18 CHAPTER 1 BLE 1.1 Summary of major morphogenic processes regulated by mesenchymal and epithelial ce Example MESENCHYMAL CELLS Condensation Mesenchyme becomes Cartilage mesenchyme Cell division Mitosis produces more tl mb mesenchyme Cell death Cells die interdigital chyme Migration Cells move at particular Heart mesenchyme atrix secretion and Synthesis or removal of Cartilage mesenchym degradation Growth Cells get large Fat cells EPITHELIAL CELLS opers Epithelium becomes 9a Mullerian duct mesenchyme(entire degeneration structure) Delamination Chick hypoblast structure Shape change or growth Cells remain attached as Neurulation morphology is altered 国+迎 Cell migration(intercalation) Rows of epithelia merge to form fewer rows 图图A+ Vertebrate gastrulation Cell division Mitosis within row or colum at- ccam Vertebrate gastrulation Matrix secretion and Synthesis or removal CErn Vertebrate organ degradation of extracellular matrix formation Migration Formation of free edges → Chick ectoderm Changes in the composition of the cell membrane or secreted allow the migration of their neighboring cells. Extracel- products. Cell membranes and secreted cell products lular matrices made by other cell types will prohibit the influence the behavior of neighboring cells. For instance, migration of the same set of cells. In this way, "paths and extracellular matrices secreted by one set of cells will guiderails"are established for migrating cells
18 CHAPTER 1 TABLE 1.1 Summary of major morphogenic processes regulated by mesenchymal and epithelial cells Process MESENCHYMAL CELLS Condensation Cell division Cell death Action Mesenchyme becomes epithelium Mitosis produces more cells (hyperplasia) Cells die Morphology <*V £* ^ rrf". Example Cartilage mesenchyme Limb mesenchyme Interdigital mesenchyme Migration Cells move at particular times and places c w \ /*• Heart mesenchyme Matrix secretion and degradation Synthesis or removal of extracellular layer •ss^ Cartilage mesenchyme Growth Cells get larger (hypertrophy) -SET Fat cells EPITHELIAL CELLS Dispersal Delamination Epithelium becomes mesenchyme (entire structure) Epithelium becomes mesenchyme (part of structure) <3>|j • Qja Mullerian duct Sj%<3J degeneration v ,3 ®> " Chick hypoblast Shape change or growth Cells remain attached as morphology is altered EEEEE Neurulation Cell migration (intercalation) Rows of epithclia merge to form fewer rows '133 Vertebrate gastrulation Cell division Mitosis within row or column CZCHXZJ C3GSZE0 3 Vertebrate gastrulation Matrix secretion and degradation Migration Synthesis or removal of extracellular matrix Formation of free edges :'..::' J - Vertebrate organ formation Chick ectoderm Changes in the composition of the cell membrane or secreted products. Cell membranes and secreted cell products influence the behavior of neighboring cells. For instance, extracellular matrices secreted by one set of cells will allow the migration of their neighboring cells. Extracellular matrices made by other cell types will prohibit the migration of the same set of cells. In this way, "paths and guiderails" are established for migrating cells
DEVELOPMENTAL ANATOMY 19 Fate maps gram, and some of the results can be controversial. Recent examples of controversial fate maps include the map for Given such a dynamic situation, one of the most important the region of the frog embryo that specifies heart and blood programs of descriptive embryology became the tracing of cell precursors (Lane and Sheets 2006), and that for the cell lineages: following individual cells to see what those region of the embryonic turtle that becomes the bones of cells become. In many organisms, resolution of individual the plastron( Cebra-Thomas et al. 2007). As we will see later cells is not possible, but one can label groups of embryonic in this book, researchers are currently constructing, refin- cells to see what that area becomes in the adult organism. ing, and arguing about the fate maps of mammalian By bringing such studies together, one can construct a fate embryos map. These diagrams " map"larval or adult structures onto the region of the embryo from which they arose. Fate maps Direct observation of living embryos constitute an important foundation for experimental embry ology, providing researchers with information on which Some embryos have relatively few cells, and the cytoplasm rtions of the embryo normally become which larval or in each of the early blastomeres has a different pigment In tebrate embryos at the early gastrula stage. the microscope and trace the descendants of a particular ate maps can be generated in several ways, and the cell into the organs they generate. E. G. Conklin patiently technology has changed greatly over the past few years. followed the fates of each early cell of Styela partita, a tuni- Construction of these maps is an ongoing research pro- cate(sea squirt) that resides in waters off the coast of Mass- achusetts(Conklin 1905). The muscle-forming cells of the embryo always had a yellow color, derived from a region Zebrafish of cytoplasm found in the B4. 1 blastomere( Figure 1.12) Epiderma Conklin's fate map was confirmed by cell-removal expe iments. Removal of the B4.1 cell(which according to Con Neural klin's map should produce all the tail musculature)in fact resulted in a larva with no tail muscles(Reverberi and Min ganti 1946 mesoderm See WEBSITE 1.1 Conklin's art and science See vAdE MECUM The compound microscope Endoderm. Dye marking Mouse Chick Epidermal Most embryos are not so accommodating as to have cells of different colors. In the early years of the twentieth cen- Neural tury, Vogt(1929) traced the fates of different areas of ectoderm amphibian eggs by applying vital dyes to the region of interest. Vital dyes stain cells but do not kill them. Vogt notochord mixed such dyes with agar and spread the agar on a micro- Mesoderm scope slide to dry. The ends of the dyed agar were very thin. Vogt cut chips from these ends and placed them on a frog embryo. After the dye stained the cells, he removed the agar chips and could follow the stained cells'move- mesoderm ments within the embryo( Figure 1.13) One problem with vital dyes is that as they become IGURE 1 11 Fate maps of vertebrates at the carly gastrula stage All are dorsal surface views(looking" down"on the embryo at more diluted with each cell division, they become difficult to detect. One way around this is to use fluorescent dyes what will become its back). Despite the different appearances of that are so intense that once injected into individual cells the adult animals, fate maps of these four vertebrates show numerous similarities among the embryos. The cells that will form divisions later. Fluorescein-conjugated dextran, for exam the notochord occupy a central dorsal position, while the precur sors of the neural system lie immediately anterior to it. The neural ple, can be injected into a single cell of an early embryo, ectoderm is surrounded by less dorsal ectoderm, which will form and the descendants of that cell can be seen by examining the epidermis of the skin. A indicates the anterior end of the the embryo under ultraviolet light( Figure 1.14) P the posterior end. The dashed green lines indicate the See VADE MECUM site of ingression--the path cells will follow as they migrate from he exterior to the interior of the embryo Histotechniques
DEVELOPMENTAL ANATOMY 19 Fate maps Given such a dynamic situation, one of the most important programs of descriptive embryology became the tracing of cell lineages: following individual cells to see what those cells become. In many organisms, resolution of individual cells is not possible, but one can label groups of embryonic cells to see what that area becomes in the adult organism. 3v bringing such studies together, one can construct a fate map. These diagrams "map" larval or adult structures onto the region of the embryo from which they arose. Fate maps constitute an important foundation for experimental embryology, providing researchers with information on which portions of the embryo normally become which larval or adult structures. Figure 1.11 shows fate maps of some vertebrate embryos at the early gastrula stage. Fate maps can be generated in several ways, and the technology has changed greatly over the past few years. Construction of these maps is an ongoing research proEpidermal ectoderm Neural ectoderm Notochord Mesoderm Endodcrm Extraembryonic mesoderm FIGURE 1.11 Fate maps of vertebrates at the early gastrula stage. All are dorsal surface views (looking "down" on the embryo at what will become its back). Despite the different appearances of '.he adult animals, fate maps of these four vertebrates show numerous similarities among the embryos. The cells that will form Tie notochord occupy a central dorsal position, while the precursors of the neural system lie immediately anterior to it. The neural ectoderm is surrounded by less dorsal ectoderm, which will form Tie epidermis of the skin. A indicates the anterior end of the embryo, P the posterior end. The dashed green lines indicate the site of ingression—the path cells will follow as they migrate from the exterior to the interior of the embryo. gram, and some of the results can be controversial. Recent examples of controversial fate maps include the map for the region of the frog embryo that specifies heart and blood cell precursors (Lane and Sheets 2006), and that for the region of the embryonic turtle that becomes the bones of the plastron (Cebra-Thomas et al. 2007). As we will see later in this book, researchers are currentlv constructing, refining, and arguing about the fate maps of mammalian embryos. Direct observation of living embryos Some embryos have relatively few cells, and the cvtoplasm in each of the early blastomeres has a different pigment. Tn such fortunate cases, it is actually possible to look through the microscope and trace the descendants of a particular cell into the organs they generate. E. G. Conklin patiently followed the fates of each early cell of Styela partita, a tunicate (sea squirt) that resides in waters off the coast of Massachusetts (Conklin 1905). The muscle-forming cells of the embryo always had a yellow color, derived from a region of cytoplasm found in the B4.1 blastomere (Figure 1.12). Conklin's fate map was confirmed by cell-removal experiments. Removal of the B4.1 cell (which according to Conklin's map should produce all the tail musculature) in fact resulted in a larva with no tail muscles (Reverberi and Minganti 1946). See WEBSITE 1.1 Conklin's art and science See VADE MECUM The compound microscope Dye marking Most embryos are not so accommodating as to have cells of different colors. In the early years of the twentieth century, Vogt (1929) traced the fates of different areas of amphibian eggs by applying vital dyes to the region of interest. Vital dyes stain cells but do not kill them. Vogt mixed such dyes with agar and spread the agar on a microscope slide to dry. The ends of the dyed agar were very thin. Vogt cut chips from these ends and placed them on a frog embryo. After the dye stained the cells, he removed the agar chips and could follow the stained cells' movements within the embryo (Figure 1.13). One problem with vital dyes is that as they become more diluted with each cell division, they become difficult to detect. One way around this is to use fluorescent dyes that are so intense that once injected into individual cells, they can still be detected in the progeny of these cells many divisions later. Fluorescein-conjugated dextran, for example, can be injected into a single cell of an early embryo, and the descendants of that cell can be seen by examining the embryo under ultraviolet light (Figure 1.14). See VADE MECUM Histotechniques
20 CHAPTER 1 (A) Animal pole Nervous b4.2 neural Anterior Posterior A4.1 B4,1 Muscle Endoderm mesenchyme 2-cell 4-cell 8-cell 64-cel stage stage ge Derivative A7. 1 Endoderm FIGURE 1.12 Fate map of the tunicate embryo. (A)Confocal sec- A6.1 A7. 2 Endoderm on through a larva of the tunicate Ciona savigny The notochord cells are stained green; the cell boundaries are stained white. The A62CA.nOtochord endoderm is blue, the muscles red, the neural tube yellow, and A4.1 the epidermis magenta. (B) Zygote of Styela partita(left), shown A7.5 Endoderm shortly before the first cell division, with the fate of the cytoplas- mic regions indicated The 8-cell embryo on the right shows these A5.2 regions after three cell divisions. (C)A linear version of the S partita fate map, showing the fates of each cell of the embryo A7.8 caudal muscle Throughout this book, we will use the color onventions of developmental anatomy a710 blue for ectoderm red for mesoderm and a ellow for endoderm. (A from Veeman et al a6.6 →+a7.l1 Palps B after Nishida 1987 and Reverberi a7. 12 Epidermis Animal Minganti 1946: C after Conklin 1905 Nishida 1987 →a6.7 + a7. 14 Epide a7,1 Genetic labeling Half-embryo B71 Endoderm One way of permanently marking cells →B7.2 Endoderm and following their fates is to create embryos in which the same organism B6.2 B7.3 Mesenchyme contains cells with different genetic B7 4 Muscle B4.1 constitutions. In the 1920s, the German embryologists Hilde Mangold and B7.6 Hans Spemann performed some of the Mesenchyme most important experiments in the his +B7.8 Muscle i tory of embryology when they trans B3-Posterior planted embryonic tissues from one b7.9 caudal muscle species of newt into the embryo of a b7.10 Epidermis different newt species. These chimeric b5.3 ermis embryosembryos made from tissues of more than one genetic source- epidermis b4.2- Animal enabled Mangold and Spemann to tell b7.13 Epidermis b6.7 which structures arose from donor tis- b7.14 em →b5.4 sue and which from host tissue(see b7.15 Epiderm Figures 7 16 and 7. 17) b6.8rb7.16Epidermis
20 CHAPTER 1 Animal pole a4.2 ..J b4.2 Ectoderm Nervous system Anterior Notochord' Muscle \ A4.1 J B4.1 Posterior Endoderm Mesenchyme Vegetal pole 4-cell 8-cell stage stage FIGURE 1.12 Fate map of the tunicate embryo. (A) Confocal section through a larva of the tunicate Ciona savignyi. The notochord cells are stained green; the cell boundaries are stained white. The endoderm is blue, the muscles red, the neural tube yellow, and ^_ , . , the epidermis magenta. (B) Zygote of Styela partita (left), shown shortly before the first cell division, with the fate of the cytoplasmic regions indicated. The 8-cell embryo on the right shows these regions after three cell divisions. (C) A linear version of the 5. partita fate map, showing the fates of each cell of the embryo. i *• A3—{ Anterior Throughout this book, we will use the color conventions of developmental anatomy: blue for ectoderm, red for mesoderm, and yellow for endoderm. (A from Veeman et al. 2008; B after Nishida 1987 and Reverberi and Minganti 1946; C after Conklin 1905 and Nishida 1987.) Genetic labeling One way of permanently marking cells and following their fates is to create embryos in which the same organism contains cells with different genetic constitutions. In the 1920s, the German embryologists Hilde Mangold an d Hans Spemann performed some of the most important experiments in the history of embryology whe n they transplanted embryonic tissues from one species of newt into the embryo of a different newt species. These chimeric embryos—embryos made from tissues of more than one genetic source— enabled Mangold and Spemann to tell which structures arose from donor tissue and which from host tissue (see Figures 7.16 and 7.17). AB2 a4.2 Half-embryo B3 B4.1 Posterior b4.2 16-cell stage Endoderm 32-cell stage •Muscle Mesenchyme a5.3 Animal a5.4 a6.5 a6.6 a6.7 a6.8 64-ccll stage A7.1 A7.2 A7.3 A7.4 A7.5 A7.6 A7.8- a7.9 a7.10 a7.11 a7.12 a7.13 a7.14 a7.15 a7.16 B7.1 B7.2 B7.3 B7.4 B7.5 B7.6 B7.7 B7.8 b7.9 b7.10 b7.11 b7.12 b7.13 b7.14 b7.15 b7.16 Derivative Endoderm Endoderm Notochord Brain stem Endoderm Notochord Notochord f Spinal cord, \^caudal muscle Brain Brain Palps Epidermis Sense organ Epidermis Epidermis Epidermis Endoderm Endoderm Mesenchyme Muscle Muscle Muscle Mesenchyme Muscle Epidermis, caudal muscle Epidcrmis Epidermis Epidermis Epidermis Epidermis Epidermis Epidermis
DEVELOPMENTAL ANATOMY 21 Agar chil Dye stains Section plane with dye (B) (D) Dorsal lip of blastopore FIGURE 1. 13 Vital dye staining method for marking specific cells egg, and the chick that hatches will have quail cells in p e of amphibian embryos. (A) Vogt's can be performed on an embryo while it is still inside of the embryonic surface with ticular sites, depending on where the graft was placed vital dyes (B-D) Dorsal surface views of stain on successively later embryos. (E) Newt embryo First, the quail nucleus has condensed DNA (heterochro dissected in a medial sagittal sec- matin)concentrated around the nucleoli, making quail tion to show the stained cells nuclei easily distinguishable from chick nuclei. Second, the interior. (After Vogt 1929) cell-specific antigens that are quail-specific can be used to find individual quail cells, even if they are"hidden"with in a large population of chick cells. In this way, fine-struc ture maps of the chick brain and skeletal system have been struction of chimeric embryos by gratting quail cells ins produced (Figu One of the best examples of this technique is the con- produced(Figure 1. 15; Le Douarin 1969; Le D ouarn an millet 1973) a chick embryo. Chicks and quail embryos develop in a In addition, the chick-quail chimeras dramatically con- similar manner(especially during the early stages devel- firmed the extensive cell migrations taken by neural crest pment), and the grafted quail cells become integrated into ells during vertebrate development. Mary Rawles (1940) the chick embryo and participate in the construction of the showed that the pigment cells(melanocytes)of the chick various organs. The substitution of quail cells for chick cells originate in the neural crest, a transient band of cells that euna Midbrain Hindbrain ③朝 forebrain forebrain FIGURE 1. 14 Fate mapping using a fluorescent dye. (A) Specific cells of a zebrafish embryo were injected with a fluorescent dye that will not diffuse from the cells. the dye was the activated by laser in a small region (about 5 cells)of the late cleavage stage embryo. (B)After formation of the central nervous system had begun, cells that expressed the active dye were visualized by fluorescent light. The fluorescent dye is seen in particular cells that generate the forebrain and midbrain. ( C) Fate map of the zebrafish central nervous system. Dye was injected into cells 6 hours after fertilization(left), and the results are color-coded onto the hatched fish(right). Overlapping colors indicate that cells from these regions of the 6-hour embryo contribute to two or more regions. (A, B from Kozlowski et al. 1998, photographs courtesy of E. Weinberg: C after Woo and Fraser 1995.)
DEVELOPMENTAL ANATOMY 21 Agar chips with dye Dye stains on embryo • Section plane ', of view (E) Dorsal lip of blastopore (where cells begin to enter the embryo) (E) FIGURE 1.13 Vital dye staining of amphibian embryos. (A) Vogt's method tor marking specific cells of the embryonic surface with vital dyes. (B-D) Dorsal surface views of stain on successively later embryos. (E) Newt embryo dissected in a medial sagittal section to show the stained cells in the interior. (After Vogt 1929.) One of the best examples of this technique is the construction of chimeric embryos by grafting quail cells inside a chick embryo. Chicks and quail embryos develop in a similar manner (especially during the early stages development), and the grafted quail cells become integrated into the chick embryo and participate in the construction of the various organs. The substitution of quail cells for chick cells can be performed on an embryo while it is still inside the egg, and the chick that hatches will have quail cells in particular sites, depending on where the graft was placed. Quail cells differ from chick cells in two important ways. First, the quail nucleus has condensed DNA {heterochromatin) concentrated around the nucleoli, making quail nuclei easily distinguishable from chick nuclei. Second, cell-specific antigens that are quail-specific can be used to find individual quail cells, even if they are "hidden" within a large population of chick cells. In this w'ay, fine-structure maps of the chick brain and skeletal system have been produced (Figure 1.15; Le Douarin 1969; Le Douarin and Teillet 1973). In addition, the chick-quail chimeras dramatically confirmed the extensive cell migrations taken by neural crest cells during vertebrate development. Mary Rawles (1940) showed that the pigment cells (melanocytes) of the chick originate in the neural crest, a transient band of cells that (Rl (C) Ventral Animal pole (eg; viewed from top) FIGURE 1.14 Fate mapping using a fluorescent dye. (A) Specific cells of a zebratish embryo were injected with a fluorescent dye that will not diffuse from the cells. The dye was then activated by laser in a small region (about 5 cells) of the late cleavage stage embryo. (B) After formation of the central nervous system had begun, cells that expressed the active dye were visualized by fluorescent light. The fluorescent dye is seen in particular cells that generate the forebrain and midbrain. (C) Fate map of the zebrafish central nervous system. Dye was injected into cells 6 hours after fertilization (left), and the results are color-coded onto the hatched fish (right). Overlapping colors indicate that cells from these regions of the 6-hour embryo contribute to two or more regions. (A,B from Kozlowski et al. 1998, photographs courtesy of E. Weinberg; C after Woo and Frascr 1995.) Retina Midbrain Hindbrain
22 CHAPTER 1 24 h(host) Chick cells Quail cells o riment wherein the cells from a particular regione/oay 3 URE 1.15 Genetic markers as cell lineage tracers. (A) Grafti embryo have been placed into a similar region of a 1-day chick embryo. After several days, the quail cells can be seen by using an antibody to quail-specific proteins This region of th lay embryo produces cells that populate the neural tube. (B)Chick and quail cells can also be distinguished by the heterochromatin of their nuclei. The quail cells have a single large nucleolus(dense purple), distinguishing them from the diffuse nuclei of the chick Chick embryo with region of quail cells on the neural tube (From Darnell and Schoenwolf 1997, courtesy of the autho joins the neural tube to the epidermis. When she trans- plant cells from a genetically modified organism. In suc a pigmented strain of chickens into a similar position in an only to those cells that express it. One version is to infect embryo from an unpigmented strain of chickens, the the cells of an embryo with a virus whose genes have been migrating pigment cells entered the epidermis and later altered such that they express the gene for a fluorescently entered the feathers(Figure 1.16). Ris(1941)used similar active protein such as GFP. *(This type of altered gene is echniques to show that while almost all of the external pigment of the chic embryo came from the migrating neu- *GFP-gre orescent protein-occurs naturally in certain jelly na itself and was not dependent on the migrating neural light and i green fluorescence when exposed to ultraviolet used as a transgenic label for cells in develop- crest cells. This pattern was confirmed in chick-quail mental and other research hybrids, in which the quail neural crest cells produced their own pigment and pattern in the chick feathers See VADE MECUm Chick-quail chimeras Transgenic DNA chimeras In most animals, it is difficult to meld a chimera from two species. One way of circumventing this problem is to trans- E 1.16 Chick resulting from transplantation of a trunk neural crest region from an of a pigmented strain of chickens into the same region of an embryo of an unpig q mented strain. The neural crest cells that gave rise to the pigment migrated into the wing epidermis and feathers. (From the archives of BH.Willie
22 CHAPTER 1 (B) Quail embryo 24 h (donor) Quail cells . • - - ] -a?-ijs? I U ***<* * "* * ..-..•. .... :^*,v.%> r - . . . '" t . •- . ."• > '.,... ...*:••• :• •••- Quail cells - ^ -Chick cells •• Chick embryo with region of quail cells on the neural tube FIGURE 1.15 Genetic markers as cell lineage tracers. (A) Grafting experiment wherein the cells from a particular region of a 1-day quail embryo have been placed into a similar region of a 1-day chick embryo. After several days, the quail cells can be seen by using an antibody to quail-specific proteins. This region of the 3- day embryo produces cells that populate the neural tube. (B) Chick and quail cells can also be distinguished by the heterochromatin of their nuclei. The quail cells have a single large nucleolus (dense purple), distinguishing them from the diffuse nuclei of the chick. (From Darnell and Schoenwolf 1997, courtesy of the authors.) joins the neural tube to the epidermis. When she transplanted small regions of neural crest-containing tissue from a pigmented strain of chickens into a similar position in an embryo from an unpigmented strain of chickens, the migrating pigment cells entered the epidermis and later entered the feathers (Figure 1.16). Ris (1941) used similar techniques to show that while almost all of the external pigment of the chick embryo came from the migrating neural crest cells, the pigment of the retina formed in the retina itself and was not dependent on the migrating neural crest cells. This pattern was confirmed in chick-quail hybrids, in which the quail neural crest cells produced their own pigment and pattern in the chick feathers. See VADE MECUM Chick-quail chimeras Transgenic DNA chimeras In most animals, it is difficult to meld a chimera from two species. One way of circumventing this problem is to transplant cells from a genetically modified organism. In such a technique, the genetic modification can then be traced only to those cells that express it. One version is to infect the cells of an embryo with a virus whose genes have been altered such that they express the gene for a fluorescently active protein such as GFR* (This type of altered gene is *GFP—green fluorescent protein—occurs naturally in certain jellyfish. It emits bright green fluorescence when exposed to ultraviolet light and is widely used as a transgenic label for cells in developmental and other research. FIGURE 1.16 Chick resulting from transplantation of a trunk neural crest region from an embryo of a pigmented strain of chickens into the same region of an embryo of an unpigmented strain. The neural crest cells that gave rise to the pigment migrated into the wing epidermis and feathers. (From the archives of B. H. Willier.)