological section of fetus inside the chorionic yolk sac. Hematoxylin-eosin stain (20X). D) Magnetic resonance image. Sagittal section. E) Ultrasound image. 1: Placenta; 2: Lateral ventricle (telencephalon); 3: Diencephalon; 4: Mesencephalon; 5: Fourth ventricle (rombencephalon); 6: Trigeminal ganglion; 7: Tongue; 8: Meckel’s cartilage; 9: Nasal cavity; 10: Upper lip; 11: Jejunum (physiological umbilical hernia); 12: Umbilical cord; 13: Genital tubercle; 14: Liver; 15: Duodenum; 16: Heart; 17: Eyeball; 18: Ribs; 19: Humerus; 20: Femur; 21: Tail; 22: Yolk sac; 23: Yolk sac cavity.
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Figure 10-11. Fetus of fifteen days of gestation (E15). Magnetic resonance images. Transverse sections. 1: Telencephalon; 2: Lateral ventricles; 3: Diencephalon; 4: Third ventricle; 5: Junction between mesencephalon and pons; 6: Fourth ventricle; 7: Rombencephalon; 8: Spinal cord; 9: Trigeminal ganglion; 10: External ear; 11: Internal ear; 12: Eyeball; 13: Left lung; 14: Right lung; 15: Left ventricle; 16: Right ventricle; 17: Left atrium; 18: Right atrium; 19: Left lateral lobe of liver; 20: Right lateral lobe of liver; 21: Caudate lobe (liver); 22: Stomach; 23: Genital tubercle; 24: Thoracic aorta; 25: Abdominal aorta; 26: Left forepaw; 27: Right forepaw; 28: Left hindpaw; 29: Right hindpaw; 30: Tail; 31: Umbilical cord; 32: Yolk sac cavity; 33: Yolk sac.
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Figure 10-12. Sex identification in fetus of fifteen days of gestation (E15). Note the greater distance between the anus and the genital tubercle in the male fetus. 1: Anus; 2: Genital tubercle; 3: Tail; 4: Right hindpaw; 5: Left hindpaw.
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Figure 10-13. Fetus of fifteen days of gestation (E15). Magnetic resonance images. Horizontal sections. 1: Telencephalon; 2: Lateral ventricle; 3: Diencephalon; 4: Mesencephalon; 5: Aqueduct of mesencephalon; 6: Rombencephalon; 7: Fourth ventricle; 8: Spinal cord; 9: Eyeball; 10: Tongue; 11: Nasal cavity; 12: Right atrium; 13: Right cranial vena cava; 14: Left cranial vena cava; 15: Right external jugular vein: 16: Left external jugular vein; 17: Caudal vena cava; 18: Right lateral lobe of liver; 19: Left lateral lobe of liver; 20: Stomach; 21: Right kidney; 22: Left kidney; 23: Thoracic aorta; 24: Right thoracic limb; 25: Left thoracic limb; 26: Right pelvic limb; 27: Left pelvic limb; 28: Ribs; 29: Tail; 30: Genital tubercle; 31: Umbilical cord; 32: Placenta; 33: Labyrinth; 34: Spogiotrophoblast; 35: Decidua; 36: Yolk sac cavity.
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Figure 10-14. Umbilical cord connecting fetus and placenta (E15). The yolk sac was removed and the umbilical cord was stretched along its length.
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Figure 10-15. Placenta of seven and half days of gestation. A) Ectoplacental cone. Hematoxylin-eosin stain (200X). B) Magnification of Reichert’s membrane (1,000X). 1: Ectoplacental cone; 2: Yolk sac cavity; 3: Ectoplacental cavity; 4: Extraembryonic mesoderm; 5: Visceral endoderm (extraembryonic); 6: Parietal endoderm (extraembryonic); 7: Trophoblast; 8: Reichert’s membrane; 9: Exocoelomic cavity; 10: Chorion; 11: Maternal blood.
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Figure 10-16. Placenta of twelve and half days of gestation. A) Transverse section. Hematoxylin-eosin stain (20X). B) Labyrinth (200X). C) Spongiotrophoblast (100X). D) Magnification of the fetal and maternal erythrocytes (1,000X). E) Magnification of giant cells (1,000X). 1: Yolk sac cavity; 2: Chorionic plate; 3: Labyrinth; 4: Spongiotrophoblast; 5: Giant cells; 6: Decidua; 7: Fetal erythrocytes (nucleated); 8: Maternal erythrocytes (anucleated); 9: Glycogen trophoblast cells; 10: Syncytiotrophoblast.
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Figure 10-17. Placenta of fifteen days of gestation. A) Transverse section. Hematoxylin-eosin stain (20X). B) Labyrinth (400X). C) Spongiotrophoblast (400X). D) Decidua (400X). 1: Decidua; 2: Spongiotrophoblast; 3: Labyrinth; 4. Spiral arteries; 5: Central arterial canal; 6: Fetal erythrocytes (nucleated); 7: Maternal erythrocytes (anucleated); 8: Syncytiotrophoblast; 9: Chorionic plate.
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Figure 10-18. Placenta of fifteen days of gestation. A) Transverse section. Staining with Lycopersicon esculentum lectin revealed with DAB (brown). Nuclei counterstained with Harris’ hematoxylin (20X). B) Labyrinth (400X). C) Spongiotrophoblast (400X). D) Decidua (400X). 1: Decidua; 2: Spongiotrophoblast; 3: Labyrinth; 4: Spiral arteries; 5: Chorionic plate.
The purpose of this chapter is not to present an exhaustive overview of the mouse embryo morphology. There are currently several monographs describing specifically and in great detail the morphological changes that occur during ontogenetic development of the mouse (see the bibliography). The intention of the authors is, therefore, to present only some of the key stages of development. Special interest is given to correlating morphological changes of the embryo and the placenta with the images obtained using ultrasound technology and nuclear magnetic resonance.
■ ■ ONTOGENIC DEVELOPMENT The ontogenic development of an individual, also called embryonic development, corresponds to a continuous succession of morphological and functional changes, which do not end with birth. Many important developmental changes occur after birth, such as the invasion of blood
10
vessels in the retina, which is not vascularized when the animal is born. Various chronological systems have been proposed to describe the ontogenetic development in the mouse. In this chapter, embryological (E) age in days is used, which is equivalent to days post-conception (dpc). This method considers that the morning of the first day of gestation (E0.5) is the morning of the day when the vaginal plug is found. This convention assumes that, in mice housed in standard conditions, with a cycle of 12 hours of light and 12 hours dark, ovulation and fertilization occur in the middle of the night. However, it is important to keep in mind that ovulation can last over an hour and that this factor may explain, at least partially, the degree of variability observed in the development of individuals from the same litter and between litters. Alternatively, the chronological method of Theiler (1989), which is based on the acquisition of specific morphological features during development, does not have this disadvantage. The average length of gestation in mice is 19.5 days.
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Morphological Mouse Phenotyping: Anatomy, Histology and Imaging
The prenatal stages of development in the mouse are: fertilization, segmentation, gastrulation, the embryonic period, and the fetal period. The stages of segmentation and gastrulation together are also known as the germinal period, with germ being the common term to refer to the individual at this particular time of development. Fertilization of the oocyte in the mouse occurs in the ampulla of the uterine tube. The fertilization process is complex and consists of several phases, including contact with the spermatazoa, oocyte activation, acrosome reaction, and penetration, amongst others. From the moment that one spermatozoon enters the oocite, the new entity formed is known as a zygote (Fig. 10-1). About 4-5 hours after oocyte activation, it is already possible to observe the male and female pronuclei in the cytoplasm of the zygote, with the male pronucleus sometimes appearing up to double the size of the female pronucleus (Fig. 10-1). The zygote is surrounded by the zona pellucida and the perivitelline space, which is located between the zygote and the zona pellucida. In the perivitelline space it is also possible to see the second polar body and the remains of the first polar body (Fig. 10-1). Development essentially begins with the segmentation period, where the zygote undergoes a series of mitotic divisions, which result in the formation of blastomeres. The first division occurs about 16-18 hours after fertilization, giving rise to a germ composed of 2 cells, the two-celled egg (Fig. 10-2). After two synchronous divisions, about 36 hours after the first division, a germ of 8 cells is formed, the eight-celled egg (Fig. 10-3). From that moment the divisions become increasingly asynchronous, and the morula is formed. The most important process of segmentation is compaction, during which the morula is condensed and the apparent individuality of blastomeres is lost. After compaction (E3-4), fluid begins to accumulates in the intercellular spaces of the morula (Fig. 10-4). This liquid eventually forms a unique cavity, the blastocoel, which will expand to form the blastocyst (Fig. 10-4). The expansion of the blastocyst, in combination with the action of proteolytic enzymes of the uterus, facilitates the breakdown of the zona pellucida and the release (eclosion) of the blastocyst (Fig. 10-4). This process is a prerequisite for implantation (E4.5). Within the blastocyst are distinguished two cell populations: the inner cell mass, which will give rise to the embryo, and the trophoblast or trophectoderm, which surrounds the inner cell mass (polar trophoblast) and the blastocoele (mural trophoblast). From the trophoblast are derived the extraembryonic organs, including the fetal portion of the placenta (Fig. 10-4). The blastocyst develops to give the gastrula (E7.5), which elongates to form a distinct structure known as the «egg cylinder» (Fig. 10-5). To form the «egg cylinder», the cells of the inner cell mass multiply and grow in the opposite direction to the implantation site. In addition, the primitive endoderm cells, which originate as a delamination of the inner cell mass, migrate to cover the mural trophoblast. When the migration of endodermal cells is complete, the blastocoel becomes the yolk sac cavity.
The cells that remain in the inner cell mass are the primitive ectoderm, which cavitates to form the proamniotic cavity. In turn, this cavity becomes split by the amniotic folds, forming the ectoplacentaria cavity, which is located below the ectoplacental cone and the definitive amniotic cavity. A third cavity, the exocoelomic cavity, subsequently appears from a cavitation in the caudal amniotic fold. Therefore, the mouse gastrula consists of three fluid-filled chambers: the ectoplacentaria cavity, exocoelomic cavity, and amniotic cavity (Fig. 10-5). During gastrulation, ectoderm cells migrate through the primitive groove to overlie the endoderm and thereby form the mesoderm, which is the third layer of cells that forms the gastrula (Fig. 10-5). The ectoderm, mesoderm and endoderm are the essential cell source necessary for the formation of the organs. The first of the embryonic organs that appears is the neural tube, which is the precursor of the central nervous system. The process by which the neural tube is formed is called neurulation and it is a precise chronological boundary that delimitates the germinal period and the embryonic period. During the embryonic period, the embryo undergoes a process of rotation («embryo turning»), whereby the endoderm located in the outer surface of the embryo ends forming the primitive gut and the ectoderm, which was originally located internally, eventually becomes the epidermis. As a representative of this embryonic period, an embryo of twelve and a half days of gestation (E12.5) is presented (Fig. 10-6 to 10-8). During the fetal period the fetus undergoes extensive growth and tissular differentiation, whereby the primary organs growth considerably to form the definitive fully functional organs. The morphological landmark for the beginning of the fetal period is the apparition of the humerus bone marrow. In the mouse, this arbitrary convention decided on by embryologists occurs between the fourteenth and fifteenth days of gestation (E14-15). The fetal period ends with delivery, although ontogeny last longer. As a representative of this fetal period a fetus of fifteen days of gestation (E15) is presented (Figs. 10-9 to 10-14).
■ ■ PLACENTA Mammalian development requires implantation or nidation of the embryo in the endometrium and the formation of a placenta (Fig. 10-14), which physically connects the embryo to their mother. Establishing this connection is a priority for the survival of the embryo and to ensure this it requires that the extra-embryonic tissues differentiate much faster than the embryo itself. The extra-embryonic tissues are the precursors of the placenta and are specialized in different functions, including invading the uterine epithelium, mediating the interaction between the fetal and maternal environment, and protecting and nourishing the embryo. The cells that invade the endometrium are the trophoblast cells of the blastocyst (Fig. 10-4). When the trophoblast is lined by primitive endoderm, it becomes what is known as the extraembryonic endoderm, which itself gives rise to
10. Anatomy of development
two sheets, the parietal endoderm and the visceral endoderm. The primary yolk sac cavity is located between these two sheets of endoderm (Fig. 10-15). Visceral endoderm cells are cylindrical, with a large number of vacuoles and numerous villi, and they are joined by tight junctions. Initially, parietal endoderm cells appear flattened and elongated and similar in appearance to fibroblasts. Subsequently, they become rounded and are no longer in contact together with the trophoblast basement membrane. In the mouse embryo the parietal endoderm cells synthesize and secrete large quantities of extracellular matrix components such as laminin, collagen IV, proteoglycans, etc. These extracellular proteins form the Reichert’s membrane, which is a thick sheet that is located between the parietal endoderm cells and the trophoblast (Fig. 10-15). Trophoblast cells become primary giant cells when they enter into contact with the uterine lining (E4.5), where their role is to erode and dissolve the endometrium. Trophoblast cells, which are in contact with the inner cell mass, form a compact cellular cap, the ectoplacental cone (Fig. 10-15). The ectoplacental cone will form the placental layers, the labyrinth, spongiotrophoblast, and secondary giant cells (Fig. 10-16). The maternal part of the placenta, the decidua, is formed by the proliferation and differentiation of endometrial cells, in a process known as decidualization. Consequently, this process results in the thickening of the uterine wall. Trophoblast giant cells, which are in contact with the decidua, express several angiogenic factors (such as VEGF, proliferin) and vasodilators (such as nitric oxide, adrenomedullin). These factors increase uterine vascularization at the implantation site, which leads to increased blood flow, an essential process for embryo survival. The radial arteries, branches of the uterine and ovarian arte-
ries (Fig. 9-10), divide into the myometrium, to give rise to the spiral arteries, which enter the decidua. Often, the spiral arteries are larger in diameter than the radial arteries from which they originate. Once they have crossed the decidua, the spiral arteries merge to form between 1 and 4 arterial channels, which cross the labyrinth (Fig. 10-18). Surrounding these channels are cells rich in cytokeratin. The endothelium of the maternal blood vessels undergoes a transition as it approaches the fetal part of the placenta, where the endothelial cells are replaced by trophoblast cells. Thus trophoblast cells are in contact with maternal blood (hemochorial placenta). The spongiotrophoblast and the secondary giant cells are fetal components of the placenta and they are in direct contact with the maternal decidua. They consist of different types of trophoblast cells, which are a source of steroid hormones and signaling factors essential for embryo development. These cells are classified into three groups: (1) spongiotrophoblast cells, (2) glycogenic cells, and (3) secondary giant cells. The giant cells are usually situated at the boundary between the decidua and spongiotrophoblast, but can also be seen associated with the spiral arteries and sinusoids of the labyrinth. The labyrinth is the largest area of the placenta and it is where exchange of gas and nutrients between mother and embryo takes place. Consequently, within the labyrinth both maternal and embryonic blood vessels can be found. The process of vascular growth and branching in the labyrinth is maintained until birth (Fig. 10-18). Placenta matures around the fourteenth day of gestation (E14) (Figs. 10-17 and 10-18), when the labyrinth zone represents more than half of the total size of the placenta and giant cells tend to disappear.
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Figure 10-1. Zygote (E0.5). A) Differential interference contrast microscopy image. B and C) Confocal laser microscopy images obtained from the same specimen. Transmission mode and mitochondria labeled with MitoTracker® (green), respectively.
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Figure 10-2. Two-celled egg (E1-1.5). A) Differential interference contrast microscopy image. B and C) Confocal laser microscopy images obtained from the same specimen. Transmission mode and mitochondria labeled with MitoTracker® (green), respectively. 1: Blastomere; 2: Cell nucleus; 3: Second polar body; 4: Pellucid zone; 5: Perivitelline space.
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Figure 10-4. Morula and blastocyst. Differential interference contrast microscopy images. A) Morula in cavitation phase (E3) (early blastocyst). B) Expanded blastocyst without zona pellucida (E4). 1: Pellucid zone; 2: Blastocoel in expansion; 3: Definitive blastocoel; 4: Polar trophoblast; 5: Mural trophoblast; 6: Inner cell mass.
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Figure 10-5. Egg cylinder (E7.5). A) Histological section of the gravid uterus. Immunodetection of a-SMA revealed with DAB (brown). Harris’ hematoxylin counterstain (20X). B) Magnification of egg cylinder. Longitudinal section, hematoxylin-eosin stain (100X). C and C’) Magnification of egg cylinder. Transverse sections. Hematoxylin-eosin stain (200X). D and E) Magnetic resonance images. Tranvese and dorsal sections, respectively. F) Ultrasound image. 1: Egg cilynder; 2: Ectoplacental cone; 3: Myometrium; 4: Stratum submucosum (myometrium); 5: Stratum subserosum (myometrium); 6: Decidua (endometrium); 7: Amniotic cavity; 8: Exocoelomic cavity; 9: Ectoplacental cavity; 10: Chorion; 11: Embryonic ectoderm; 12: Embryonic mesoderm; 13: Visceral endoderm; 14: Amnion; 15: Yolk sac cavity; 16: Parietal endoderm and Reichert’s membrane; 17: Lumbar vertebra; 18: Wing of ilium; 19: Epaxial muscles; 20: Cecum; 21: Horn of uterus.
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Figure 10-6. Uterus of twelve and half days of gestation. A) The abdominal muscles were removed. B) The intestines were displaced cranially to observe the uterine topography. C and E) Magnetic resonance images. Horizontal and transverse sections, respectively. D) Isolated gravid uterus. 1: Right lateral lobe of liver; 2: Left lateral lobe of liver; 3: Stomach; 4: Duodenum; 5: Jejunum; 6: Cecum; 7: Right horn of uterus; 8: Left horn of uterus; 9: Right ovary; 10: Left ovary; 11: Lumbar vertebra; 12: Free border; 13: Mesometrial border; 14: Wing of ilium; 15: Epaxial muscles.
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Dorsal Lateral Figure 10-7. Embryo and yolk sac of twelve and half days of gestation (E12.5). A) Embryo within the horn of uterus. B) Histological section of the horn of gravid uterus. Immunodetection of a-SMA revealed with DAB (brown). Harris’ hematoxylin counterstain (20X). C) Isolated embryo within the yolk sac and placenta. D) Histological section of the embryo and placenta. Hematoxylin-eosin stain (20X). E) Magnetic resonance image. F) Ultrasound image. 1: Myometrium; 2: Placenta; 3: Labyrinth; 4: Spongiotrophoblast; 5: Decidua; 6: Embryo; 7: Amnion; 8: Yolk sac cavity; 9: Yolk sac; 10: Umbilical cord.
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Figure 10-8. Embryo of twelve and half days of gestation (E12.5). A) Embryo within the amnion. B) Sagittal histological section of isolated embryo. Hematoxylin-eosin stain (20X). C) Magnetic resonance image. Sagittal section. D and E) Magnetic resonance images. Transverse sections. 1: Telencephalon; 2: Diencephalon; 3: Mesencephalon; 4: Rombencephalon; 5: Spinal cord; 6: Nasal cavity; 7: Vomeronasal organ; 8: Internal ear; 9: Mandibular process (first branchial arch); 10: Ventricle; 11: Atrium; 12: Lung; 13: Liver; 14: Umbilical cord; 15:Amnion; 16: Yolk sac cavity; 17: Forelimb bud; 18: Hindlimb bud; 19: Placenta.
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6 5 Figure 10-9. Uterus of fifteen days of gestation. A) The gravid uterus was removed from the abdomen. B and C) Magnetic resonance images. Horizontal and transverse sections, respectively. 1: Caudal vena cava; 2: Right ovarian vein; 3: Uterine vein; 4: Radial veins; 5: Fetuses; 6: Placenta; 7: Descending colon; 8: Stomach; 9: Cecum; 10: Lumbar vertebra; 11: Wing of ilium; 12: Epaxial muscles.
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E 22 17 Figure 10-10. Fetus of fifteen days of gestation (E15). A) Fetus inside of the horn of uterus. B) Fetus inside of yolk sac. C) Sagittal histological section of fetus inside the chorionic yolk sac. Hematoxylin-eosin stain (20X). D) Magnetic resonance image. Sagittal section. E) Ultrasound image. 1: Placenta; 2: Lateral ventricle (telencephalon); 3: Diencephalon; 4: Mesencephalon; 5: Fourth ventricle (rombencephalon); 6: Trigeminal ganglion; 7: Tongue; 8: Meckel’s cartilage; 9: Nasal cavity; 10: Upper lip; 11: Jejunum (physiological umbilical hernia); 12: Umbilical cord; 13: Genital tubercle; 14: Liver; 15: Duodenum; 16: Heart; 17: Eyeball; 18: Ribs; 19: Humerus; 20: Femur; 21: Tail; 22: Yolk sac; 23: Yolk sac cavity.
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Figure 10-11. Fetus of fifteen days of gestation (E15). Magnetic resonance images. Transverse sections. 1: Telencephalon; 2: Lateral ventricles; 3: Diencephalon; 4: Third ventricle; 5: Junction between mesencephalon and pons; 6: Fourth ventricle; 7: Rombencephalon; 8: Spinal cord; 9: Trigeminal ganglion; 10: External ear; 11: Internal ear; 12: Eyeball; 13: Left lung; 14: Right lung; 15: Left ventricle; 16: Right ventricle; 17: Left atrium; 18: Right atrium; 19: Left lateral lobe of liver; 20: Right lateral lobe of liver; 21: Caudate lobe (liver); 22: Stomach; 23: Genital tubercle; 24: Thoracic aorta; 25: Abdominal aorta; 26: Left forepaw; 27: Right forepaw; 28: Left hindpaw; 29: Right hindpaw; 30: Tail; 31: Umbilical cord; 32: Yolk sac cavity; 33: Yolk sac.
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Figure 10-12. Sex identification in fetus of fifteen days of gestation (E15). Note the greater distance between the anus and the genital tubercle in the male fetus. 1: Anus; 2: Genital tubercle; 3: Tail; 4: Right hindpaw; 5: Left hindpaw.
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Figure 10-13. Fetus of fifteen days of gestation (E15). Magnetic resonance images. Horizontal sections. 1: Telencephalon; 2: Lateral ventricle; 3: Diencephalon; 4: Mesencephalon; 5: Aqueduct of mesencephalon; 6: Rombencephalon; 7: Fourth ventricle; 8: Spinal cord; 9: Eyeball; 10: Tongue; 11: Nasal cavity; 12: Right atrium; 13: Right cranial vena cava; 14: Left cranial vena cava; 15: Right external jugular vein: 16: Left external jugular vein; 17: Caudal vena cava; 18: Right lateral lobe of liver; 19: Left lateral lobe of liver; 20: Stomach; 21: Right kidney; 22: Left kidney; 23: Thoracic aorta; 24: Right thoracic limb; 25: Left thoracic limb; 26: Right pelvic limb; 27: Left pelvic limb; 28: Ribs; 29: Tail; 30: Genital tubercle; 31: Umbilical cord; 32: Placenta; 33: Labyrinth; 34: Spogiotrophoblast; 35: Decidua; 36: Yolk sac cavity.
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Figure 10-14. Umbilical cord connecting fetus and placenta (E15). The yolk sac was removed and the umbilical cord was stretched along its length.
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Figure 10-15. Placenta of seven and half days of gestation. A) Ectoplacental cone. Hematoxylin-eosin stain (200X). B) Magnification of Reichert’s membrane (1,000X). 1: Ectoplacental cone; 2: Yolk sac cavity; 3: Ectoplacental cavity; 4: Extraembryonic mesoderm; 5: Visceral endoderm (extraembryonic); 6: Parietal endoderm (extraembryonic); 7: Trophoblast; 8: Reichert’s membrane; 9: Exocoelomic cavity; 10: Chorion; 11: Maternal blood.
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Figure 10-16. Placenta of twelve and half days of gestation. A) Transverse section. Hematoxylin-eosin stain (20X). B) Labyrinth (200X). C) Spongiotrophoblast (100X). D) Magnification of the fetal and maternal erythrocytes (1,000X). E) Magnification of giant cells (1,000X). 1: Yolk sac cavity; 2: Chorionic plate; 3: Labyrinth; 4: Spongiotrophoblast; 5: Giant cells; 6: Decidua; 7: Fetal erythrocytes (nucleated); 8: Maternal erythrocytes (anucleated); 9: Glycogen trophoblast cells; 10: Syncytiotrophoblast.
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Figure 10-17. Placenta of fifteen days of gestation. A) Transverse section. Hematoxylin-eosin stain (20X). B) Labyrinth (400X). C) Spongiotrophoblast (400X). D) Decidua (400X). 1: Decidua; 2: Spongiotrophoblast; 3: Labyrinth; 4. Spiral arteries; 5: Central arterial canal; 6: Fetal erythrocytes (nucleated); 7: Maternal erythrocytes (anucleated); 8: Syncytiotrophoblast; 9: Chorionic plate.
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Figure 10-18. Placenta of fifteen days of gestation. A) Transverse section. Staining with Lycopersicon esculentum lectin revealed with DAB (brown). Nuclei counterstained with Harris’ hematoxylin (20X). B) Labyrinth (400X). C) Spongiotrophoblast (400X). D) Decidua (400X). 1: Decidua; 2: Spongiotrophoblast; 3: Labyrinth; 4: Spiral arteries; 5: Chorionic plate.